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8 Virtually all the microbial traits you have read about in earlier chapters are controlled or influenced by heredity. The inherited characteristics of microbes include shape, structural features, metabolism, ability to move, and interactions with other organisms. Individual organisms transmit these characteristics to their offspring through genes.

The development of antibiotic resistance in microorganisms is often carried on plasmids such as those in the photo, which are readily transferred between bacterial cells. They are responsible for the emergence of methicillin-resistant Staphylococcus aureus and the recent emergence of carbapenem- resistant Klebsiella pneumoniae. The emergence of vancomycin- resistant S. aureus (VRSA) poses a serious threat to patient care. In this chapter you will see how VRSA acquired this characteristic.

Emerging diseases provide another reason why it is important to understand genetics. New diseases are the results of genetic changes in some existing organism; for example, E. coli O157:H7 acquired the genes for Shiga toxin from Shigella.

Currently, microbiologists are using genetics to study unculturable microbes and the relationship between hosts and microbes.

The Big Picture on pages 206–207 highlights key principles of genetics that are explained in greater detail throughout the chapter.

In the Clinic As a nurse at a U.S. military hospital, you treat service members injured in the recent Middle East conflicts. You notice that wounds infected by Acinetobacter baumannii are not responding to antibiotics. The Centers for Disease Control and Prevention reports that the antibiotic- resistance genes found in A. baumannii are the same as those in Pseudomonas, Salmonella, and Escherichia. Cephalosporin-resistance genes are on the chromosome, tetracycline resistance is encoded by a plasmid, and streptomycin resistance is associated with a transposon. Can you suggest mechanisms by which Acinetobacter acquired this resistance?

Hint: Read about genetic recombination on pages 229–235.

Microbial Genetics

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▶ Plasmids exist in cells separate from chromosomes.

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CHAPTER 8 Microbial Genetics 205

Structure and Function of the Genetic Material LEARNING OBJECTIVES

8-1 Define genetics, genome, chromosome, gene, genetic code, genotype, phenotype, and genomics.

8-2 Describe how DNA serves as genetic information.

8-3 Describe the process of DNA replication.

8-4 Describe protein synthesis, including transcription, RNA processing, and translation.

8-5 Compare protein synthesis in prokaryotes and eukaryotes.

Genetics is the science of heredity. It includes the study of genes: how they carry information, how they replicate and pass to sub- sequent generations of cells or between organisms, and how the expression of their information within an organism determines its characteristics. The genetic information in a cell is called the genome. A cell’s genome includes its chromosomes and plas- mids. Chromosomes are structures containing DNA that physi- cally carry hereditary information; the chromosomes contain the genes. Genes are segments of DNA (except in some viruses, in which they are made of RNA) that code for functional prod- ucts. Usually these products are proteins, but they can also be RNAs (ribosomal RNA, transfer RNA, or microRNA).

We saw in Chapter 2 that DNA is a macromolecule composed of repeating units called nucleotides. Each nucleotide consists of a nucleobase (adenine, thymine, cytosine, or guanine), deoxyri- bose (a pentose sugar), and a phosphate group (see Figure 2.16, page 45). The DNA within a cell exists as long strands of nucle- otides twisted together in pairs to form a double helix. Each strand has a string of alternating sugar and phosphate groups (its sugar-phosphate backbone), and a nitrogenous base is attached to each sugar in the backbone. The two strands are held together by hydrogen bonds between their nitrogenous bases. The base pairs always occur in a specific way: adenine always pairs with thymine, and cytosine always pairs with guanine. Because of this specific base pairing, the base sequence of one DNA strand determines the base sequence of the other strand. The two strands of DNA are thus complementary.

The structure of DNA helps explain two primary features of biological information storage. First, the linear sequence of bases provides the actual information. Genetic information is encoded by the sequence of bases along a strand of DNA, in much the same way as our written language uses a linear sequence of letters to form words and sentences. The genetic language, however, uses an alphabet with only four letters—the four kinds of nucleobases in DNA (or RNA). But 1000 of these four bases, the number contained in an average-sized gene, can be arranged in 41000 different ways. This astronomically large number explains how genes can be varied enough to provide all the information a cell needs to grow and perform its func- tions. The genetic code, the set of rules that determines how a

nucleotide sequence is converted into the amino acid sequence of a protein, is discussed in more detail later in this chapter.

Second, the complementary structure allows for the pre- cise duplication of DNA during cell division. Each offspring cell receives one of the original strands from the parent, thus ensuring one strand that functions correctly.

Much of cellular metabolism is concerned with translating the genetic message of genes into specific proteins. A gene is usually copied to make a messenger RNA (mRNA) molecule, which ultimately results in the formation of a protein. When the ultimate molecule for which a gene codes (a protein, for example) has been produced, we say that the gene has been expressed. The flow of genetic information can be shown as flowing from DNA to RNA to proteins, as follows:

RNA ProteinDNA

This theory was called the central dogma by Francis Crick in 1956, when he first proposed that the sequence of nucleotides in DNA deter- mines the sequence of amino acids in a protein.

Genotype and Phenotype The genotype of an organism is its genetic makeup—all its DNA—the information that codes for all the particular char- acteristics of the organism. The genotype represents potential

ASM: Although the central dogma is universal in all cells, the processes differ

in prokaryotes and eukaryotes, as we shall see in this chapter.

205 224 227 234

CLINICAL CASE Where There’s Smoke

Marcel DuBois, a 70-year-old grandfather of 12, quietly hangs up the phone. His doctor has just called him with the results of his stool DNA test that he undertook at the Mayo Clinic last week. Marcel’s doctor suggested this new, noninvasive screening tool for colorectal cancer because Marcel is not comfortable with the colonoscopy procedure and usually tries to postpone getting one. The stool DNA test, however, uses stool samples, which contain cells that have been shed from the colon lining. The DNA from these cells is tested for DNA markers that may indicate the presence of precancerous polyps or cancerous tumors. Marcel makes an appointment to come in to see his doctor the next afternoon.

Once in the office, the doctor explains to Marcel and his wife, Janice, that the stool DNA test detected the presence of serrated colorectal polyps. This type of polyp is usually difficult to see with a colonoscopy because it is not raised and can be the same color as the colon wall.

How can DNA show whether a person has cancer? Read on to find out.

206

GeneticsBIG PICTURE Genetics is the science of heredity. It includes the study of genes: how they are replicated, expressed, and passed on from one generation to another. The central dogma of molecular biology describes how, typically, DNA is transcribed to messenger RNA, which, in turn, is translated into proteins that carry out vital cellular functions. Mutations introduce change into this process—ultimately leading to new or lost functions.

How mutations alter a genome

DNA

mRNA

Protein

Function

Mutated DNA

Altered mRNA

Altered protein

Altered function

Typical chain of events described by

central dogma

Mutations can be caused by base substitutions or frameshift mutations.

In base substitution mutations, a single DNA base pair is altered.

T A C T T C A

A U G A A G T

T A A T T C A

A U A A G TT

In frameshift mutations, DNA base pairs are added or removed from the sequence, causing a shift in the sequence reading.

T A C T T C A

A U G A A G T

T A T T C A

A U A A G T

Groups of genes in operons can be inducible or repressible.

Active repressor

DNA

Inactive repressor

Inducer

“OFF” (gene not expressed)

“ON” (gene expressed)

An inducible operon includes genes that are in the “off” mode, with the repressor bound to the DNA, and is turned “on” by the environmental inducer.

“ON” (gene expressed)

“OFF” (gene not expressed)

A repressible operon includes genes that are in the “on” mode, without the repressor bound to the DNA, and is turned “off” by the environmental corepressor and repressor.

DNA

Inactive repressor

Active repressor

Corepressor

AFM 7 nmAtomic force micrograph showing DNA molecules.

207

Alteration of bacterial genes and/or gene expression may cause disease, prevent disease treatment, or be manipulated for human benefit.

TEM 0.4 mm

Diseases: Many bacterial diseases are caused by the presence of toxic proteins that damage human tissue. These toxic proteins are coded for by genes. Vibrio cholerae, shown above, produces an enterotoxin that causes diarrhea and severe dehydration, which can be fatal if left untreated.

Antibiotic resistance: Mutations in the bacterial genome are one of the first steps toward the development of antibiotic resistance. This process has occurred with Staphylococcus aureus, which is currently resistant to beta-lactam antibiotics such as penicillin. Methicillin was introduced to treat penicillin-resistant S. aureus. Methicillin-resistant S. aureus (MRSA), shown in purple above, is now a leading cause of healthcare-associated infections.

SEM 0.3 mm

Biofilms: Biofilms, such as the one seen here growing on a toothbrush bristle, are produced by altered bacterial gene expression when populations are large enough. Various Streptococcus species, including S. mutans, form biofilms on teeth and gums, contributing to the development of dental plaque and dental caries.

SEM 5 mm

Biotechnology: Scientists can alter a microorganism’s genome, adding genes that will produce human proteins used in treating disease. Insulin, used for treatment of diabetes, is produced in this manner.

DNA expression leads to cell function via the production of proteins.

Genes in operons are turned on or off together.

Mutations alter DNA sequences.

DNA mutations can change bacterial function.

●

KEY CONCEPTS

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208 PART ONE Fundamentals of Microbiology

properties, but not the properties themselves. Phenotype refers to actual, expressed properties, such as the organism’s ability to perform a particular chemical reaction. Phenotype, then, is the manifestation of genotype. For example, E. coli with the stx gene can produce the stx (Shiga toxin) protein.*

In a sense, an organism’s phenotype is its collection of pro- teins, because most of a cell’s properties derive from the struc- tures and functions of proteins. In microbes, most proteins are either enzymatic (catalyze particular reactions) or structural (participate in large functional complexes such as membranes or flagella). Even phenotypes that depend on structural mac- romolecules such as lipids or polysaccharides rely indirectly on proteins. For instance, the structure of a complex lipid or polysaccharide molecule results from catalytic activities of enzymes that synthesize, process, and degrade those molecules. Thus, saying that phenotypes are due to proteins is a useful simplification.

DNA and Chromosomes Bacteria typically have a single circular chromosome consist- ing of a single circular molecule of DNA with associated pro- teins. The chromosome is looped and folded and attached at one or several points to the plasma membrane. The DNA of E. coli has about 4.6 million base pairs and is about 1 mm long—1000 times longer than the entire cell (Figure 8.1). How- ever, the chromosome takes up only about 10% of the cell’s volume because the DNA is twisted, or supercoiled.

The entire genome does not consist of back-to-back genes. Noncoding regions called short tandem repeats (STRs) occur in most genomes, including that of E. coli. STRs are repeating sequences of two- to five-base sequences. These are used in DNA fingerprinting (discussed on page 258).

Now, the complete base sequences of chromosomes can be determined. Computers are used to search for open reading frames, that is, regions of DNA that are likely to encode a protein. As you will see later, these are base sequences between start and stop codons. The sequencing and molecular charac- terization of genomes is called genomics. The use of genomics to track Zika virus is described in the Clinical Focus box on page 218.

The Flow of Genetic Information DNA replication makes possible the flow of genetic informa- tion from one generation to the next. This is called vertical gene transfer. As shown in Figure 8.2, the DNA of a cell repli- cates before cell division so that each offspring cell receives a chromosome identical to the parent’s. Within each metaboliz- ing cell, the genetic information contained in DNA also flows in another way: it is transcribed into mRNA and then trans- lated into protein. We describe the processes of transcription and translation later in this chapter.

Chromosome

1 mm TEM

Figure 8.1 A prokaryotic chromosome.

How many times longer than the 2-mm cell is the chromosome? Q

CHECK YOUR UNDERSTANDING

✓ 8-1 Give a clinical application of genomics.

✓ 8-2 Why is the base pairing in DNA important?

DNA Replication In DNA replication, one “parental” double-stranded DNA mol- ecule is converted to two identical offspring molecules. The complementary structure of the nitrogenous base sequences in the DNA molecule is the key to understanding DNA replica- tion. Because the bases along the two strands of double-helical DNA are complementary, one strand can act as a template for the production of the other strand (Figure 8.3a).

DNA replication requires the presence of several cellular proteins that direct a particular sequence of events. Enzymes involved in DNA replication and other processes are listed in Table 8.1. When replication begins, the supercoiling is relaxed by topoisomerase or gyrase. The two strands of parental DNA are unwound by helicase and separated from each other in one small DNA segment after another. Free nucleotides present in the cell cytoplasm are matched up to the exposed bases of the single-stranded parental DNA. Where thymine is present on the original strand, only adenine can fit into place on the new strand; where guanine is present on the original strand, only cytosine can fit into place, and so on. Any bases that are improperly base-paired are removed and *Gene names are italicized, but the protein name is not italicized.

CHAPTER 8 Microbial Genetics 209

●

Genetic information can be transferred horizontally between cells of the same generation.

Genetic information can be transferred vertically to the next generation of cells.

Genetic information is used within a cell to produce the proteins needed for the cell to function.

New combinations of genes

Offspring cells

Transcription

DNA

Parent cell

Cell metabolizes and grows Recombinant cell

Translation

expression recombination replication

DNA is the blueprint for a cell’s proteins, including enzymes.

DNA is obtained either from another cell in the same generation or from a parent cell during cell division.

DNA can be expressed within a cell or transferred to another cell through recombination and replication.

KEY CONCEPTS

The Flow of Genetic Information FOUNDATION

FIGURE 8.2

replaced by replication enzymes. Once aligned, the newly added nucleotide is joined to the growing DNA strand by an enzyme called DNA polymerase. Then the parental DNA is unwound a bit further to allow the addition of the next nucleotides. The point at which replication occurs is called the replication fork.

As the replication fork moves along the parental DNA, each of the unwound single strands combines with new nucleotides. The original strand and this newly synthesized daughter strand then rewind. Because each new double-stranded DNA molecule contains one original (conserved) strand and one new strand, the process of replication is referred to as semiconservative replication.

Before looking at DNA replication in more detail, let’s dis- cuss the structure of DNA (see Figure 2.16 on page 45 for an overview). It is important to understand that the paired DNA strands are oriented in opposite directions (antiparallel) rela- tive to each other. The carbon atoms of the sugar component of each nucleotide are numbered 1′ (pronounced “one prime”)

to 5′. For the paired bases to be next to each other, the sugar components in one strand are upside down relative to the other. The end with the hydroxyl attached to the 3′ carbon is called the 3′ end of the DNA strand; the end having a phos- phate attached to the 5′ carbon is called the 5′ end. The way in which the two strands fit together dictates that the 5′ S 3′ direction of one strand runs counter to the 5′ S 3′ direction of the other strand (Figure 8.3b). This structure of DNA affects the replication process because DNA polymerases can add new nucleotides to the 3′ end only. Therefore, as the replication fork moves along the parental DNA, the two new strands must grow in different directions.

One new strand, called the leading strand, is synthesized con- tinuously in the 5′ S 3′ direction (from a template parental strand running 3′ S 5′). In contrast, the lagging strand of the new DNA is synthesized discontinuously in fragments of about 1000 nucleotides, called Okazaki fragments. These must be joined later to make the continuous strand.

209

210 PART ONE Fundamentals of Microbiology

Parental strand

3¿ end Daughter

strand forming

5¿ end Daughter

strand Parental strand

Parental strand

Replication fork

3¿ end

5¿ end

Deoxyribose sugar

Phosphate

(a) The replication fork

(b) The two strands of DNA are antiparallel. The sugar-phosphate backbone of one strand is upside down relative to the backbone of the other strand. Turn the book upside down to demonstrate this.

T A

KEY

1 The double helix of the parental DNA separates as weak hydrogen bonds between the nucleotides on opposite strands break in response to the action of replication enzymes.

Hydrogen bonds form between new complementary nucleotides and each strand of the parental template to form new base pairs.

Enzymes catalyze the formation of sugar-phosphate bonds between sequential nucleotides on each resulting daughter strand.

O O

–O

H2C

5¿ end

3¿ end

5¿ end

3¿ end

O O

O–

CH2

O O

HO O

C G

CG G

G C

T AT

T T

Adenine ThymineA T

Guanine CytosineG C

Figure 8.3 DNA replication.

What is the advantage of semiconservative replication? Q

TABLE 8.1 Important Enzymes in DNA Replication, Expression, and Repair

DNA Gyrase Relaxes supercoiling ahead of the replication fork

DNA Ligase Makes covalent bonds to join DNA strands; Okazaki fragments, and new segments in excision repair

DNA Polymerases Synthesize DNA; proofread and facilitate repair of DNA

Endonucleases Cut DNA backbone in a strand of DNA; facilitate repair and insertions

Exonucleases Cut DNA from an exposed end of DNA; facilitate repair

Helicase Unwinds double-stranded DNA

Methylase Adds methyl group to selected bases in newly made DNA

Photolyase Uses visible light energy to separate UV-induced pyrimidine dimers

Primase An RNA polymerase that makes RNA primers from a DNA template

Ribozyme RNA enzyme that removes introns and splices exons together

RNA Polymerase Copies RNA from a DNA template

snRNP RNA-protein complex that removes introns and splices exons together

Topoisomerase or Gyrase Relaxes supercoiling ahead of the replication fork; separates DNA circles at the end of DNA replication

Transposase Cuts DNA backbone, leaving single-stranded “sticky ends”

CHAPTER 8 Microbial Genetics 211

G C

C OH

Sugar

Phosphate

OHOH

C G C G

P P P

A T A T

A T A

G C

New strand

Template strand

When a nucleoside triphosphate bonds to the sugar, it loses two phosphates.

Hydrolysis of the phosphate bonds provides the energy for the reaction.

P P i

Figure 8.4 Adding a nucleotide to DNA.

Why is one strand “upside down” relative to the other strand? Why can’t both strands “face” the same way?

Energy Needs DNA replication requires a great deal of energy. The energy is supplied from the nucleotides, which are actually nucleoside triphosphates. You already know about ATP; the only differ- ence between ATP and the adenine nucleotide in DNA is the sugar component. Deoxyribose is the sugar in the nucleosides used to synthesize DNA, and nucleoside triphosphates with ribose are used to synthesize RNA. Two phosphate groups are removed to add the nucleotide to a growing strand of DNA; hydrolysis of the nucleoside is exergonic and provides energy to make the new bonds in the DNA strand (Figure 8.4).

Figure 8.5 provides more detail about the many steps that go into this complex process.

DNA replication by some bacteria, such as E. coli, goes bidirectionally around the chromosome (Figure 8.6). Two repli- cation forks move in opposite directions away from the origin of replication. Because the bacterial chromosome is a closed loop, the replication forks eventually meet when replication is completed. The two loops must be separated by a topoisomer- ase. Much evidence shows an association between the bacterial plasma membrane and the origin of replication. After dupli- cation, if each copy of the origin binds to the membrane at

Enzymes unwind the parental double helix.

Proteins stabilize the unwound parental DNA.

DNA polymerase

The leading strand is synthesized continuously from the primer by DNA polymerase.

Replication fork

The lagging strand is synthesized discontinuously. Primase, an RNA polymerase, synthesizes a short RNA primer, which is then extended by DNA polymerase.

4 DNA polymerase digests RNA primer and replaces it with DNA.

DNA polymerase

Primase RNA primer

DNA ligase joins the discontinuous fragments of the lagging strand.

DNA polymerase

DNA ligaseOkazaki fragment

Parental strand

5¿

3¿

5¿

3¿

REPLICATION

Figure 8.5 A summary of events at the DNA replication fork.

Why is one strand of DNA synthesized discontinuously? Q

212 PART ONE Fundamentals of Microbiology

20 nmSEM(a) An E. coli chromosome in the process of replicating

(b) Bidirectional replication of a circular bacterial DNA molecule

Origin of replication

Replication fork

Daughter strands

Parental strand

Termination of replication

Replication fork

REPLICATION

Replication fork

Replication fork Figure 8.6 Replication of bacterial DNA.

What is the origin of replication? Q

opposite poles, then each offspring cell receives one copy of the DNA molecule—that is, one complete chromosome.

DNA replication is an amazingly accurate process. Typi- cally, mistakes are made at a rate of only 1 in every 10 billion bases incorporated. Such accuracy is largely due to the proof- reading capability of DNA polymerase. As each new base is added, the enzyme evaluates whether it forms the proper com- plementary base-pairing structure. If not, the enzyme excises the improper base and replaces it with the correct one. In this way, DNA can be replicated very accurately, allowing each daughter chromosome to be virtually identical to the parental DNA.

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CHECK YOUR UNDERSTANDING

✓ 8-3 Describe DNA replication, including the functions of DNA gyrase, DNA ligase, and DNA polymerase.

RNA and Protein Synthesis How is the information in DNA used to make the proteins that control cell activities? In the process of transcription, genetic information in DNA is copied, or transcribed, into a comple- mentary base sequence of RNA. The cell then uses the infor- mation encoded in this RNA to synthesize specific proteins through the process of translation. We now take a closer look at these two processes as they occur in a bacterial cell.

Transcription in Prokaryotes Transcription is the synthesis of a complementary strand of RNA from a DNA template. We will discuss transcription in prokaryotic cells here. Transcription in eukaryotes is discussed on page 215.

Ribosomal RNA (rRNA) forms an integral part of ribo- somes, the cellular machinery for protein synthesis. Transfer RNA is also involved in protein synthesis, as we will see. Messenger RNA (mRNA) carries the coded information for making specific proteins from DNA to ribosomes, where pro- teins are synthesized.

During transcription, a strand of mRNA is synthesized using a specific portion of the cell’s DNA as a template. In other words, the genetic information stored in the sequence of nucleobases of DNA is rewritten so that the same information appears in the base sequence of mRNA.

As in DNA replication, a guanine (G) in the DNA template dictates a cytosine (C) in the mRNA being made, and a C in the DNA template dictates a G in the mRNA. Likewise, a thymine (T) in the DNA template dictates an adenine (A) in the mRNA.

CHAPTER 8 Microbial Genetics 213

However, an adenine in the DNA template dictates a uracil (U) in the mRNA, because RNA contains uracil instead of thymine. (Uracil has a chemical structure slightly different from thy- mine, but it base-pairs in the same way.) If, for example, the template portion of DNA has the base sequence 3’-ATGCAT, the newly synthesized mRNA strand will have the complemen- tary base sequence 5’-UACGUA.

The process of transcription requires both an enzyme called RNA polymerase and a supply of RNA nucleotides (Figure 8.7). Transcription begins when RNA polymerase binds to the DNA at a site called the promoter. Only one of the two DNA strands serves as the template for RNA synthesis for a given gene. Like DNA, RNA is synthesized in the 5′ S 3′ direction. RNA synthesis continues until RNA polymerase reaches a site on the DNA called the terminator.

Transcription allows the cell to produce short-term copies of genes that can be used as the direct source of information for protein synthesis. Messenger RNA acts as an intermedi- ate between the permanent storage form, DNA, and the process that uses the infor- mation, translation.

Translation We have seen how the genetic information in DNA transfers to mRNA during transcription. Now we will see how mRNA serves as the source of information for the synthesis of pro- teins. Protein synthesis is called translation because it involves decoding the “language” of nucleic acids and converting it into the “language” of proteins.

AU U U

UG G

AA O

A TT

T T T

T T T T A

A A

C C

A A

C C

G C

G G

T A

RNA polymerase binds to the promoter, and DNA unwinds at the beginning of a gene.

RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA.

The site of synthesis moves along DNA; DNA that has been transcribed rewinds.

Transcription reaches the terminator.

RNA and RNA polymerase are released, and the DNA helix re-forms.

DNA

mRNA

Protein

TRANSCRIPTION

10 nm AFMRNA polymerase bound to DNA

DNA

RNA polymerase

RNA synthesis

Complete RNA strand

Promoter (gene begins) RNA polymerase

RNA Terminator (gene ends)

RNA

RNA nucleotides RNA polymerase

Template strand of DNA

Promoter

Figure 8.7 The process of transcription. The orienting diagram indicates the relationship of transcription to the overall flow of genetic information within a cell.

When does transcription stop? Q

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214 PART ONE Fundamentals of Microbiology

The language of mRNA is in the form of codons, groups of three nucleotides, such as AUG, GGC, or AAA. The sequence of codons on an mRNA molecule determines the sequence of amino acids that will be in the protein being synthesized. Each codon “codes” for a particular amino acid. This is the genetic code (Figure 8.8).

Codons are written in terms of their base sequence in mRNA. Notice in Figure 8.8 that there are 64 possible codons but only 20 amino acids. This means that most amino acids are signaled by several alternative codons, a situation referred to as the degeneracy of the code. For example, leucine has six codons, and alanine has four codons. Degeneracy allows for a certain amount of misreading of, or mutation in, the DNA without affecting the protein ultimately produced.

Of the 64 codons, 61 are sense codons, and 3 are nonsense codons. Sense codons code for amino acids, and nonsense codons (also called stop codons) do not. Rather, the nonsense codons— UAA, UAG, and UGA—signal the end of the protein molecule’s synthesis. The start codon that initiates the synthesis of the pro- tein molecule is AUG, which is also the codon for methionine. In bacteria, the start AUG codes for formylmethionine rather than the methionine found in other parts of the protein. The initiat- ing methionine is often removed later, so not all proteins contain methionine.

During translation, codons of an mRNA are “read” sequen- tially; and, in response to each codon, the appropriate amino acid is assembled into a growing chain. The site of translation is the ribosome, and transfer RNA (tRNA) molecules both rec- ognize the specific codons and transport the required amino acids.

Each tRNA molecule has an anticodon, a sequence of three bases that is complementary to a codon. In this way, a tRNA molecule can base-pair with its associated codon. Each tRNA can also carry on its other end the amino acid encoded by the codon that the tRNA recognizes. The functions of the ribo- some are to direct the orderly binding of tRNAs to codons and to assemble the amino acids brought there into a chain, ulti- mately producing a protein.

Figure 8.9 shows the details of translation. The two ribo- somal subunits, a tRNA with the anticodon UAC, and the mRNA molecule to be translated, along with several addi- tional protein factors, all assemble. This sets up the start codon (AUG) in the proper position to allow translation to begin. After the ribosome joins the first two amino acids with a peptide bond, the first tRNA molecule leaves the ribosome. The ribosome then moves along the mRNA to the next codon. As the proper amino acids are brought into line one by one, peptide bonds are formed between them, and a polypeptide chain results. (Also see Figure 2.14, page 42.) Translation

Second position

Fi rs

t p os

iti on

Th ir

d po

si tio

C A G

UUU

UUC

UUA

UUG

Phe

Leu

UCU

UCC

UCA

UCG

Ser

Tyr Cys U

CUU

CUC

CUA

CUG

Leu

CCU

CCC

CCA

CCG

Pro

CAU

CAC

CAA

CAG

His

Gln

CGU

CGC

CGA

CGG

Arg

Ile

ACU

ACC

ACA

ACG

Thr

AAU

AAC

AAA

AAG

Asn

Lys

AGU

AGC

AGA

AGG

Ser

Arg

GUU

GUC

GUA

GUG

Val

GCU

GCC

GCA

GCG

Ala

GAU

GAC

GAA

GAG

Asp

Glu

GGU

GGC

GGA

GGG

Gly

UAU

UAC

UAA

UAG

Stop

Stop Trp

UGU

UGC

UGA

UGG

Stop

AUU

AUC

AUA

AUG Met/start

Figure 8.8 The genetic code. The three nucleotides in an mRNA codon are designated, respectively, as the first position, second position, and third position of the codon on the mRNA. Each set of three nucleotides specifies a particular amino acid, represented by a three-letter abbreviation (see Table 2.5, page 41). The codon AUG, which specifies the amino acid methionine, is also the start of protein synthesis. The word Stop identifies the nonsense codons that signal the termination of protein synthesis.

What is the advantage of the degeneracy of the genetic code? Q

ends when one of the three nonsense codons in the mRNA is reached. The ribosome then comes apart into its two subunits, and the mRNA and newly synthesized polypeptide chain are released. The ribosome, the mRNA, and the tRNAs are then available to be used again.

The ribosome moves along the mRNA in the 5′ S 3′ direc- tion. As a ribosome moves along the mRNA, it will soon allow the start codon to be exposed. Additional ribosomes can then assemble and begin synthesizing protein. In this way, there are usually a number of ribosomes attached to a single mRNA, all at various stages of protein synthesis. In prokaryotic cells, the translation of mRNA into protein can begin even before

CHAPTER 8 Microbial Genetics 215

transcription is complete (Figure 8.10). Because mRNA is pro- duced in the cytoplasm in prokaryotes, the start codons of an mRNA being transcribed are available to ribosomes before the entire mRNA molecule is even made.

Transcription in Eukaryotes In eukaryotic cells, transcription takes place in the nucleus. The mRNA must be completely synthesized and moved through the nuclear membrane to the cytoplasm before translation can begin. In addition, the RNA undergoes processing before it leaves the nucleus. In eukaryotic cells, the regions of genes that code for proteins are often interrupted by noncoding DNA. Thus, eukary- otic genes are composed of exons, the regions of DNA expressed, and introns, the intervening regions of DNA that do not encode protein. In the nucleus, RNA polymerase synthesizes a molecule called an RNA transcript that contains copies of the introns. Particles called small nuclear ribonucleoproteins, abbreviated snRNPs and pronounced “snurps,” remove the introns and splice the exons together. In some organisms, the introns act as ribo- zymes to catalyze their own removal (Figure 8.11).

* * *

To summarize, genes are the units of biological information encoded by the sequence of nucleotide bases in DNA. A gene is expressed, or turned into a product within the cell, through the processes of transcription and translation. The genetic informa- tion carried in DNA is transferred to a temporary mRNA mol- ecule by transcription. Then, during translation, the mRNA directs the assembly of amino acids into a polypeptide chain: a ribosome attaches to mRNA, tRNAs deliver the amino acids to the ribosome as directed by the mRNA codon sequence, and the ribosome assembles the amino acids into the chain that will be the newly synthe- sized protein.

CHECK YOUR UNDERSTANDING

✓ 8-4 What is the role of the promoter, terminator, and mRNA in transcription?

✓ 8-5 How does mRNA production in eukaryotes differ from the process in prokaryotes?

A cell’s genetic and metabolic machineries are integrated and inter- dependent. The bacterial cell carries out an enormous number of metabolic reactions (see Chapter 5). The common feature of all metabolic reactions is that they are catalyzed by enzymes that are proteins synthesized via transcription and translation. Feedback inhibition stops a cell from performing unneeded chemical reac- tions (Chapter 5, page 116) by stopping enzymes that have already been synthesized. We will now look at mechanisms to prevent synthesis of enzymes that are not needed.

Because protein synthesis requires a huge amount of energy, cells save energy by making only those proteins needed at a particular time. Next we look at how chemical reactions are regulated by controlling gene expression.

Many genes, perhaps 60–80%, are not regulated but are instead constitutive, meaning that their products are con- stantly produced at a fixed rate. Usually these genes, which are effectively turned on all the time, code for enzymes that the cell needs in fairly large amounts for its major life processes. Glycolysis enzymes are examples. The produc- tion of other enzymes is regulated so that they are present only when needed. Trypanosoma, the protozoan parasite that causes African sleeping sickness, has hundreds of genes cod- ing for surface glycoproteins. Each protozoan cell turns on only one glycoprotein gene at a time. As the host’s immune system kills parasites with one type of surface molecule, parasites expressing a different surface glycoprotein can con- tinue to grow.

Pre-transcriptional Control Two genetic control mechanisms known as repression and induction regulate the transcription of mRNA and, conse- quently, the synthesis of enzymes from them. These mecha- nisms control the formation and amounts of enzymes in the cell, not the activities of the enzymes.

The Operon Model of Gene Expression Details of the control of gene expression by induction and repression are described by the operon theory formulated in the 1960s by François Jacob and Jacques Monod. An operon is a group of genes that are transcribed together and controlled by one promoter. We’ll look first at an inducible operon, in which transcription must be turned on. In E. coli, the enzymes of the lac operon are needed to metabolize lactose. In addition to b-galactosidase, these enzymes include lac permease, which is involved in the transport of lactose into the cell, and trans- acetylase, which metabolizes certain disaccharides other than lactose.

The genes for the three enzymes involved in lactose uptake and utilization are next to each other on the bacterial

Play Translation: Overview, Genetic Code, Process @MasteringMicrobiology

ASM: The regulation of gene expression is influenced by external and internal

molecular cues and/or signals.

The Regulation of Bacterial Gene Expression LEARNING OBJECTIVES

8-6 Define operon.

8-7 Explain pre-transcriptional regulation of gene expression in bacteria.

8-8 Explain post-transcriptional regulation of gene expression.

Play Interactive Microbiology @MasteringMicrobiology See how operons affect a patient’s health

216 PART ONE Fundamentals of Microbiology

Met

U ACA A U A U G G GU U A

mRNA

The second codon of the mRNA pairs with a tRNA carrying the second amino acid at the A site. The first amino acid joins to the second by a peptide bond. This attaches the polypeptide to the tRNA in the P site.

Peptide bond forms

A site

E site

Leu

Met Phe

The ribosome moves along the mRNA until the second tRNA is in the P site. The next codon to be translated is brought into the A site. The first tRNA now occupies the E site.

U U UAA AA

U U C

C A G G G

U A C

mRNA

Ribosome moves along mRNA

Leu

When the ribosome reaches a stop codon, the polypeptide is released.

Phe

Met

U C U

A AG U AG

Polypeptide released

mRNA

Stop codon

Met

7 Finally, the last tRNA is released, and the ribosome comes apart. The released polypeptide forms a new protein.

Gly

GlyGly Leu

Leu

Arg Leu P

Phe

Met

Gl y

Gly

Leu

Arg

LeuU C

mRNA

New protein

Gly

chromosome and are regulated together (Figure 8.12). These genes, which determine the structures of proteins, are called structural genes to distinguish them from an adjoining control region on the DNA. When lactose is introduced into the cul- ture medium, the lac structural genes are all transcribed and translated rapidly and simultaneously. We will now see how this regulation occurs.

In the control region of the lac operon are two relatively short segments of DNA. One, the promoter, is the segment where RNA polymerase initiates transcription. The other is the operator, which is like a traffic light that acts as a go or

Met Leu

On the assembled ribosome, a tRNA carrying the first amino acid is paired with the start codon on the mRNA. The place where this first tRNA sits is called the P site. A tRNA carrying the second amino acid approaches.

U A C

A U G

A U

U U A

Start codon

Second codon

mRNA

2Components needed to begin translation come together.

A C

A U G

tRNA

mRNA

Anticodon

Ribosomal subunit

C A

U A

Ribosome

P Site

The second amino acid joins to the third by another peptide bond, and the first tRNA is released from the E site.

U U A U U U

A AG G U

tRNA released

Gly

A A U

Leu

Met

The ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide.

U G

G G U U A U GU U U U A

Growing polypeptide chain

mRNA

AA A

CA U

Met

Met Leu

Gly

Phe

Phe Met

DNA

mRNA

Protein

TRANSLATION

Figure 8.9 The process of translation. The overall goal of translation is to produce proteins using mRNAs as the source of biological information. The complex cycle of events illustrated here shows the primary role of tRNA and ribosomes in the decoding

of this information. The ribosome acts as the site where the mRNA-encoded information is decoded, as well as the site where individual amino acids are connected into polypeptide chains. The tRNA molecules act as the actual “translators”—one end of each tRNA

recognizes a specific mRNA codon, while the other end carries the amino acid encoded by that codon.

Q When does translation stop?

stop signal for transcription of the structural genes. A set of operator and promoter sites and the structural genes they con- trol define an operon; thus, the combination of the three lac structural genes and the adjoining control regions is called the lac operon.

A regulatory gene called the I gene encodes a repressor pro- tein that switches inducible and repressible operons on or off. The lac operon is an inducible operon (see Figure 8.12). In the absence of lactose, the repressor binds to the operator site, thus preventing transcription. If lactose is present, the repressor binds

CHAPTER 8 Microbial Genetics 217

Met

U ACA A U A U G G GU U A

mRNA

Peptide bond forms

A site

E site

Leu

Met Phe

The ribosome moves along the mRNA until the second tRNA is in the P site. The next codon to be translated is brought into the A site. The first tRNA now occupies the E site.

U U UAA AA

U U C

C A G G G

U A C

mRNA

Ribosome moves along mRNA

Leu

When the ribosome reaches a stop codon, the polypeptide is released.

Phe

Met

U C U

A AG U AG

Polypeptide released

mRNA

Stop codon

Met

7 Finally, the last tRNA is released, and the ribosome comes apart. The released polypeptide forms a new protein.

Gly

GlyGly Leu

Leu

Arg Leu P

Phe

Met

Gl y

Gly

Leu

Arg

LeuU C

mRNA

New protein

Gly

Figure 8.9 The process of translation. (continued)

DNA

mRNA

Protein

TRANSLATION

Direction of transcription

Polyribosome

Peptide

RNA polymerase

Direction of translation

Ribosome

DNA

mRNA

5¿

60 nm TEM

Figure 8.10 Simultaneous transcription and translation in bacteria. Many molecules of mRNA are being synthesized simultaneously. The longest mRNA molecules were the first to be transcribed at the promoter. Note the ribosomes attached to the newly forming mRNA. The micrograph shows a polyribosome (many ribosomes) in a single bacterial gene.

Why can translation begin before transcription is complete in prokaryotes but not in eukaryotes?

Met Leu

U A C

A U G

A U

U U A

Start codon

Second codon

mRNA

2Components needed to begin translation come together.

A C

A U G

tRNA

mRNA

Anticodon

Ribosomal subunit

C A

U A

Ribosome

P Site

The second amino acid joins to the third by another peptide bond, and the first tRNA is released from the E site.

U U A U U U

A AG G U

tRNA released

Gly

A A U

Leu

Met

The ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide.

U G

G G U U A U GU U U U A

Growing polypeptide chain

mRNA

AA A

CA U

Met

Met Leu

Gly

Phe

Phe Met

DNA

mRNA

Protein

TRANSLATION

218 PART ONE Fundamentals of Microbiology CLINICAL FOCUS Tracking Zika Virus

to a metabolite of lactose instead of to the operator, and lactose- digesting enzymes are transcribed.

In repressible operons, the structural genes are tran- scribed until they are turned off (Figure 8.13). The genes for the enzymes involved in the synthesis of tryptophan are reg- ulated in this manner. The structural genes are transcribed and translated, leading to tryptophan synthesis. When excess

In 2014, Brazilian physicians reported clusters of patients with fever and rash. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect dengue, chikungunya, West Nile, and Zika viruses. Public health officials were relieved when the cause was identified as Zika virus (ZIKV) because ZIKV had never made anyone sick enough to go to the hospital. ZIKV is an arbovirus (arthropod-borne virus) that is spread between susceptible vertebrate hosts by blood-feeding arthropods, such as mosquitoes. At the same time, local health officials saw a fourfold increase in microcephaly—fetal brains were not developing at the same rate as the body. Some mothers reported having rashes and achy joints, but these symptoms weren’t long-lasting, and Zika virus disease was common.

Adriana Melo, an obstetrician, sent samples of amniontic fluid from two patients to be tested. RT-PCR confirmed the presence of ZIKV. By 2016, nearly 5000 cases of microcephaly had been reported in Brazil.

This Old World flavivirus was first identified in 1947 in monkeys in the Zika Forest of Uganda. Prior to 2000, only 14 human cases had been documented in the world. In 2007, an outbreak occurred on the island of Yap in Micronesia. Over 70% of Yap residents were infected with

ZIKV. However, no deaths or neurological complications were reported.

The ZIKV genome consists of a positive, single-stranded RNA consisting of 10,794 base pairs. (Positive RNA can act as mRNA and be translated.) The polyprotein encoded by the genome is cut to produce the proteins

that make up the virus. The virus has acquired several mutations, and researchers are looking for clues in these mutations to determine the virus’s journey around the world.

1. Using the portions of the genomes (shown below) that encode viral proteins, can you determine how similar these viruses are? Can you figure out its movement around the world? Determine the amino acids encoded, and group the viruses based on percentage of similarity to the Uganda strain.

2. Based on amino acids, there are two groups called clades. Can you identify the two groups?

3. The two clades are the African and Asian. Calculate the percentage of difference between nucleotides to see how the viruses are related within their clade.

4. The virus in the Americas is most closely related to the Asian strain that circulated in French Polynesia.

Source: GenBank genome sequences.

TEM 40 mmZika virus.

Brazil K K R R S A E T S G L L L T A M A V S K

Colombia K K R R S A E T S G L L L T A M A V N K

French Polynesia K K R R G A D T S L L L L T A M A V S K

Haiti K K R R G A D T S L L L L T A M A I S K

Mexico K K R R S A E T S L L L L T A M A V N E

Micronesia K K R R G A D T S L L L L T A M A I S K

Nigeria R K R R G A D T S L L L L T V M A I S K

Uganda 1947 R K R R G A D A S L L L L T V M A I S K

United States K K R R G A E T S L L L L T A M A V S K

tryptophan is present, the tryptophan acts as a corepressor binding to the repressor protein. The repressor protein can now bind to the operator, stop- ping further tryptophan synthesis.

218

Play Operons: Overview, Induction, Repression @MasteringMicrobiology

CHAPTER 8 Microbial Genetics 219

CHECK YOUR UNDERSTANDING

✓ 8-6 Use the following metabolic pathway to answer the questions that follow it.

enzyme a enzyme b Substrate A Intermediate B End-product C

a. If enzyme a is inducible and is not being synthesized at present, a (1) protein must be bound tightly to the (2) site. When the inducer is present, it will bind to the (3) so that (4) can occur.

b. If enzyme a is repressible, end-product C, called a (1) , causes the (2) to bind to the (3) . What causes derepression?

Regulatory gene

Promoter Operator

I P O Z Y A

Control region

Operon

Structural genes

I P Z Y A

RNA polymerase

2 Repressor active, operon off. The repressor protein binds with the operator, preventing transcription from the operon.

3 Repressor inactive, operon on. When the inducer allolactose binds to the repressor protein, the inactivated repressor can no longer block transcription. The structural genes are transcribed, ultimately resulting in the production of the enzymes needed for lactose catabolism.

Repressor mRNA

Transcription

Translation

Active repressor protein

I P O Z Y A

Transcription

Translation

Operon mRNA

Allolactose (inducer)

b-Galactosidase

Inactive repressor protein

Permease Transacetylase

Structure of the operon. The operon consists of the promoter (P) and operator (O ) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I ).

DNA

What causes transcription of an inducible enzyme? Q

Figure 8.12 An inducible operon. Lactose-digesting enzymes are produced in the presence of lactose. In E. coli, the genes for the three enzymes are in the lac operon. b-galactosidase is encoded by lacZ. The lacY gene encodes the lac permease, and lacA encodes transacetylase, whose function in lactose metabolism is still unclear.

Positive Regulation Regulation of the lactose operon also depends on the level of glucose in the medium, which in turn controls the intracel- lular level of the small molecule cyclic AMP (cAMP), a sub- stance derived from ATP that serves as a cellular alarm signal. Enzymes that metabolize glucose are constitutive, and cells grow at their maximal rate with glucose as their carbon source because they can use it most efficiently (Figure 8.14). When glucose is no longer available, cAMP accumulates in the cell. The cAMP binds to the allosteric site of catabolic activator protein (CAP). CAP then binds to the lac promoter, which ini- tiates transcription by making it easier for RNA polymerase to bind to the promoter. Thus transcription of the lac operon requires both the presence of lactose and the absence of glu- cose (Figure 8.15).

Cyclic AMP is an example of an alarmone, a chemical alarm signal that promotes a cell’s response to environmental or nutritional stress. (In this case, the stress is the lack of glucose.)

Exon Intron Exon Intron Exon

mRNA

RNA trans- cript

DNA

Nucleus

Cytoplasm

1 In the nucleus, a gene composed of exons and introns is transcribed to RNA by RNA polymerase.

2 Processing involves snRNPs in the nucleus to remove the intron-derived RNA and splice together the exon-derived RNA into mRNA.

3 After further modification, the mature mRNA travels to the cytoplasm, where it directs protein synthesis.

Figure 8.11 RNA processing in eukaryotic cells.

Why can’t the RNA transcript be used for translation? Q

220 PART ONE Fundamentals of Microbiology

OI P E D C B A

P E D C B A

2 Repressor inactive, operon on. The repressor is inactive, and transcription and translation proceed, leading to the synthesis of tryptophan.

3 Repressor active, operon off. When the corepressor tryptophan binds to the repressor protein, the activated repressor binds with the operator, preventing transcription from the operon.

RNA polymerase

Active repressor protein

Inactive repressor protein

Polypeptides comprising the enzymes for tryptophan synthesis

Repressor mRNA

Transcription

Translation

Operon mRNA

I P E D C B A

Tryptophan (corepressor)

11 Structure of the operon. The operon consists of the promoter (P ) and operator (O ) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I ).

Regulatory gene

Promoter Operator

Control region

Operon

Structural genes

DNA

Figure 8.13 A repressible operon. Tryptophan, an amino acid, is produced by anabolic enzymes encoded by five structural genes. Accumulation of tryptophan represses transcription of these genes, preventing further synthesis of tryptophan. The E. coli trp operon is shown here.

What causes transcription of a repressible enzyme? Q

The same mechanism involving cAMP allows the cell to use other sugars. Inhibition of the metabolism of alternative car- bon sources by glucose is termed catabolite repression (or the glucose effect). When glucose is available, the level of cAMP in the cell is low, and consequently CAP is not bound.

Epigenetic Control Eukaryotic and bacterial cells can turn genes off by methyl- ating certain nucleotides—that is, by adding a methyl group 1¬ CH32. The methylated (off) genes are passed to offspring cells. Unlike mutations, this isn’t permanent, and the genes can be turned on in a later generation. This is called epigene- tic inheritance (epigenetic = on genes). Epigenetics may explain why bacteria behave differently in a biofilm.

Post-transcriptional Control Some regulatory mechanisms stop protein synthesis after tran- scription has occurred. A part of an mRNA molecule, called a riboswitch, that binds to a substrate can change the mRNA structure. Depending on the type of change, translation can be initiated or stopped. Both eukaryotes and prokaryotes use riboswitches to control expression of some genes.

Lo g 1

0 o

f n um

be r

of c

el ls

Lo g 1

0 o

f n um

be r

of c

el ls

(a) Bacteria growing on glucose as the sole carbon source grow faster than on lactose.

(b) Bacteria growing in a medium containing glucose and lactose first consume the glucose and then, after a short lag time, the lactose. During the lag time, intra- cellular cAMP increases, the lac operon is transcribed, lactose is transported into the cell, and d-galacto- sidase is synthesized to break down lactose.

Time

Glucose

Lactose

All glucose consumed

Glucose used Lag

time

Lactose used

Figure 8.14 The growth rate of E. coli on glucose and lactose.

When both glucose and lactose are present, why will cells use glucose first?

CHAPTER 8 Microbial Genetics 221

lacI lacZ

RNA polymerase can bind and transcribe

CAP-binding site

CAP-binding site RNA polymerase can't bind

Active CAP

Inactive CAP

cAMP

DNA

Promoter

Inactive lac repressor

Operator

(a) Lactose present, glucose scarce (cAMP level high). If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for lactose digestion.

lacI lacZ

Inactive CAP

Promoter

Inactive lac repressor

Operator

(b) Lactose present, glucose present (cAMP level low). When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.

Figure 8.15 Positive regulation of the lac operon.

Will transcription of the lac operon occur in the presence of lactose and glucose? In the presence of lactose and the absence of glucose? In the presence of glucose and the absence of lactose?

Single-stranded RNA molecules of approximately 22 nucleotides, called microRNAs (miRNAs), inhibit protein production in eukaryotic cells. In humans, miRNAs pro- duced during development allow different cells to produce different proteins. Heart cells and skin cells have the same genes, but the cells in each organ produce different proteins because of miRNAs produced in each cell type during devel- opment. Similar short RNAs in bacteria enable the cell to cope with environmental stresses, such as low temperature or oxidative damage. An miRNA base-pairs with a comple- mentary mRNA, forming a double-stranded RNA. This double-stranded RNA is enzymatically destroyed so that the mRNA-encoded protein is not made (Figure 8.16). The action of another type of RNA, siRNA, is similar and is discussed on page 256.

1 Transcription of miRNA occurs.

2 miRNA binds to target mRNA that has at least six complementary bases.

3 mRNA is degraded.

miRNA

DNA

mRNA

Figure 8.16 MicroRNAs control a wide range of activities in cells.

In mammals, some miRNAs hybridize with viral RNA. What would happen if a mutation occurred in the miRNA gene?

CHECK YOUR UNDERSTANDING ✓ 8-7 What is the role of cAMP in regulating gene expression?

✓ 8-8 How does miRNA stop protein synthesis?

Changes in Genetic Material LEARNING OBJECTIVES

8-9 Classify mutations by type.

8-10 Describe two ways mutations can be repaired.

8-11 Describe the effect of mutagens on the mutation rate.

8-12 Outline the methods of direct and indirect selection of mutants.

8-13 Identify the purpose of and outline the procedure for the Ames test.

A cell’s DNA can be changed by mutations and horizontal gene transfer. Changes in DNA result in genetic variations that can impact microbial function (e.g., biofilm formation, pathoge- nicity, and antibiotic resistance). Survival and reproduction of

222 PART ONE Fundamentals of Microbiology

encoded by the gene. Silent mutations commonly occur when one nucleotide is substituted for another in the DNA, espe- cially at a location corresponding to the third position of the mRNA codon. Because of the degeneracy of the genetic code, the resulting new codon might still code for the same amino acid. Even if the amino acid is changed, the function of the protein may not change if the amino acid is in a nonvital por- tion of the protein, or is chemically very similar to the original amino acid.

The most common type of mutation involving single base pairs is base substitution (or point mutation), in which a single base at one point in the DNA sequence is replaced with a dif- ferent base. When the DNA replicates, the result is a substituted base pair (Figure 8.17). For example, AT might be substituted for GC, or CG for GC. If a base substitution occurs within a gene that codes for a protein, the mRNA transcribed from the gene will carry an incorrect base at that position. When the mRNA is translated into protein, the incorrect base may cause the inser- tion of an incorrect amino acid in the protein. If the base substi- tution results in an amino acid substitution in the synthesized

the bacteria with a new genotype can be favored by natural and human-influenced environments and result in a huge diversity of microorganisms. The survival of new genotypes is called natural selection.

Mutation A mutation is a permanent change in the base sequence of DNA. Such a change will sometimes cause a change in the product encoded by that gene. For example, when the gene for an enzyme mutates, the enzyme encoded by the gene may become inactive or less active because its amino acid sequence has changed. Such a change in genotype may be disadvantageous, or even lethal, if the cell loses a phenotypic trait it needs. However, a mutation can be beneficial if, for instance, the altered enzyme encoded by the mutant gene has a new or enhanced activity that benefits the cell. See the Clinical Focus box in Chapter 26, page 771.

Types of Mutations Many simple mutations are silent (neutral); the change in DNA base sequence causes no change in the activity of the product

During DNA replication, a thymine is incorporated opposite guanine by mistake.

T TA

A T

G C

A T

U UG U UA U UG GU U

A T

G T

Parental DNA

Daughter DNA

Granddaughter DNA

2 If not corrected, in the next round of replication, adenine pairs with the new thymine, yielding an AT pair in place of the original GC pair.

3 When mRNA is transcribed from the DNA containing this substitution, a codon is produced that, during translation, encodes a different amino acid: tyrosine instead of cysteine.

Daughter DNA

mRNA

Amino acids

Transcription

Translation

Replication

Cysteine Tyrosine Cysteine Cysteine

Figure 8.17 Base substitutions. This mutation leads to an altered protein in a grandchild cell.

Does a base substitution always result in a different amino acid? Q

CHAPTER 8 Microbial Genetics 223

results in the change from glutamic acid to valine in the protein. This causes the shape of the hemoglobin molecule to change under conditions of low oxygen, which, in turn, alters the shape of the red blood cells.

By creating a nonsense (stop) codon in the middle of an mRNA molecule, some base substitutions effectively prevent the synthesis of a complete functional protein; only a fragment is synthesized. A base substitution resulting in a nonsense codon is thus called a nonsense mutation (Figure 8.18c).

Besides base-pair mutations, there are also changes in DNA called frameshift mutations, in which one or a few nucleotide pairs are deleted or inserted in the DNA (Figure 8.18d). This mutation can shift the “translational reading frame”—that is, the three-by-three grouping of nucleotides recognized as codons by the tRNAs during translation. For example, delet- ing one nucleotide pair in the middle of a gene causes changes in many amino acids downstream from the site of the original mutation. Frameshift mutations almost always result in a long stretch of altered amino acids and the production of an inac- tive protein from the mutated gene. In most cases, a nonsense codon will eventually be encountered and thereby terminate translation.

Base substitutions and frameshift mutations may occur spontaneously because of occasional mistakes made during DNA replication. These spontaneous mutations apparently occur in the absence of any mutation-causing agents.

protein, this change in the DNA is known as a missense mutation (Figure 8.18a and Figure 8.18b).

The effects of such mutations can be dramatic. For example, sickle cell disease is caused by a single change in the gene for globin, the protein component of hemoglobin. Hemoglobin is primarily responsible for transporting oxygen from the lungs to the tissues. A single change from an A to a T at a specific site

T A C T T C A A A C C G A T T

G AA U G A A G U U U G C U A

T A C T T C A A A C G A T T

G AA U G A A G U U U C U A

T A C T C A A A C G A T T

G AA U G A G U U U C U A

U G

T A C T C A A C G A T T

Met Lys Phe Gly

DNA (template strand)

mRNA

Amino acid sequence

(a) Normal DNA molecule

Met Lys Phe Ser

DNA (template strand)

mRNA

Amino acid sequence

(b) Missense mutation

Met

G AA U G A G U U U A

(d) Frameshift mutation

Lys Leu Ala

Transcription

Translation

Stop

Met

Figure 8.18 Types of mutations and their effects on the amino acid sequences of proteins.

Q What happens if base 9 in (a) is changed to a C?

CHECK YOUR UNDERSTANDING

✓ 8-9 How can a mutation be beneficial?

Mutagens

Chemical Mutagens Agents in the environment, such as certain chemicals and radiation, that directly or indirectly bring about mutations are called mutagens.

One of the many chemicals known to be a mutagen is nitrous acid. Figure 8.19 shows how exposing DNA to nitrous acid can convert the base adenine to a form that pairs with cytosine instead of the usual thymine. When DNA contain- ing such modified adenines replicates, one daughter DNA molecule will have a base-pair sequence different from that of the parent DNA. Eventually, some AT base pairs of the parent will have been changed to GC base pairs in a granddaughter cell. Nitrous acid makes a specific base-pair change in DNA. Like all mutagens, it alters DNA at random locations.

Another type of chemical mutagen is the nucleoside analog. These molecules are structurally similar to normal nitrogenous bases, but they have slightly altered base-pairing

224 PART ONE Fundamentals of Microbiology

CH2OH

H H

N H H

N H

N CH2OH O

H H H

C T

A T HNO2

HNO2

Normal parent DNA

Replication

Normal daughter DNA

C T

Altered parent DNA

C T

Altered daughter DNA

C T

Mutated granddaughter DNA

C T

Altered granddaughter DNA

C T

A Replication

(a) Adenosine nucleoside normally base-pairs by hydrogen bonds with an oxygen and a hydrogen of a thymine or uracil nucleotide.

(b) The altered adenine pairs with cytosine instead of thymine.

Altered adenine will hydrogen bond with a hydrogen and a nitrogen of a cytosine nucleotide.

Figure 8.19 Oxidation of nucleotides makes a mutagen. The nitrous acid emitted into the air by burning fossil fuels oxidizes adenine.

Q What is a mutagen?

205 224 227 234

CLINICAL CASE

A person’s DNA can undergo mutations. One improper nucleotide in DNA creates a mutation, which could alter the function of the gene. Cancer is abnormal cell growth caused by mutations. These mutations can be inherited.

As Marcel and his wife, Janice, drive home from the doctor’s office, they review Marcel’s family history. Marcel’s brother, Robert, passed away from colon cancer 10 years ago, but Marcel has always been the picture of health. Even at 70, he hasn’t given a thought to retiring from his Memphis barbeque restaurant that he once co-owned with his brother until Robert’s death.

What factors may have contributed to Marcel’s colon cancer?

properties. Examples, 2-aminopurine and 5-bromouracil, are shown in Figure 8.20. When nucleoside analogs are given to growing cells, the analogs are randomly incorporated into cellular DNA in place of the normal bases. Then, during DNA replication, the analogs cause mistakes in base pairing. The

(a) The 2-aminopurine is incorporated into DNA in place of adenine but can pair with cytosine, so an AT pair becomes a CG pair.

(b) The 5-bromouracil is used as an anticancer drug because it is mistaken for thymine by cellular enzymes but pairs with cytosine. In the next DNA replication, an AT pair becomes a GC pair.

Adenine nucleoside

Normal nitrogenous base

2-Aminopurine nucleoside

Analog

Thymine nucleoside 5-Bromouracil nucleoside

CH2OH

H H

N H H

N N

NH2

NCH2OH O

H H H

CH2OH

H H

CH3

CH2OH

H H

Figure 8.20 Nucleoside analogs and the nitrogenous bases they replace. A nucleoside is phosphorylated, and the resulting nucleotide used to synthesize DNA.

Q Why do these drugs kill cells?

Play MicroFlix 3D Animation @MasteringMicrobiology

CHAPTER 8 Microbial Genetics 225

DNA that is complementary to the correct strand. For many years biologists questioned how the incorrect base could be distinguished from the correct base if it was not physically dis- torted like a thymine dimer. In 1970, Hamilton Smith provided

incorrectly paired bases will be copied during subsequent rep- lication of the DNA, resulting in base-pair substitutions in the progeny cells. Some antiviral and antitumor drugs are nucleo- side analogs, including AZT (azidothymidine), used to treat HIV infection.

Still other chemical mutagens cause small deletions or insertions, which can result in frameshifts. For instance, under certain conditions, benzopyrene, which is present in smoke and soot, is an effective frameshift mutagen. Aflatoxin— produced by Aspergillus flavus (a-sper-JIL-lus FLĀ-vus), a mold that grows on peanuts and grain—is a frameshift mutagen. Frameshift mutagens usually have the right size and chemical properties to slip between the stacked base pairs of the DNA double helix. They may work by slightly offsetting the two strands of DNA, leaving a gap or bulge in one strand or the other. When the staggered DNA strands are copied during DNA synthesis, one or more base pairs can be inserted or deleted in the new double-stranded DNA. Interestingly, frameshift mutagens are often potent carcinogens.

Radiation X rays and gamma rays are forms of radiation that are potent mutagens because of their ability to ionize atoms and mole- cules. The penetrating rays of ionizing radiation cause electrons to pop out of their usual shells (see Chapter 2). These electrons bombard other molecules and cause more damage, and many of the resulting ions and free radicals (molecular fragments with unpaired electrons) are very reactive. Some of these ions oxidize bases in DNA, resulting in errors in DNA replication and repair that produce mutations (see Figure 8.19). An even more serious outcome is the breakage of covalent bonds in the sugar-phosphate backbone of DNA, which causes physical breaks in chromosomes.

Another form of mutagenic radiation is ultraviolet (UV) light, a nonionizing component of ordinary sunlight. However, the most mutagenic component of UV light (wavelength 260 nm) is screened out by the ozone layer of the atmo- sphere. The most important effect of direct UV light on DNA is the formation of harmful covalent bonds between pyrimidine bases. Adjacent thymines in a DNA strand can cross-link to form thymine dimers. Such dimers, unless repaired, may cause serious damage or death to the cell because it cannot properly transcribe or replicate such DNA.

Bacteria and other organisms have enzymes that can repair UV-induced damage. Photolyases, also known as light-repair enzymes, use visible light energy to separate the dimer back to the original two thymines. Nucleotide excision repair, shown in Figure 8.21, is not restricted to UV-induced damage; it can repair mutations from other causes as well. Enzymes cut out the incorrect base and fill in the gap with newly synthesized

T T

1 Exposure to ultraviolet light causes adjacent thymines to become cross-linked, forming a thymine dimer and disrupting their normal base pairing.

2 An endonuclease cuts the DNA, and an exonuclease removes the damaged DNA.

3 DNA polymerase fills the gap by synthesizing new DNA, using the intact strand as a template.

4 DNA ligase seals the remaining gap by joining the old and new DNA.

Thymine dimer

Ultraviolet light

T T

New DNA

Figure 8.21 The creation and repair of a thymine dimer caused by ultraviolet light. After exposure to UV light, adjacent thymines can become cross-linked, forming a thymine dimer. In the absence of visible light, the nucleotide excision repair mechanism is used in a cell to repair the damage.

Q How do excision repair enzymes “know” which strand is incorrect?

226 PART ONE Fundamentals of Microbiology

the answer with the discovery of methylases. These enzymes add a methyl group to selected bases soon after a DNA strand is made. A repair endonuclease then cuts the nonmethylated strand.

The Frequency of Mutation The mutation rate is the probability that a gene will mutate when a cell divides. The rate is usually stated as a power of 10, and because mutations are very rare, the exponent is always a negative number. For example, if there is one chance in a mil- lion that a gene will mutate when the cell divides, the mutation rate is 1/1,000,000, which is expressed as 10-6. Spontaneous mistakes in DNA replication occur at a very low rate, perhaps only once in 109 replicated base pairs (a mutation rate of one in a billion). Because the average gene has about 103 base pairs, the spontaneous rate of mutation is about one in 106 (a mil- lion) replicated genes.

Mutations usually occur more or less randomly along a chromosome. The occurrence of random mutations at low fre- quency is an essential aspect of the adaptation of species to their environment, for evolution requires that genetic diver- sity be generated randomly and at a low rate. For example, in a bacterial population of significant size—say, greater than 107 cells—a few new mutant cells will always be produced in every generation. Most mutations either are harmful and likely to be removed from the gene pool when the individual cell dies or are neutral. However, a few mutations may be beneficial. For example, a mutation that confers antibiotic resistance is ben- eficial to a population of bacteria that is regularly exposed to antibiotics. Once such a trait has appeared through mutation, cells carrying the mutated gene are more likely than other cells to survive and reproduce as long as the environment stays the same. Soon most of the cells in the population will have the gene; an evolutionary change will have occurred, although on a small scale.

A mutagen usually increases the spontaneous rate of muta- tion, which is about one in 106 replicated genes, by a factor of 10 to 1000 times. In other words, in the presence of a muta- gen, the normal rate of 10-6 mutations per replicated gene becomes a rate of 10-5 to 10-3 per replicated gene. Mutagens are used experimentally to enhance the production of mutant cells for research on the genetic properties of microorganisms and for commercial purposes.

CHECK YOUR UNDERSTANDING

✓ 8-10 How can mutations be repaired? ✓ 8-11 How do mutagens affect the mutation rate?

Play Mutations: Types, Repair @MasteringMicrobiology

Identifying Mutants Mutants can be detected by selecting or testing for an altered phenotype. Whether or not a mutagen is used, mutant cells with specific mutations are always rare compared with other cells in the population. The problem is detecting such a rare event.

Experiments are usually performed with bacteria because they reproduce rapidly, so large numbers of organisms (more than 109 per milliliter of nutrient broth) can easily be used. Furthermore, because bacteria generally have only one copy of each gene per cell, the effects of a mutated gene are not masked by the presence of a normal version of the gene, as in many eukaryotic organisms.

Positive (direct) selection involves the detection of mutant cells by rejection of the unmutated parent cells. For exam- ple, suppose we were trying to find mutant bacteria that are resistant to penicillin. When the bacterial cells are plated on a medium containing penicillin, the mutant can be identi- fied directly. The few cells in the population that are resistant (mutants) will grow and form colonies, whereas the normal, penicillin-sensitive parental cells cannot grow.

To identify mutations in other kinds of genes, negative (indirect) selection can be used. This process selects a cell that cannot perform a certain function, using the technique of replica plating. For example, suppose we wanted to use replica plating to identify a bacterial cell that has lost the ability to synthesize the amino acid histidine (Figure 8.22). First, about 100 bacterial cells are inoculated onto an agar plate. This plate, called the master plate, contains a medium with histidine on which all cells will grow. After 18 to 24 hours of incubation, each cell reproduces to form a colony. Then a pad of sterile material, such as latex, filter paper, or velvet, is pressed over the master plate, and some of the cells from each colony adhere to the velvet. Next, the velvet is pressed down onto two (or more) sterile plates. One plate contains a medium with- out histidine, and one contains a medium with histidine on which the original, nonmutant bacteria can grow. Any colony that grows on the medium with histidine on the master plate but that cannot synthesize its own histidine will not be able to grow on the medium without histidine. The mutant colony can then be identified on the master plate. Of course, because mutants are so rare (even those induced by mutagens), many plates must be screened with this technique to isolate a spe- cific mutant.

Replica plating is a very effective means of isolating mutants that require one or more new growth factors. Any mutant microorganism having a nutritional requirement that is absent in the parent is known as an auxotroph. For example, an auxotroph may lack an enzyme needed to synthesize a par- ticular amino acid and will therefore require that amino acid as a growth factor in its nutrient medium.

CHAPTER 8 Microbial Genetics 227

Identifying Chemical Carcinogens Many known mutagens have been found to be carcinogens, substances that cause cancer in animals, including humans. In recent years, chemicals in the environment, the workplace, and the diet have been implicated as causes of cancer in humans. Animal testing procedures are time-consuming and expensive, so some faster and less expensive procedures for preliminary screening of potential carcinogens that do not use animals have been developed. One of these, called the Ames test, uses bacteria as carcinogen indicators.

The Ames test is based on the observation that exposure of mutant bacteria to mutagenic substances may cause new mutations that reverse the effect (the change in phenotype) of the original mutation. These are called reversions. Specifi- cally, the test measures the reversion of histidine auxotrophs of Salmonella (so-called his- cells, mutants that have lost the ability to synthesize histidine) to histidine-synthesizing cells (his+) after treatment with a mutagen (Figure 8.23). Bacteria are incubated in both the presence and absence of the substance being tested. Because animal enzymes must activate many chemicals into forms that are chemically reactive for mutagenic or carcinogenic activity to appear, the chemical to be tested

1 Sterile velvet is pressed on the grown colonies on the master plate.

Handle

Velvet surface (sterilized)

2 Cells from each colony are transferred from the velvet to new plates.

3 Plates are incubated.

4 Growth on plates is compared. A colony that grows on the medium with histidine but could not grow on the medium without histidine is auxotrophic (histidine-requiring mutant).

Petri plate with medium lacking histidine

Colony missing

Petri plate with medium containing histidine

Master plate with medium containing histidine

Auxotrophic mutant

Figure 8.22 Replica plating. In this example, the auxotrophic mutant cannot synthesize histidine. The plates must be carefully marked (with an X here) to maintain orientation so that colony positions are known in relation to the original master plate.

Q What is an auxotroph?

205 224 227 234

CLINICAL CASE

Not all mutations are inherited; some are induced by genotoxins, that is, chemicals that damage a cell’s genetic material. Marcel is not overweight and has never smoked. Researchers have known since the 1970s that people who consume cooked meat and meat products are more likely to develop colon cancer. The suspect cancer-causing chemicals are aromatic amines that form during high-heat cooking.

Marcel has owned his Memphis barbeque restaurant for over 50 years. He is a hands-on type of employer and is always in the kitchen overseeing the cooking process. All of his barbequed meat is seared over high heat and then slow- cooked for hours. Marcel is considered the expert in this technique, but now it seems as if his profession could be a factor in his disease.

What test can be used to determine whether a chemical is genotoxic?

228 PART ONE Fundamentals of Microbiology

color change of the pH indicator. The Ames test is routinely used to evaluate new chemicals and air and water pollutants.

About 90% of the substances found by the Ames test to be mutagenic have also been shown to be carcinogenic in ani- mals. By the same token, the more mutagenic substances have generally been found to be more carcinogenic.

and the mutant bacteria are incubated together with rat liver extract, a rich source of activation enzymes. If the substance being tested is mutagenic, it will cause the reversion of his- bacteria to his+ bacteria at a rate higher than the spontaneous reversion rate. The number of observed revertants indicates the degree to which a substance is mutagenic and therefore pos- sibly carcinogenic.

The Ames test can be performed in liquid media with a pH indicator in a 96-well plate. Several potential mutagens or different concentations of mutagens can be qualitatively tested in different wells. Bacterial growth is determined by a

Suspected mutagen

Rat liver extract

Experimental sample

Incubation

Experimental plate

Colonies of revertant bacteria

Rat liver extract

Control (no suspected mutagen added)

Control plate

Cultures of histidine-dependent Salmonella Media lacking histidine

1 2 3 4Two cultures are pre- pared of Salmonella bacteria that have lost the ability to synthesize histidine (histidine- dependent).

The suspected mutagen is added to the experimental sample only; rat liver extract (an activator) is added to both samples.

Each sample is poured onto a plate of medium lacking histidine. The plates are then incubated at 378C for two days. Only bacteria whose histidine-dependent phenotype has mutated back (reverted) to histidine- synthesizing will grow into colonies.

The numbers of colonies on the experimental and control plates are compared. The control plate may show a few spontaneous histidine-synthesizing revertants. The test plates will show an increase in the number of histidine-synthesizing revertants if the test chemical is indeed a mutagen and potential carcinogen. The higher the concentration of mutagen used, the more revertant colonies will result.

Incubation

Figure 8.23 The Ames reverse gene mutation test.

Q Do all mutagens cause cancer?

CHECK YOUR UNDERSTANDING

✓ 8-12 How would you isolate an antibiotic-resistant bacterium? An antibiotic-sensitive bacterium?

✓ 8-13 What is the principle behind the Ames test?

CHAPTER 8 Microbial Genetics 229

Like mutation, genetic recombination contributes to a population’s genetic diversity, which is the source of variation in evolution. In highly evolved organisms such as present-day microbes, recombination is more likely to be beneficial than mutation because recombi- nation will less likely destroy a gene’s function and may bring together combinations of genes that enable the organism to carry out a valuable new function.

The major protein that constitutes the flagella of Salmonella is also one of the primary proteins that causes our immune sys- tems to respond. However, these bacteria have the capability of producing two different flagellar proteins. As our immune system mounts a response against those cells containing one form of the flagellar protein, those organisms producing the second are not affected. Which flagellar protein is produced is determined by a recombination event that apparently occurs somewhat randomly within the chromosomal DNA. Thus, by altering the flagellar pro- tein produced, Salmonella can better avoid the defenses of the host.

Vertical gene transfer occurs when genes are passed from an organism to its offspring. Plants and animals transmit their genes by vertical transmission. Bacteria can pass their genes not only to their offspring, but also laterally, to other microbes of the same generation. This is known as horizontal gene transfer (see Figure 8.2). Horizontal gene transfer between nor- mal microbiota and pathogens may be important in the spread of antibiotic resistance. Horizontal gene transfer between bacteria occurs in several ways. In all of the mechanisms, the transfer involves a donor cell that gives a portion of its total DNA to a recipient cell. Once transferred, part of the donor’s DNA can be incorporated into the recipient’s DNA; the remain- der is degraded by cellular enzymes. The recipient cell that incorporates donor DNA into its own DNA is called a recombinant. The transfer of genetic material between bacteria is by no means a frequent event; it may occur in only 1% or less of an entire population. Let’s examine in detail the specific types of genetic transfer.

Plasmids and Transposons Plasmids and transposons are genetic elements that exist out- side chromosomes. They occur in both prokaryotic and eukary- otic organisms, but this discussion focuses on their role in genetic change in prokaryotes. Plasmids and transposons are called mobile genetic elements because they can move from one chromosome to another or from one cell to another.

Plasmids

Recall from Chapter 4 (page 90) that plasmids are self- replicating, gene-containing, circular pieces of DNA about 1–5% the size of

Genetic Transfer and Recombination LEARNING OBJECTIVES

8-14 Describe the functions of plasmids and transposons.

8-15 Differentiate horizontal and vertical gene transfer.

8-16 Compare the mechanisms of genetic recombination in bacteria.

Genetic recombination refers to the exchange of genes between two DNA molecules to form new combinations of genes on a chromosome. Figure 8.24 shows one mechanism for genetic recombination. If a cell picks up foreign DNA (called donor DNA in the figure), some of it could insert into the cell’s chromosome—a process called crossing over—and some of the genes carried by the chromosomes are shuffled. The DNA has recombined, so that the chromosome now carries a portion of the donor’s DNA.

If A and B represent DNA from different individuals, how are they brought close enough together to recombine? In eukary- otes, genetic recombination is an ordered process that usually occurs as part of the sexual cycle of the organism. Crossing over generally takes place during the formation of reproductive cells, such that these cells contain recombinant DNA. In bacteria, genetic recombination can happen in a number of ways, which we will discuss in the following sections.

1 DNA from one cell aligns with DNA in the recipient cell. Notice that there is a nick in the donor DNA.

Recipient chromosome

RecA protein

Donor DNA

2 DNA from the donor aligns with complementary base pairs in the recipient’s chromosome. This can involve thousands of base pairs.

3 RecA protein catalyzes the joining of the two strands.

4 The result is that the recipient’s chromosome contains new DNA. Complementary base pairs between the two strands will be resolved by DNA polymerase and ligase. The donor DNA will be destroyed. The recipient may now have one or more new genes.

Figure 8.24 Genetic recombination by crossing over. Foreign DNA can be inserted into a chromosome by breaking and rejoining the chromosome. This can insert one or more new genes into the chromosome. A photograph of RecA protein is shown in Figure 3.11a, page 60.

Q What type of enzyme breaks the DNA?

ASM: Genetic variations can impact microbial functions (e.g., in

biofilm formation, pathogenicity, and drug resistance).

Play Horizontal Gene Transfer: Overview @MasteringMicrobiology

The number of antibiotic-resistant bacteria in our intestinal microbiome increases with age. The reason: exposure to antibiotics. In the presence of a bacteria-killing drug, a resistant

mutant will grow while the nonresistant, or susceptible, bacteria die off. So over the human life span, which includes many episodes of illnesses and treatments, we end up populated with more and more antibiotic-resistant microbes.

At first, this seems like a desirable effect. For instance, if beneficial intestinal microbes survive a course of drugs meant to treat your pneumonia, then you may not experience medication side effects such as GI discomfort or diarrhea. Unfortunately, recent evidence shows that a drug-resistant microbiome may actually threaten us in ways we previously didn’t understand.

Scientists suspect that drug resistance in pathogenic bacteria often originates from drug-resistant normal microbiota. Nearly half of the resistance genes identified in intestinal bacteria are identical to resistance genes found in pathogens. Swapping of genes between species that come in contact with each other (horizontal gene transfer) happens easily in the

intestines, where large numbers of different microbes mingle. In one study, Escherichia coli bacteria that were resistant to the drugs sulfonamide and ampicillin were found residing in volunteers who ingested E. coli bacteria that were susceptible to these antibiotics. How could this be? The researchers traced the drug-resistance genes to a plasmid found in E. coli that had resided in the volunteers before the study— the resistant bacteria had transferred the plasmid to the drug-susceptible bacteria once the different strains met up in the intestine. Likewise, resistance to the drug vancomycin is believed to have transferred from the commensal bacterium Enterococcus faecalis to pathogenic strains of Staphylococcus aureus. The resistance gene was found on a conjugative plasmid in both species.

Antibiotics remain an essential part of modern health care. However, these days, weighing whether an antibiotic is truly needed is all the more important.

EXPLORING THE MICROBIOME Horizontal Gene Transfer and the Unintended Consequences of Antibiotic Usage

Plasmids can be transferred between unrelated bacteria through cytoplasmic bridges between cells.

the bacterial chromosome (Figure 8.25). They are found mainly in bacteria but also in some eukaryotic microorganisms, such as Saccharomyces cerevisiae. The F factor is a conjugative plasmid that carries genes for sex pili and for the transfer of the plasmid to another cell. Although plasmids are usually dispensable, under certain conditions genes carried by plasmids can be crucial to the survival and growth of the cell. For example, dissimilation plasmids code for enzymes that trigger the catabolism of certain unusual sugars and hydrocarbons. Some species of Pseudomonas can actually use such exotic substances as toluene, camphor, and petroleum as primary carbon and energy sources because they have catabolic enzymes encoded by genes carried on plasmids. Such specialized capabilities permit the survival of those micro- organisms in very diverse and challenging environments. Because of their ability to degrade and detoxify a variety of unusual com- pounds, many of them are being investigated for possible use in the cleanup of environmental wastes.

Other plasmids code for proteins that enhance the patho- genicity of a bacterium. The strain of E. coli that causes infant diarrhea and traveler’s diarrhea carries plasmids that code for

toxin production and for bacterial attachment to intestinal cells. Without these plasmids, E. coli is a harmless resident of the large intestine; with them, it is pathogenic. Other plasmid-encoded toxins include the exfoliative toxin of Staphylococcus aureus, Clostridium tetani neurotoxin, and toxins of Bacillus anthracis. Still other plasmids contain genes for the synthesis of bacteriocins, toxic proteins that kill other bacteria. These plasmids have been found in many bacterial genera, and they are useful markers for the identification of certain bacteria in clinical laboratories.

Resistance factors (R factors) are plasmids that have signif- icant medical importance. They were first discovered in Japan in the late 1950s after several dysentery epidemics. In some of these epidemics, the infectious agent was resistant to the usual antibiotic. Following isolation, the pathogen was also found to be resistant to a number of different antibiotics. In addition, other normal bacteria from the patients (such as E. coli) proved to be resistant as well. Researchers soon discovered that these bacteria acquired resistance through the spread of genes from one organism to another. The plasmids that mediated this transfer are R factors.

230

CHAPTER 8 Microbial Genetics 231

In some cases, the accumulation of resistance genes on a single plasmid is quite remarkable. For example, Figure 8.25a shows a genetic map of resistance plasmid R100. This particular plasmid can be transferred between a number of enteric genera, including Escherichia, Klebsiella, and Salmonella.

R factors present very serious problems for treating infec- tious diseases with antibiotics. The widespread use of anti- biotics in medicine and agriculture (see the box in Chapter 20 on page 583) has led to the preferential survival (selec- tion) of bacteria that have R factors, so populations of resis- tant bacteria grow larger and larger. The transfer of resistance between bacterial cells of a population, and even between bacteria of different genera, also contributes to the problem. The ability to reproduce sexually with members of its own species defines a eukaryotic species. However, a bacterial species can conjugate and transfer plasmids to other species. Neisseria may have acquired its penicillinase-producing plasmid from Streptococcus, and Agrobacterium can transfer plasmids to plant cells (see Figure 9.20, page 262). Noncon- jugative plasmids may be transferred from one cell to another by inserting themselves into a conjugative plasmid or a chro- mosome or by transformation when released from a dead cell. Insertion is made possible by an insertion sequence, which will be discussed shortly.

Plasmids are an important tool for genetic engineering, dis- cussed in Chapter 9 (pages 243–247).

Transposons Transposons are small segments of DNA that can move (be “transposed”) from one region of a DNA molecule to another. These pieces of DNA are 700 to 40,000 base pairs long.

In the 1950s, American geneticist Barbara McClintock dis- covered transposons in corn, but they occur in all organisms and have been studied most thoroughly in microorganisms. They may move from one site to another site on the same chro- mosome or to another chromosome or plasmid. As you might imagine, the frequent movement of transposons could wreak havoc inside a cell. For example, as transposons move about on chromosomes, they may insert themselves within genes, inacti- vating them. Fortunately, transposition occurs relatively rarely. The frequency of transposition is comparable to the spontane- ous mutation rate that occurs in bacteria—that is, from 10-5 to 10-7 per generation.

All transposons contain the information for their own transposition. As shown in Figure 8.26a, the simplest transpo- sons, also called insertion sequences (IS), contain only a gene that codes for an enzyme (transposase, which catalyzes the cut- ting and resealing of DNA that occurs in transposition) and recognition sites. Recognition sites are short inverted repeat sequences of DNA that the enzyme recognizes as recombina- tion sites between the transposon and the chromosome.

R factors carry genes that confer upon their host cell resis- tance to antibiotics, heavy metals, or cellular toxins. Many R factors contain two groups of genes. One group is called the resistance transfer factor (RTF) and Includes genes for plasmid replication and conjugation. The other group, the r-determinant, has the resistance genes; it codes for the production of enzymes that inactivate certain drugs or toxic substances (Figure 8.25a). Different R factors, when present in the same cell, can recombine to produce R factors with new combinations of genes in their r-determinants.

R T

r-determ inant

Origin of replication

Mercury resistance

Sulfonamide resistance

Streptomycin resistance

Pilus and conjugation

proteins

Origin of transfer

Chloram- phenicol resistance

Tetracycline resistance

(a)

(b) SEM 20 nm

Figure 8.25 R factor, a type of plasmid. (a) A diagram of an R factor, which has two parts: the RTF contains genes needed for plasmid replication and transfer of the plasmid by conjugation, and the r-determinant carries genes for resistance to four different antibiotics and mercury (sul = sulfonamide resistance, str = streptomycin resistance, cml = chloramphenicol resistance, tet = tetracycline resistance, mer = mercury resistance); numbers are base pairs * 1000. (b) Plasmids from E. coli bacteria.

Q Why are R factors important in the treatment of infectious diseases?

232 PART ONE Fundamentals of Microbiology

Transposons with antibiotic resistance genes are of practi- cal interest, but there is no limitation on the kinds of genes that transposons can have. Thus, transposons provide a natu- ral mechanism for the movement of genes from one chromo- some to another. Furthermore, because they may be carried between cells on plasmids or viruses, they can also spread from one organism—or even species—to another. For example, van- comycin resistance was transferred from Enterococcus faecalis to Staphylococcus aureus via a transposon called Tn1546. Trans- posons are thus a potentially powerful mediator of evolu- tion in organisms.

Kanamycin resistance

Tn5

IS1

Transposase gene

IS1

Inverted repeat

IS1

A T

G A

CTA A

A 1 Transposase cuts DNA, leaving sticky ends.

2 Sticky ends of transposon and target DNA anneal.

T C

Kanam ycin resistance

(a) An insertion sequence (IS), the simplest transposon, contains a gene for transposase, the enzyme that catalyzes transposition. The tranposase gene is bounded at each end by inverted repeat sequences that function as recognition sites for the transposon. IS1 is one example of an insertion sequence, shown here with simplified IR sequences.

(b) Complex transposons carry other genetic material in addition to transposase genes. The example shown here, Tn5, carries the gene for kanamycin resistance and has complete copies of the insertion sequence IS1 at each end.

Figure 8.26 Transposons and insertion.

Q Why are transposons sometimes referred to as “jumping genes”?

Complex transposons also carry other genes not connected with the transposition process. For example, bacterial transpo- sons may contain genes for enterotoxin or for antibiotic resis- tance (Figure 8.26b). Plasmids such as R factors are frequently made up of a collection of transposons (Figure 8.26c).

Play Transposons: Overview, Insertion Sequences, Complex Transposons @MasteringMicrobiology

CHECK YOUR UNDERSTANDING

✓ 8-14 What types of genes do plasmids carry?

Transformation in Bacteria During the process of transformation, genes are transferred from one bacterium to another as “naked” DNA in solution. This process was first demonstrated over 70 years ago, although it was not understood at the time. Not only did transforma- tion show that genetic material could be transferred from one bacterial cell to another, but study of this phenomenon even- tually led to the conclusion that DNA is the genetic material. The initial experiment on transformation was performed by Frederick Griffith in England in 1928 while he was working with two strains of Streptococcus pneumoniae. One, a virulent strain, has a polysaccharide capsule that prevents phagocy- tosis. The bacteria grow and cause pneumonia. The other, an avirulent strain, lacks the capsule and does not cause disease.

Griffith was interested in determining whether injections of heat-killed bacteria of the encapsulated strain could be used to vaccinate mice against pneumonia. As he expected, injections of living encapsulated bacteria killed the mouse (Figure 8.27a); injections of live nonencapsulated bacteria (Figure 8.27b) or dead encapsulated bacteria (Figure 8.27c) did not kill the mouse. However, when the dead encapsulated bacteria were mixed with live nonencapsulated bacteria and injected into the mice, many of the mice died. In the blood of the dead mice, Griffith found living, encapsulated bacteria. Hereditary material (genes) from the dead bacteria had entered the live cells and changed them genetically so that their progeny were encapsulated and there- fore virulent (Figure 8.27d).

Subsequent investigations based on Griffith’s research revealed that bacterial transformation could be carried out without mice. A broth was inoculated with live nonencapsu- lated bacteria. Dead encapsulated bacteria were then added to the broth. After incubation, the culture was found to contain living bacteria that were encapsulated and virulent. The nonen- capsulated bacteria had been transformed; they had acquired

CHAPTER 8 Microbial Genetics 233

1 Living encapsulated bacteria injected into mouse.

2 Mouse died.

1 Living nonencapsulated bacteria injected into mouse.

1 Heat-killed encapsulated bacteria injected into mouse.

1 Living nonencapsulated and heat-killed encapsulated bacteria injected into mouse.

2 Mouse remained healthy. 2 Mouse died.

3 Colonies of encapsulated bacteria were isolated from dead mouse.

3 A few colonies of nonencap- sulated bacteria were isolated from mouse; phagocytes destroyed nonencapsulated bacteria.

3 No colonies were isolated from mouse.

3 Colonies of encapsulated bacteria were isolated from dead mouse.

2 Mouse remained healthy.

(a) (b) (c) (d)

RECOMBINATION

Figure 8.27 Griffith’s experiment demonstrating genetic transformation. (a) Living encapsulated bacteria caused disease and death when injected into a mouse. (b) Living nonencapsulated bacteria are readily destroyed by the phagocytic defenses of the host, so the mouse remained healthy after injection. (c) After being killed by heat,

encapsulated bacteria lost the ability to cause disease. (d) However, the combination of living nonencapsulated bacteria and heat- killed encapsulated bacteria (neither of which alone causes disease) did cause disease. Somehow, the live nonencapsulated bacteria were transformed by the dead encapsulated bacteria so that they acquired the ability

to form capsules and therefore cause disease. Subsequent experiments proved the transforming factor to be DNA.

Q Why did encapsulated bacteria kill the mouse while nonencapsulated bacteria did not? What killed the mouse in (d)?

a new hereditary trait by incorporating genes from the killed encapsulated bacteria.

The next step was to extract various chemical components from the killed cells to determine which component caused the transformation. These crucial experiments were performed in the United States by Oswald T. Avery and his associates Colin M. MacLeod and Maclyn McCarty. After years of research, they announced in 1944 that the component responsible for transforming harmless S. pneumoniae into virulent strains was DNA. Their results provided one of the conclusive indications that DNA was indeed the carrier of genetic information.

Since the time of Griffith’s experiment, considerable infor- mation has been gathered about transformation. In nature, some bacteria, perhaps after death and cell lysis, release their

DNA into the environment. Other bacteria can then encounter the DNA and, depending on the particular species and growth conditions, take up fragments of DNA and integrate them into their own chromosomes by recombination. A protein called RecA binds to the cell’s DNA and then to donor DNA causing the exchange of strands. A recipient cell with this new combination of genes is a kind of hybrid, or recombi- nant cell (Figure 8.28). All the descendants of such a recom- binant cell will be identical to it. Transformation occurs naturally among very few genera of bacteria, including Bacillus, Haemophilus (hē-MAH-fil-us), Neisseria, Acinetobacter (a-sin-E- tō-bak9ter), and certain strains of the genera Streptococcus and Staphylococcus.

234 PART ONE Fundamentals of Microbiology

Conjugation differs from transformation in two major ways. First, conjugation requires direct cell-to-cell contact. Second, the conjugating cells must generally be of opposite mating type; donor cells must carry the plasmid, and recipi- ent cells usually do not. In gram-negative bacteria, the plasmid carries genes that code for the synthesis of sex pili, projec- tions from the donor’s cell surface that contact the recipient and help bring the two cells into direct contact (Figure 8.29a). Gram- positive bacterial cells produce sticky surface molecules that cause cells to come into direct contact with each other. In the process of conjugation, the plasmid is replicated during the transfer of a single-stranded copy of the plasmid DNA to the recipient, where the complementary strand is synthesized (Figure 8.29b).

Because most experimental work on conjugation has been done with E. coli, we will describe the process in this organ- ism. In E. coli, the F factor (fertility factor) was the first plasmid observed to be transferred between cells during con- jugation. Donors carrying F factors (F+ cells) transfer the plas- mid to recipients (F- cells), which become F+ cells as a result (Figure 8.30a). In some cells carrying F factors, the factor

Even though only a small portion of a cell’s DNA is trans- ferred to the recipient, the molecule that must pass through the recipient cell wall and membrane is still very large. When a recipient cell is in a physiological state in which it can take up the donor DNA, it is said to be competent. Competence results from alterations in the cell wall that make it permeable to large DNA molecules.

Conjugation in Bacteria Another mechanism by which genetic material is transferred from one bacterium to another is known as conjugation. Conjugation is mediated by a conjugative plasmid, (discussed on page 229).

1 Recipient cell takes up donor DNA.

2 Donor DNA aligns with complementary bases.

a b c d

Recipient cell

Chromosomal DNADNA fragments from donor cells

3 Recombination occurs between donor DNA and recipient DNA.

Genetically transformed cell

Degraded unrecombined DNA

a b c d

3¿5¿

A D

b c d

A D

a D

B C

5¿ 3¿

Figure 8.28 The mechanism of genetic transformation in bacteria. Some similarity is needed for the donor and recipient to align. Genes a, b, c, and d may be mutations of genes A, B, C, and D.

Q What type of enzyme cuts the donor DNA?

Play Transformation @MasteringMicrobiology

205 224 227 234

CLINICAL CASE Resolved

The Ames test allows rapid screening of chemicals for genotoxicity. The his- mutant Salmonella bacteria used in the Ames test are spread over glucose–minimal salts agar plates. A paper disk saturated with 2-aminofluorene (2-AF), an aromatic amine, is placed on the culture. The figure, for example, shows that reversion of the his- mutation allowed the Salmonella to grow. This indicates that the chemical is mutagenic and is therefore potentially carcinogenic.

There are studies indicating that 2-AF activated by enzymes is more damaging than 2-AF alone, suggesting that the interaction between diet and intestinal microbiota is more likely to cause cancer than just diet. Variations in diet produce little change in the kinds of bacteria in the intestine, but they produce dramatic changes in the metabolic activity of the bacteria.

The detection of serrated colorectal polyps from Marcel’s stool DNA test led to a diagnosis of an early, rather than late, stage of colorectal cancer. The offending polyps are found and removed, and Marcel undergoes chemotherapy to kill any missed cancer cells in his colon.

CHAPTER 8 Microbial Genetics 235

Sex pilus

F+ cell

F– cell

Mating bridge

(a) Sex pilus (b) Mating bridge TEMSEM 0.3 mm1.5 mm

Figure 8.29 Bacterial conjugation.

Q What is an F+ cell?

integrates into the chromosome, converting the F+ cell to an Hfr cell (high frequency of recombination) (Figure 8.30b). When conjugation occurs between an Hfr cell and an F- cell, the Hfr cell’s chromosome (with its integrated F factor) repli- cates, and a parental strand of the chromosome is transferred to the recipient cell (Figure 8.30c). Replication of the Hfr chro- mosome begins in the middle of the integrated F factor, and a small piece of the F factor leads the chromosomal genes into the F- cell. Usually, the chromosome breaks before it is com- pletely transferred. Once within the recipient cell, donor DNA can recombine with the recipient’s DNA. (Donor DNA that is not integrated is degraded.) Therefore, by conjugation with an Hfr cell, an F- cell may acquire new versions of chromo- somal genes (just as in transformation). However, it remains an F- cell because it did not receive a complete F factor during conjugation.

Conjugation is used to map the location of genes on a bac- terial chromosome (Figure 8.31). The genes for the synthesis of threonine (thr) and leucine (leu) are first, reading clockwise from 0. Their locations were determined by conjugation experiments. Assume that conjugation is allowed for only 1 minute between an Hfr strain that is his+, pro+, thr+, and leu+, and an F- strain that is his-, pro-, thr-, and leu-. If the F- acquired the ability to syn- thesize threonine, then the thr gene is located early in the chro- mosome, between 0 and 1 minute. If after 2 minutes the F- cell now becomes thr+ and leu+, the order of these two genes on the chromosome must be thr, leu.

Transduction in Bacteria A third mechanism of genetic transfer between bacteria is transduction. In this process, bacterial DNA is transferred from a donor cell to a recipient cell inside a virus that infects bacte- ria, called a bacteriophage, or phage. (Phages will be discussed further in Chapter 13.)

To understand how transduction works, we will consider the life cycle of one type of transducing phage of E. coli; this phage carries out generalized transduction (Figure 8.32).

During phage reproduction, phage DNA and proteins are synthesized by the host bacterial cell. The phage DNA should be packaged inside the phage protein coat. However, bacterial DNA, plasmid DNA, or even DNA of another virus may be packaged inside a phage pro- tein coat.

All genes contained within a bacterium infected by a generalized transducing phage are equally likely to be packaged in a phage coat and transferred. In another type of transduction, called specialized transduction, only certain bacterial genes are transferred (see page 375). In one type of specialized transduction, the phage codes for certain toxins produced by their bacterial hosts, such as diphtheria toxin for Corynebacterium diphtheriae (kor9Ī-nē-bak-TI-rē-um dif- THI-rē-Ī), erythrogenic toxin for Streptococcus pyogenes, and Shiga toxin for E. coli O157:H7.

CHECK YOUR UNDERSTANDING

✓ 8-15 Differentiate horizontal and vertical gene transfer. ✓ 8-16 Compare conjugation between the following pairs: F + *

F -, Hfr * F -.

Play Conjugation: Overview, F Factor, Hfr Conjugation, Chromosome Mapping @MasteringMicrobiology

Play Transduction: Generalized Transduction @MasteringMicrobiology

236 PART ONE Fundamentals of Microbiology

Recombination between F factor and chromosome, occurring at a specific site on each

Insertion of F factor into chromosome

Hfr cellF+ cell

Integrated F factor

F+ cell F– cell

Replication and transfer of F factor

F+ cell F+ cell

(a) When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F–), the F– cell is converted to an F+ cell.

(b) When an F factor becomes integrated into the chromosome of an F+ cell, it makes the cell a high frequency of recombination (Hfr) cell.

Replication and transfer of part of the chromosome

In the recipient, recombination between the Hfr chromosome fragment and the F– chromosome

Hfr cell

Recombinant F– cell

Hfr cell F– cell

Bacterial chromosome

F factor

Mating bridge

RECOMBINATION

Figure 8.30 Conjugation in E. coli.

Q Do bacteria reproduce during conjugation?

CHAPTER 8 Microbial Genetics 237

Bacterial DNA

Phage DNA

Recipient bacterial DNA

Many cell divisions

Donor bacterial DNA

1 A phage infects the donor bacterial cell.

3 Occasionally during phage assembly, pieces of bacterial DNA are pack- aged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA.

Phage protein coat

Bacterial chromosome

Donor cell

Phage DNA

Recipient cell

Recombinant cell reproduces normally

2 Phage DNA and proteins are made, and the bacterial chromosome is broken into pieces.

4 A phage carrying bacterial DNA infects a new host cell, the recipient cell.

5 Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells.

RECOMBINATION

Figure 8.32 Transduction by a bacteriophage. Shown here is generalized transduction, in which any bacterial DNA can be transferred from one cell to another.

Q How could E. coli acquire the Shiga toxin gene?

Genes and Evolution LEARNING OBJECTIVE

8-17 Discuss how genetic mutation and recombination provide material for natural selection to act upon.

We have now seen how gene activity can be controlled by the cell’s internal regulatory mechanisms and how genes them- selves can be altered or rearranged by mutation, transposition, and recombination. All these processes provide diversity in the descendants of cells. Diversity provides the raw material for evo- lution, and natural selection provides its driving force. Natural selection will act on diverse populations to ensure the survival of those fit for that particular environment. The different kinds of microorganisms that exist today are the result of a long history of evolution. Microorganisms have continually changed by alter- ations in their genetic properties and acquisition of adaptations

Play Interactive Microbiology @MasteringMicrobiology See how the selection of antibiotic-resistant microbes affects a patient’s health

CHECK YOUR UNDERSTANDING

✓ 8-17 Natural selection means that the environment favors survival of some genotypes. From where does diversity in genotypes come?

Amino acid metabolism

DNA replication and repair

Lipid metabolism

KEY

Carbohydrate metabolism

Membrane synthesis

50 60

3480 kbp

bp1

1160 kbp

2320 kbp

Figure 8.31 A genetic map of the chromosome of E. coli. This map is made by observing recombinant cells after conjugation. The numbers inside the circle indicate the number of minutes it takes to transfer the genes during mating between two cells; the numbers in colored boxes indicate the number of base pairs. 1 kbp = 1000 base pairs.

Q How many minutes of conjugation would be needed to transfer genes for membrane synthesis on this chromosome?

to many different habitats. See Exploring the Microbiome on page 230 and the box on antibiotic resistance in Chapter 26, page 771, for examples of natural selection.

238 PART ONE Fundamentals of Microbiology

Structure and Function of the Genetic Material (pp. 205–217) 1. Genetics is the study of what genes are, how they carry

information, how their information is expressed, and how they are replicated and passed to subsequent generations or other organisms.

2. DNA in cells exists as a double-stranded helix; the two strands are held together by hydrogen bonds between specific nitrogenous base pairs: AT and CG.

3. A gene is a sequence of nucleotides, that encodes a functional product, usually a protein.

4. The DNA in a cell is duplicated before the cell divides, so each offspring cell receives the same genetic information.

Genotype and Phenotype (pp. 205, 208) 5. Genotype is the genetic composition of an organism, its entire

complement of DNA.

6. Phenotype is the expression of the genes: the proteins of the cell and the properties they confer on the organism.

DNA and Chromosomes (p. 208) 7. The DNA in a chromosome exists as one long double helix

associated with various proteins that regulate genetic activity.

8. Genomics is the molecular characterization of genomes.

The Flow of Genetic Information (p. 208) 9. Following cell division, each offspring cell receives a chromosome

that is virtually identical to the parent’s.

10. Information contained in the DNA is transcribed into RNA and translated into proteins.

DNA Replication (pp. 208–212) 11. During DNA replication, the two strands of the double helix

separate at the replication fork, and each strand is used as a template by DNA polymerases to synthesize two new strands of DNA according to the rules of complementary base pairing.

12. The result of DNA replication is two new strands of DNA, each having a base sequence complementary to one of the original strands.

13. Because each double-stranded DNA molecule contains one original and one new strand, the replication process is called semiconservative.

14. DNA is synthesized in one direction designated 5’ S 3’. At the replication fork, the leading strand is synthesized continuously and the lagging strand discontinuously.

15. DNA polymerase proofreads new molecules of DNA and removes mismatched bases before continuing DNA synthesis.

RNA and Protein Synthesis (pp. 212–217) 16. During transcription, the enzyme RNA polymerase synthesizes a

strand of RNA from one strand of double-stranded DNA, which serves as a template.

17. RNA is synthesized from nucleotides containing the bases A, C, G, and U, which pair with the bases of the DNA strand being transcribed.

18. RNA polymerase binds the promoter; transcription begins at AUG; the region of DNA that is the end point of transcription is the terminator; RNA is synthesized in the 5’ S 3’ direction.

19. Translation is the process in which the information in the nucleotide base sequence of mRNA is used to dictate the amino acid sequence of a protein.

20. The mRNA associates with ribosomes, which consist of rRNA and protein.

21. Three-base codons of mRNA specify amino acids.

22. The genetic code refers to the relationship among the nucleotide base sequence of DNA, the corresponding codons of mRNA, and the amino acids for which the codons code.

23. Specific amino acids are attached to molecules of tRNA. Another portion of the tRNA has a base triplet called an anticodon.

24. The base pairing of codon and anticodon at the ribosome results in specific amino acids being brought to the site of protein synthesis.

25. The ribosome moves along the mRNA strand as amino acids are joined to form a growing polypeptide; mRNA is read in the 5’ S 3’ direction.

26. Translation ends when the ribosome reaches a stop codon on the mRNA.

The Regulation of Bacterial Gene Expression (pp. 217–221)

1. Regulating protein synthesis at the gene level is energy-efficient because proteins are synthesized only as they are needed.

2. Constitutive genes are expressed at a fixed rate. Examples are genes for the enzymes in glycolysis.

Pre-transcriptional Control (pp. 217–220) 3. In bacteria, a group of coordinately regulated structural genes with

related metabolic functions, plus the promoter and operator sites that control their transcription, is called an operon.

4. In the operon model for an inducible system, a regulatory gene codes for the repressor protein.

5. When the inducer is absent, the repressor binds to the operator, and no mRNA is synthesized.

6. When the inducer is present, it binds to the repressor so that it cannot bind to the operator; thus, mRNA is made, and enzyme synthesis is induced.

7. In repressible systems, the repressor requires a corepressor in order to bind to the operator site; thus, the corepressor controls enzyme synthesis.

8. Transcription of structural genes for catabolic enzymes (such as b-galactosidase) is induced by the absence of glucose. Cyclic AMP and CRP must bind to a promoter in the presence of an alternative carbohydrate.

9. Methylated nucleotides are not transcribed in epigenetic control.

Post-transcriptional Control (pp. 220–221) 10. mRNA as a riboswitch regulates translation.

11. MicroRNAs combine with mRNA; the resulting double-stranded RNA is destroyed.

Study Outline Go to @MasteringMicrobiology for Interactive Microbiology, In the Clinic videos, MicroFlix, MicroBoosters, 3D animations, practice quizzes, and more.

CHAPTER 8 Microbial Genetics 239

2. In crossing over, genes from two chromosomes are recombined into one chromosome containing some genes from each original chromosome.

3. Vertical gene transfer occurs during reproduction when genes are passed from an organism to its offspring.

4. Horizontal gene transfer in bacteria involves a portion of the cell’s DNA being transferred from donor to recipient.

5. When some of the donor’s DNA has been integrated into the recipient’s DNA, the resultant cell is called a recombinant.

Plasmids and Transposons (pp. 229–232) 6. Plasmids are self-replicating circular molecules of DNA carrying

genes that are not usually essential for the cell’s survival.

7. There are several types of plasmids, including conjugative plasmids, dissimilation plasmids, plasmids carrying genes for toxins or bacteriocins, and resistance factors.

8. Transposons are small segments of DNA that can move from one region to another region of the same chromosome or to a different chromosome or a plasmid.

9. Complex transposons can carry any type of gene, including antibiotic-resistance genes, and are thus a natural mechanism for moving genes from one chromosome to another.

Transformation in Bacteria (pp. 232–234) 10. During this process, genes are transferred from one bacterium to

another as “naked” DNA in solution.

Conjugation in Bacteria (pp. 234–235) 11. This process requires contact between living cells.

12. One type of genetic donor cell is an F+; recipient cells are F-. F cells contain plasmids called F factors; these are transferred to the F- cells during conjugation.

Transduction in Bacteria (pp. 235–237) 13. In this process, DNA is passed from one bacterium to another in a

bacteriophage and is then incorporated into the recipient’s DNA.

14. In generalized transduction, any bacterial genes can be transferred.

Genes and Evolution (p. 237) 1. Diversity is the

precondition for evolution.

2. Genetic mutation and recombination provide diversity of organisms, and the process of natural selection allows the growth of those best adapted to a given environment.

Changes in Genetic Material (pp. 221–228) 1. Mutations and horizontal gene transfer can change a bacterium’s

genotype.

Mutation (p. 222) 2. A mutation is a change in the nitrogenous base sequence of DNA;

that change causes a change in the product coded for by the mutated gene.

3. Many mutations are neutral, some are disadvantageous, and others are beneficial.

Types of Mutations (pp. 222–223) 4. A base substitution occurs when one base pair in DNA is replaced

with a different base pair.

5. Alterations in DNA can result in missense mutations, frameshift, or nonsense mutations.

6. Spontaneous mutations occur without the presence of any mutagen.

Mutagens (pp. 223–226) 7. Mutagens are agents in the environment that cause permanent

changes in DNA.

8. Ionizing radiation causes the formation of ions and free radicals that react with DNA; base substitutions or breakage of the sugar- phosphate backbone results.

9. Ultraviolet (UV) radiation is nonionizing; it causes bonding between adjacent thymines.

The Frequency of Mutation (p. 226) 10. Mutation rate is the probability that a gene will mutate when a cell

divides; the rate is expressed as 10 to a negative power.

11. A low rate of spontaneous mutations is beneficial in providing the genetic diversity needed for evolution.

Identifying Mutants (p. 226) 12. Mutants can be detected by selecting or testing for an altered

phenotype.

13. Positive selection involves the selection of mutant cells and the rejection of nonmutated cells.

14. Replica plating is used for negative selection—to detect, for example, auxotrophs that have nutritional requirements not possessed by the parent (nonmutated) cell.

Identifying Chemical Carcinogens (pp. 227–228) 15. The Ames test is a relatively inexpensive and rapid test for

identifying possible chemical carcinogens.

16. The test assumes that a mutant cell can revert to a normal cell in the presence of a mutagen and that many mutagens are carcinogens.

Genetic Transfer and Recombination (pp. 229–237) 1. Genetic recombination, the rearrangement of genes from separate

groups of genes, usually involves DNA from different organisms; it contributes to genetic diversity.

Play Interactive Microbiology @MasteringMicrobiology See how the selection of antibiotic-resistant microbes affects a patient’s health

240 PART ONE Fundamentals of Microbiology

c. Write the code for the complementary strand of DNA completed in part (a).

d. What would be the effect if C were substituted for T at base 10? e. What would be the effect if A were substituted for G at base 11? f. What would be the effect if G were substituted for T at base 14? g. What would be the effect if C were inserted between bases 9

and 10? h. How would UV radiation affect this strand of DNA? i. Identify a nonsense sequence in this strand of DNA.

5. When iron is not available, E. coli can stop synthesis of all proteins, such as superoxide dismutase and succinate dehydrogenase, that require iron. Describe a mechanism for this regulation.

6. Identify when (before transcription, after transcription but before translation, after translation) each of the following regulatory mechanisms functions. a. ATP combines with an enzyme, altering its shape. b. A short RNA is synthesized that is complementary to mRNA. c. Methylation of DNA occurs. d. An inducer combines with a repressor.

7. Which sequence is the best target for damage by UV radiation: AGGCAA, CTTTGA, or GUAAAU? Why aren’t all bacteria killed when they are exposed to sunlight?

8. You are provided with cultures with the following characteristics:

Culture 1: F+, genotype A+ B+ C+

Culture 2: F-, genotype A- B- C-

a. Indicate the possible genotypes of a recombinant cell resulting from the conjugation of cultures 1 and 2.

b. Indicate the possible genotypes of a recombinant cell resulting from conjugation of the two cultures after the F+ has become an Hfr cell.

9. Why are mutation and recombination important in the process of natural selection and the evolution of organisms?

10. NAME IT Normally a commensal in the human intestine, this bacterium became pathogenic after acquiring a toxin gene from a Shigella bacterium.

Multiple Choice Match the following terms to the definitions in questions 1and 2.

a. conjugation b. transcription c. transduction d. transformation e. translation

1. Transfer of DNA from a donor to a recipient cell by a bacteriophage.

2. Transfer of DNA from a donor to a recipient as naked DNA in solution.

3. Feedback inhibition differs from repression because feedback inhibition a. is less precise. b. is slower acting. c. stops the action of preexisting enzymes. d. stops the synthesis of new enzymes. e. all of the above

3. Match the following examples of mutagens.

Column A

_______ a. A mutagen that is incorporated into DNA in place of a normal base

_______ b. A mutagen that causes the formation of highly reactive ions

_______ c. A mutagen that alters adenine so that it base-pairs with cytosine

_______ d. A mutagen that causes insertions

_______ e. A mutagen that causes the formation of pyrimidine dimers

Column B

1. Frameshift mutagen

2. Nucleoside analog

3. Base-pair mutagen

4. Ionizing radiation

5. Nonionizing radiation

4. The following is a code for a strand of DNA.

DNA 3’ A T A T _ _ _ T T T _ _ _ _ _ _ _ _ _ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

mRNA C G U U G A

tRNA U G G

Amino Acid Met

ATAT = Promoter sequence

For answers to the Knowledge and Comprehension questions, turn to the Answers tab at the back of the textbook.

Knowledge and Comprehension

Review 1. Briefly describe the components of DNA, and explain its functional

relationship to RNA and protein.

2. DRAW IT Identify and mark each of the following on the portion of DNA undergoing replication: replication fork, DNA polymerase, RNA primer, parent strands, leading strand, lagging strand, the direction of replication on each strand, and the 5’ end of each strand.

Study Questions

5¿ 3¿

a. Using the genetic code provided in Figure 8.8, fill in the blanks to complete the segment of DNA shown.

b. Fill in the blanks to complete the sequence of amino acids coded for by this strand of DNA.

CHAPTER 8 Microbial Genetics 241

2. Replication of the E. coli chromosome takes 40 to 45 minutes, but the organism has a generation time of 26 minutes. How does the cell have time to make complete chromosomes for each offspring cell?

3. Pseudomonas has a plasmid containing the mer operon, which includes the gene for mercuric reductase. This enzyme catalyzes the reduction of the mercuric ion Hg2+ to the uncharged form of mercury, Hg0. Hg2+ is quite toxic to cells; Hg0 is not. a. What do you suppose is the inducer for this operon? b. The protein encoded by one of the mer genes binds Hg2+ in the

periplasm and brings it into the cell. Why would a cell bring in a toxin?

Clinical Applications and Evaluation 1. Ciprofloxacin, erythromycin, and acyclovir are used to treat

microbial infections. Ciprofloxacin inhibits DNA gyrase. Erythromycin binds in front of the A site on the 50S subunit of a ribosome. Acyclovir is a guanine analog. a. What steps in protein synthesis are inhibited by each drug? b. Which drug is more effective against bacteria? Why? c. Which drugs will have effects on the host’s cells? Why? d. Use the index to identify the disease for which acyclovir is

primarily used. Why is it more effective than erythromycin for treating this disease?

2. HIV, the virus that causes AIDS, was isolated from three individuals, and the amino acid sequences for the viral coat were determined. Of the amino acid sequences shown below, which two of the viruses are most closely related? How can these amino acid sequences be used to identify the source of a virus?

Patient Viral Amino Acid Sequence

A Asn Gln Thr Ala Ala Ser Lys Asn Ile Asp Ala Leu

B Asn Leu His Ser Asp Lys Ile Asn Ile Ile Leu Leu

C Asn Gln Thr Ala Asp Ser Ile Val Ile Asp Ala Leu

3. Human herpesvirus-8 (HHV-8) is common in parts of Africa, the Middle East, and the Mediterranean, but is rare elsewhere except in AIDS patients. Genetic analyses indicate that the African strain is not changing, whereas the Western strain is accumulating changes. Using the portions of the HHV-8 genomes (shown below) that encode one of the viral proteins, how similar are these two viruses? What mechanism can account for the changes? What disease does HHV-8 cause?

Western 3’-ATGGAGTTCTTCTGGACAAGA African 3’-AT A A AC TT T TTCT T GACAA CG

4. Bacteria can acquire antibiotic resistance by all of the following except a. mutation. b. insertion of transposons. c. conjugation. d. snRNPs. e. transformation.

5. Suppose you inoculate three flasks of minimal salts broth with E. coli. Flask A contains glucose. Flask B contains glucose and lactose. Flask C contains lactose. After a few hours of incubation, you test the flasks for the presence of b-galactosidase. Which flask(s) do you predict will have this enzyme? a. A b. B c. C d. A and B e. B and C

6. Plasmids differ from transposons in that plasmids a. become inserted into chromosomes. b. are self-replicated outside the chromosome. c. move from chromosome to chromosome. d. carry genes for antibiotic resistance. e. none of the above

Use the following choices to answer questions 7 and 8:

a. catabolite repression b. DNA polymerase c. induction d. repression e. translation

7. Mechanism by which the presence of glucose inhibits the lac operon.

8. The mechanism by which lactose controls the lac operon.

9. Two offspring cells are most likely to inherit which one of the following from the parent cell? a. a change in a nucleotide in mRNA b. a change in a nucleotide in tRNA c. a change in a nucleotide in rRNA d. a change in a nucleotide in DNA e. a change in a protein

10. Which of the following is not a method of horizontal gene transfer? a. binary fission b. conjugation c. integration of a transposon d. transduction e. transformation

Analysis 1. Nucleoside analogs and ionizing radiation are used in treating

cancer. These mutagens can cause cancer, so why do you suppose they are used to treat the disease?

▶ Human immunodeficiency virus (HIV) (yellow) budding from a host cell.

242

F or thousands of years, people have consumed foods produced by the action of microorganisms. Bread, chocolate, and soy sauce are some of the best-known examples. But it was only just over 100 years ago that scientists showed that microorganisms are responsible for these products. This knowledge opened the way for using microorganisms to produce other important products. Since World War I, microbes have been used to produce a variety of chemicals, such as ethanol, acetone, and citric acid. Since World War II, microorganisms have been grown to produce antibiotics. More recently, microbes and their enzymes are replacing a variety of chemical processes involved in manufacturing such products as paper, textiles, and fructose. Using microbes or their enzymes instead of chemical syntheses offers several advantages: microbes may use inexpensive, abundant raw materials; microbes work at normal temperatures and pressure, thereby avoiding the need for expensive and dangerous systems; and microbes don’t produce toxic, hard-to-treat wastes. In the past 30 years, DNA technology has been added to the tools used to make products.

In this chapter you will learn the tools and techniques that are used to research and develop a product. You will also learn how DNA technology is used to track outbreaks of infectious disease and to provide evidence for courts of law in forensic microbiology. The Clinical Case illustrates the use of DNA technology to track HIV (see the photo).

In the Clinic A crime suspect claims he is innocent. His clothing became bloodstained, he says, when he tried to resuscitate the victim. The pattern of blood on the suspect may have resulted from striking the victim, but it is also consistent with spatter from the victim’s nose and mouth during CPR. As a forensic nurse for a police department, you collect bloodstained fabric from the suspect and a blood sample from the crime scene. You request PCR for streptococci on both samples. The test is positive for the fabric, but negative for the blood at the scene. How can a PCR test detect evidence of Streptococcus bacteria in such small samples? Do these results help or hurt the suspect?

Hint: Read about the polymerase chain reaction technique on page 247.

Biotechnology and DNA Technology9

Play In the Clinic Video @MasteringMicrobiology Play In the Clinic Video @MasteringMicrobiology

CHAPTER 9 Biotechnology and DNA Technology 243

Introduction to Biotechnology LEARNING OBJECTIVES

9-1 Compare and contrast biotechnology, genetic modification, and recombinant DNA technology.

9-2 Identify the roles of a clone and a vector in making recombinant DNA.

Biotechnology is the use of microorganisms, cells, or cell com- ponents to make a product. Microbes have been used in the commercial production of foods, vaccines, antibiotics, and vitamins for years. Bacteria are also used in mining to extract valuable elements from ore (see Figure 28.12, page 822). Addi- tionally, animal cells have been used to produce viral vaccines since the 1950s. Until the 1980s, products made by living cells were all made by naturally occurring cells; the role of scientists was to find the appropriate cell and develop a method for their large-scale cultivation.

Now, microorganisms and plants are being used as “facto- ries” to produce chemicals that the organisms don’t naturally make. This is accomplished by inserting, deleting, or modify- ing genes with recombinant DNA (rDNA) technology, which is sometimes called genetic engineering. The devel- opment of rDNA technology is expanding the practical applica- tions of biotechnology almost beyond imagination.

Recombinant DNA Technology Recombination of DNA occurs naturally in microbes (see Chapter 8). In the 1970s and 1980s, scientists developed artifi- cial techniques for making rDNA.

A gene from one organism can be inserted into the DNA of a bacterium or a yeast. In many cases, the recipient can then be made to express the gene, which may code for a commercially useful product. Thus, bacteria with genes for human insulin are now being used to produce insulin for treating diabetes, and a vac- cine for hepatitis B is being made by yeast carrying a gene for part of the hepatitis virus (the yeast produces a viral coat protein). Sci- entists hope that such an approach may prove useful in produc- ing vaccines against other infectious agents, thus eliminating the need to use whole organisms, as in conventional vaccines.

The rDNA techniques can also be used to make thousands of copies of the same DNA molecule—to amplify DNA—thus generating sufficient DNA for various kinds of experimenta- tion and analysis. This technique has practical application for identifying microbes, such as viruses, that can’t be cultured.

An Overview of Recombinant DNA Procedures An overview of some of the procedures typically used for mak- ing rDNA, along with some promising applications, is shown in Figure 9.1. A vector is a DNA molecule that transports

ASM: Cell genomes can be manipulated to alter cell function.

foreign DNA into a cell. (See more on vectors on page 246.) The gene of interest is inserted into the vector DNA in vitro. In Figure 9.1, the vector is a plasmid. The DNA molecule chosen as a vector must be self-replicating, such as a plasmid or a viral genome. This recombinant vector DNA is taken up by a cell such as a bacterium, where it can multiply. The cell contain- ing the recombinant vector is then grown in culture to form a clone of many genetically identical cells, each of which carries copies of the vector, and therefore many copies of the gene of interest. This is why DNA vectors are often called gene-cloning vectors, or simply cloning vectors. (In addition to referring to a culture of identical cells, the word clone is also routinely used as a verb, to describe the entire process, as in “to clone a gene.”)

The final step varies according to whether the gene itself or the product of the gene is of interest. From the cell clone, the researcher may isolate (“harvest”) large quantities of the gene of interest, which may then be used for a variety of purposes. The gene may even be inserted into another vector for intro- duction into another kind of cell (such as a plant or animal cell). Alternatively, if the gene of interest is expressed (tran- scribed and translated) in the cell clone, its protein product can be harvested and used for a variety of purposes.

The advantages of using rDNA for obtaining such pro- teins is illustrated by one of its early successes, the produc- tion of human growth hormone (hGH) in E. coli bacteria. Some individuals don’t produce adequate amounts of hGH, so their growth is stunted. In the past, hGH had to be obtained from human pituitary glands at autopsy. (Growth hormone

CLINICAL CASE No Ordinary Checkup

Dr. B. is closing his dental practice after 20 years. Four years ago, he went to his family doctor because of debilitating exhaustion. He thought he had a flu virus that he could not shake, and he was also having night sweats. His doctor ordered a myriad of blood tests, but only one came back positive. Dr. B. had HIV. Although he immediately began an HIV treatment regimen, one year later he was diagnosed with AIDS. Now, two years later, Dr. B. is very ill and can no longer work.

Dr. B. lets his employees know the situation and suggests that they all get tested for HIV. All of Dr. B.’s employees, including the hygienists, test negative. Dr. B. also writes an open letter to his patients informing them of his decision to close his practice and why he is doing so. This letter prompts 400 former patients to be tested for HIV, seven of whom test positive for antibodies against HIV.

What type of test can determine whether these patients contracted HIV from Dr. B.? Read on to find out.

243 249 252 254 257

1 Vector, such as a plasmid, is isolated.

2 DNA containing the gene of interest from a different species is cleaved by an enzyme into fragments.

Bacterial chromosome

Plasmid

DNA containing the gene of interest

Plasmid RNA Protein product

Gene encoding protein for pest resistance is inserted into plant cells.

Gene encoding degradative enzyme to clean up toxic waste is inserted into bacterial cells.

Amylase, cellulase, and other enzymes prepare fabrics for clothing manufacture.

Human growth hormone treats stunted growth.

3 The desired gene is selected and inserted into a plasmid.

4 The plasmid is taken up by a cell, such as a bacterium.

5 Cells with the gene of interest are cloned with either of two goals in mind.

6a Create and harvest copies of a gene.

6b Create and harvest protein products of a gene.

bacterium

transformed bacterium

recombinant DNA (plasmid)

KEY CONCEPTS

●

Genes from one organism’s cells can be inserted and expressed in another organism’s cells.

Genetically modified cells can be used to create a wide variety of useful products and applications.

●

FOUNDATION FIGURE

9.1 A Typical Genetic Modification Procedure

244

CHAPTER 9 Biotechnology and DNA Technology 245

cultures to radiation, the highest-yielding variant among the survivors was selected for another exposure to a mutagen. Using mutations, biologists increased the amount of penicillin the fungus produced by over 1000 times.

Screening each mutant for penicillin production is a tedious process. Site-directed mutagenesis is more targeted and can be used to make a specific change in a gene. Suppose you determine that changing one amino acid will make a laun- dry enzyme work better in cold water. Using the genetic code (see Figure 8.8, page 214), you could, using the techniques described next, produce the sequence of DNA that encodes that amino acid and insert it into that enzyme’s gene.

The science of molecular genetics has advanced to such a degree that many routine cloning procedures are performed using prepackaged materials and procedures that are very much like cookbook recipes. Scientists have a grab bag of methods from which to choose, depending on the ultimate application of their experiments. Next we describe some of the most important tools and techniques, and later we will con- sider some applications.

Restriction Enzymes Recombinant DNA technology has its technical roots in the discovery of restriction enzymes, a special class of DNA- cutting enzymes that exist in many bacteria. First isolated in 1970, restriction enzymes in nature had actually been observed earlier, when certain bacteriophages were found to have a restricted host range. If these phages were used to infect bacteria other than their usual hosts, restriction enzymes in the new host destroyed almost all the phage DNA. Restriction enzymes protect a bacterial cell by hydrolyzing phage DNA. The bacterial DNA is protected from digestion because the cell methylates (adds methyl groups to) some of the cytosines in its DNA. The purified forms of these bacterial enzymes are used in today’s laboratories.

What is important for rDNA techniques is that a restric- tion enzyme recognizes and cuts, or digests, only one par- ticular sequence of nucleotide bases in DNA, and it cuts this sequence the same way each time. Typical restriction enzymes used in cloning experiments recognize four-, six-, or eight-base sequences. Hundreds of restriction enzymes are known, each producing DNA fragments with characteristic ends. A few restric- tion enzymes are listed in Table 9.1. You can see they are named for their bacterial source. Some of these enzymes (e.g., HaeIII) cut both strands of DNA in the same place, producing blunt ends, and others make staggered cuts in the two strands—cuts that are not directly opposite each other (Figure 9.2). These stag- gered ends, or sticky ends, are most useful in rDNA because they can be used to join two different pieces of DNA that were cut by the same restriction enzyme. The sticky ends “stick” to stretches of single-stranded DNA by complementary base pairing.

from other animals is not effective in humans.) This practice was not only expensive but also dangerous because on several occasions neurological diseases were transmitted with the hor- mone. Human growth hormone produced by genetically modi- fied E. coli is a pure and cost-effective product. Recombinant DNA techniques also result in faster production of the hor-

mone than traditional methods might allow.

CHECK YOUR UNDERSTANDING

✓ 9-1 Differentiate biotechnology and rDNA technology. ✓ 9-2 In one sentence, describe how a vector and clone are

used.

Tools of Biotechnology LEARNING OBJECTIVES

9-3 Compare selection and mutation.

9-4 Define restriction enzymes, and outline how they are used to make rDNA.

9-5 List the four properties of vectors.

9-6 Describe the use of plasmid and viral vectors.

9-7 Outline the steps in PCR, and provide an example of its use.

Research scientists and technicians isolate bacteria and fungi from natural environments such as soil and water to find, or select, the organisms that produce a desired product. The selected organism can be mutated to make more product or to make a better product.

Selection In nature, organisms with characteristics that enhance survival are more likely to survive and reproduce than are variants that lack the desirable traits. This is called natural selection. Humans use artificial selection to select desirable breeds of animals or strains of plants to cultivate. As microbiologists learned how to isolate and grow microorganisms in pure culture, they were able to select the ones that could accomplish a desired objec- tive, such as brewing beer more efficiently or producing a new antibiotic. Over 2000 strains of antibiotic-producing bacteria have been discovered by testing soil bacteria and selecting the strains that produce an antibiotic.

Mutation Mutations are responsible for much of the diversity of life (see Chapter 8). A bacterium with a mutation that confers resistance to an antibiotic will survive and reproduce in the presence of that antibiotic. Biologists working with antibiotic-producing microbes discovered that they could create new strains by exposing microbes to mutagens. After random mutations were created in penicillin-producing Penicillium by exposing fungal

246 PART ONE Fundamentals of Microbiology

sources have been produced by the action of the same restric- tion enzyme, the two pieces will have identical sets of sticky ends and can be spliced (recombined) in vitro. The sticky ends join spontaneously by hydrogen bonding (base pairing). The enzyme DNA ligase is used to covalently link the backbones of the DNA pieces, producing an rDNA molecule.

Vectors Many different types of DNA molecules can serve as vectors, provided they have certain properties. The most important property is self-replication; once in a cell, a vector must be capable of replicating. Any DNA that is inserted in the vector will be replicated in the process. Thus, vectors serve as vehicles for the replication of desired DNA sequences.

Vectors also need to be large enough to be manipulated out- side the cell during rDNA procedures. Smaller vectors are more

TABLE 9.1 Selected Restriction Enzymes Used in rDNA Technology

Enzyme Bacterial Source Recognition Sequence

BamHI Bacillus

amyloliquefaciens

G G A T C C C C T A G G

EcoRI Escherichia

coli

G A A T T C C T T A A G

HaeIII Haemophilus

aegyptius

G G C C C C G G

HindIII Haemophilus

influenzae

A A G C T T T T C G A A

Recognition sites

Cut G A A T T C

C T T A A G Cut

Cut G A A T T C

C T T A A G

A A T T C

C T T A A

Cut

C T T A A

G C T T A A

GA A T T C

G C T T A A

GA A T T C

G C T T A A

GA A T T C

A A T T C

Sticky end

G A A T T C

A A T T C

G C T T A A

AATTC

CTT AA G

G C T T A A

AATTC

CT TAA G

2 These cuts produce a DNA fragment with two sticky ends.

DNA from another source, perhaps a plasmid, cut with the same restriction enzyme

5 rDNA

DNA

A restriction enzyme cuts (red arrows) double-stranded DNA at its particular recognition sites, shown in blue.

When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing.

The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of rDNA.

4 The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur.

Figure 9.2 A restriction enzyme’s role in making rDNA.

Q Why are restriction enzymes used to make rDNA?

Play Recombinant DNA Technology @MasteringMicrobiology

Notice in Figure 9.2 that the darker base sequences on the two strands are the same but run in opposite directions. Stag- gered cuts leave stretches of single-stranded DNA at the ends of the DNA fragments. If two fragments of DNA from different

CHAPTER 9 Biotechnology and DNA Technology 247

lacZ

ori

amp pUC19

HindIII BamHI EcoRI

being used to insert corrective genes into human cells that have defective genes. Gene therapy is discussed on page 255.

CHECK YOUR UNDERSTANDING

✓ 9-3 How are selection and mutation used in biotechnology? ✓ 9-4 What is the value of restriction enzymes in rDNA technology? ✓ 9-5 What criteria must a vector meet? ✓ 9-6 Why is a vector used in rDNA technology?

Polymerase Chain Reaction The polymerase chain reaction (PCR) is a technique by which small samples of DNA can be quickly amplified, that is, increased to quantities that are large enough for analysis.

Starting with just one gene-sized piece of DNA, PCR can be used to make billions of copies in only a few hours. The PCR process is shown in Figure 9.4.

Each strand of the target DNA will serve as a template for DNA synthesis. Added to this DNA are a supply of the four nucleotides (for assembly into new DNA) and the enzyme for catalyzing the synthesis, DNA polymerase (see Chapter 8, page 209). Short pieces of nucleic acid called primers are also added to help start the reaction. The primers are complementary to the ends of the target DNA and will hybridize to the fragments to be amplified. Then, the polymerase synthesizes new com- plementary strands. After each cycle of synthesis, the DNA is heated to convert all the new DNA into single strands. Each newly synthesized DNA strand serves in turn as a template for more new DNA.

As a result, the process proceeds exponentially. All of the nec- essary reagents are added to a tube, which is placed in a thermal cycler. The thermal cycler can be set for the desired temperatures, times, and number of cycles. Use of an automated thermal cycler is made possible by the use of DNA polymerase taken from a thermophilic bacterium such as Thermus aquaticus; the enzyme from such organisms can survive the heating phase without being destroyed. Thirty cycles, completed in just a few hours, will increase the amount of target DNA by more than a billion times.

The amplified DNA can be seen by gel electrophoresis. In real-time PCR, or quantitative PCR (qPCR), the newly made DNA is tagged with a fluorescent dye, so that the levels of fluores- cence can be measured after every PCR cycle (that’s the real time aspect). Another PCR procedure called reverse-transcription (RT-PCR) uses viral RNA or a cell’s mRNA as the template. The enzyme, reverse transcriptase, makes DNA from the RNA tem- plate, and the DNA is then amplified.

Note that PCR can only be used to amplify relatively small, specific sequences of DNA as determined by the choice of primers. It cannot be used to amplify an entire genome.

PCR can be applied to any situation that requires the ampli- fication of DNA. Especially noteworthy are diagnostic tests that use PCR to detect the presence of infectious agents in situations in

Figure 9.3 A plasmid used for cloning. A plasmid vector used for cloning in the bacterium E. coli is pUC19. An origin of replication (ori) allows the plasmid to be self-replicating. Two genes, one encoding resistance to the antibiotic ampicillin (amp) and one encoding the enzyme β-galactosidase (lacZ), serve as marker genes. Foreign DNA can be inserted at the restriction enzyme sites.

Q What is a vector in rDNA technology?

easily manipulated than larger DNA molecules, which tend to be more fragile. Preservation is another important property of vectors. The DNA molecule’s circular form protects the vector’s DNA from being destroyed by its recipient. Notice in Figure 9.3 that the DNA of a plasmid is circular. Another preservation mechanism occurs when a virus’s DNA inserts itself quickly into the chromosome of the host.

When it is necessary to retrieve cells that contain the vector, a marker gene in the vector often helps make selection easy. Common selectable marker genes are for antibiotic resistance or for an enzyme that carries out an easily identified reaction.

Plasmids are one of the primary vectors in use, particularly variants of R factor plasmids. Plasmid DNA can be cut with the same restriction enzymes as the DNA that will be cloned, so that all pieces of the DNA will have the same sticky ends. When the pieces are mixed, the DNA to be cloned will be inserted into the plasmid (Figure 9.2). Note that other fragment com- binations can occur as well, including the plasmid reforming a circle with no DNA inserted.

Some plasmids are capable of existing in several different species. They are called shuttle vectors and can be used to move cloned DNA sequences among organisms, such as among bac- terial, yeast, and mammalian cells, or among bacterial, fungal, and plant cells. Shuttle vectors can be very useful in the process of genetically modifying multicellular organisms—for example, when herbicide resistance genes are inserted into plants.

A different kind of vector is viral DNA. This type of vec- tor can usually accept much larger pieces of foreign DNA than plasmids can. After the DNA has been inserted into the viral vector, it can be cloned in the virus’s host cells. The choice of a suitable vector depends on many factors, including the organ- ism that will receive the new gene and the size of the DNA to be cloned. Retroviruses, adenoviruses, and herpesviruses are

248 PART ONE Fundamentals of Microbiology

CHECK YOUR UNDERSTANDING

✓ 9-7 For what is each of the following used in PCR: primer, DNA polymerase, 94°C?

Copy of target DNA

Copies of target DNA

Incubate at 94°C for 1 minute; this temperature will separate the strands.

Incubate at 60°C for 1 minute; this allows primers to attach to single-stranded DNA.

Incubate at 72°C for 1 minute; DNA polymerase copies the target DNA at this temperature.

Repeat the cycle of heating and cooling to make two more copies of target DNA.

FIRST CYCLE

SECOND CYCLE

5¿ 3¿

3¿ 5¿5¿ 3¿

5¿ 3¿

3¿ 5¿ Copies of target DNA

5¿ 3¿

3¿ 5¿

Copy of target DNA 3¿ 5¿

5¿

3¿ 5¿

3¿

5¿ 3¿

3¿ 5¿

Target DNA

Primer DNA polymerase

Nucleotides

1 Add primers, nucleotides, and DNA polymerase.

PREPARATION 5¿ 3¿

3¿ 5¿

Figure 9.4 The polymerase chain reaction. Deoxynucleotides (dNTPs) base-pair with the target DNA: adenine pairs with thymine, and cytosine pairs with guanine.

Q How does reverse-transcription PCR differ from this figure?

which they would otherwise be undetectable. A qPCR test provides rapid identification of drug-resistant Mycobacterium tuberculosis. Otherwise, this bacterium can take up to 6 weeks to culture, leaving patients untreated for a significant period of time.

Play PCR: Overview, Components, Process @MasteringMicrobiology

Techniques of Genetic Modification LEARNING OBJECTIVES

9-8 Describe five ways of getting DNA into a cell.

9-9 Describe how a genomic library is made.

9-10 Differentiate cDNA from synthetic DNA.

9-11 Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products.

9-12 List one advantage of modifying each of the following: E. coli, Saccharomyces cerevisiae, mammalian cells, plant cells.

CHAPTER 9 Biotechnology and DNA Technology 249

glycol increases the frequency of fusion (Figure 9.5). In the new hybrid cell, the DNA derived from the two “parent” cells may undergo natural recombination. This method is especially valuable in the genetic manipulation of plant and

algal cells. A remarkable way of introducing foreign DNA into plant

cells is to literally shoot it directly through the thick cellu- lose walls using a gene gun (Figure 9.6). Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls. Some of the cells express the introduced DNA as though it were their own.

DNA can be introduced directly into an animal cell by microinjection. This technique requires the use of a glass micropipette with a diameter that is much smaller than the cell. The micropipette punctures the plasma membrane, and DNA can be injected through it (Figure 9.7).

Inserting Foreign DNA into Cells Recombinant DNA procedures require that DNA molecules be manipulated outside the cell and then returned to living cells. There are several ways to introduce DNA into cells. The choice of method is usually determined by the type of vector and host cell being used.

In nature, plasmids are usually transferred between closely related microbes by cell-to-cell contact, such as in conjuga- tion. To modify a cell, a plasmid must be inserted into a cell by transformation, a procedure during which cells can take up DNA from the surrounding environment (see Chapter 8, page 232). Many cell types, including E. coli, yeast, and mam- malian cells, do not naturally transform; however, simple chemical treatments can make all of these cell types compe- tent, or able to take up external DNA. For E. coli, the procedure for making cells competent is to soak them in a solution of calcium chloride for a brief period. Following this treatment, the now-competent cells are mixed with the cloned DNA and given a mild heat shock. Some of these cells will then take up the DNA.

There are other ways to transfer DNA to cells. A process called electroporation uses an electrical current to form microscopic pores in the membranes of cells; the DNA then enters the cells through the pores. Electroporation is generally applicable to all cells; those with cell walls often must be con- verted to protoplasts first. Protoplasts are produced by enzy- matically removing the cell wall, thereby allowing more direct access to the plasma membrane.

The process of protoplast fusion also takes advantage of the properties of protoplasts. Protoplasts in solution fuse at a low but significant rate; the addition of polyethylene

CLINICAL CASE

Reverse-transcription PCR using a primer for an HIV gene can be used to amplify DNA for analysis. The Centers for Disease Control and Prevention (CDC) interviews the seven former patients to determine whether their histories show any additional risk factors for contracting HIV. Five out of the seven have no identified risk factors for HIV other than having had invasive procedures performed on them by Dr. B. The

CDC then performs reverse- transcription PCR on DNA from white blood cells in Dr. B.’s peripheral blood and the seven HIV-positive patients (see the figure).

What can be concluded from the PCR amplification in the figure?

243 249 252 254 257

Patients

D en

tis t

B C D E F G

Bacterial cells

Protoplasts

Chromosome

Plasma membrane

Cell wall

1 Bacterial cell walls are enzymatically digested, producing protoplasts.

2 In solution, protoplasts are treated with polyethylene glycol.

3 Protoplasts fuse.

4 Segments of the two chromosomes recombine.

5 Recombinant cell grows new cell wall.

Recombinant cell

Figure 9.5 Protoplast fusion. Removal of the cell wall leaves only the delicate plasma membranes, which will fuse together, allowing the exchange of DNA.

Q What is a protoplast?

250 PART ONE Fundamentals of Microbiology

Obtaining DNA We have seen how genes can be cloned into vectors by using restriction enzymes and how genes can be transformed or transferred into a variety of cell types. But how do biologists obtain the genes they are interested in? There are two main sources of genes: (1) genomic libraries containing either natu- ral copies of genes or cDNA copies of genes made from mRNA, and (2) synthetic DNA.

Genomic Libraries Isolating specific genes as individual pieces of DNA is seldom practical. Therefore, researchers interested in genes from a particular organism start by extracting the organism’s DNA, which can be obtained from cells of any organism, whether plant, animal, or microbe, by lysing the cells and precipitating the DNA. This process results in a DNA mass that includes the organism’s entire genome. After the DNA is digested by restric- tion enzymes, the restriction fragments are then spliced into plasmid or phage vectors, and the recombinant vectors are introduced into bacterial cells. The goal is to make a collection of clones large enough to ensure that at least one clone exists for every gene in the organism. This collection of clones con- taining different DNA fragments is called a genomic library; each “book” is a bacterial or phage strain that contains a frag- ment of the genome (Figure 9.8). Such libraries are essential for

Figure 9.6 A gene gun, which can be used to insert DNA-coated “bullets” into a cell.

Q Name four other methods of inserting DNA into a cell.

80 mm LM

Figure 9.7 The microinjection of foreign DNA into an egg. The egg is first immobilized by applying mild suction to the large, blunt, holding pipette (right). Several hundred copies of the gene of interest are then injected into the nucleus of the cell through the tiny end of the micropipette (left).

Q Why is microinjection impractical for bacterial and fungal cells?

Thus, there is a great variety of restriction enzymes, vec- tors, and methods of inserting DNA into cells. But foreign DNA will survive only if it’s either present on a self-replicating vector or incorporated into one of the cell’s chromosomes by recombination.

Plasmid Library

Genome to be stored in library is cut up with restriction enzyme

Recombinant plasmid

Host cell

Recombinant phage DNA

Phage Library

Phage cloning vector

Figure 9.8 Genomic libraries. Each fragment of DNA, containing about one gene, is carried by a vector, either a plasmid within a bacterial cell or a phage.

Q Differentiate a restriction fragment from a gene.

CHAPTER 9 Biotechnology and DNA Technology 251

(Figure 9.9). This synthesis is the reverse of the normal DNA-to- RNA transcription process. A DNA copy of mRNA is produced by reverse transcriptase. Following this, the mRNA is enzymati- cally digested away. DNA polymerase then synthesizes a com- plementary strand of DNA, creating a double-stranded piece of DNA containing the information from the mRNA. Molecules of cDNA produced from a mixture of all the mRNAs from a tissue or cell type can then be cloned to form a cDNA library.

The cDNA method is the most common method of obtain- ing eukaryotic genes. A difficulty with this method is that long molecules of mRNA may not be completely reverse-transcribed into DNA; the reverse transcription often aborts, forming only parts of the desired gene.

Synthetic DNA Under certain circumstances, genes can be made in vitro with the help of DNA synthesis machines (Figure 9.10). A keyboard on the machine is used to enter the desired sequence of nucleo- tides, much as letters are entered into a word processor to com- pose a sentence. A microprocessor controls the synthesis of the DNA from stored supplies of nucleotides and the other neces- sary reagents. A short chain of about 200 nucleotides, called an oligonucleotide, can be synthesized by this method. Unless the gene is very small, at least several chains must be synthesized separately and linked together to form an entire gene.

The difficulty of this approach, of course, is that the sequence of the gene must be known before it can be synthe- sized. If the gene hasn’t already been isolated, then the only

Exon Intron Exon Intron Exon

DNA

1 A gene composed of exons and introns is transcribed to RNA by RNA polymerase.

RNA transcript

2 Processing enzymes in the nucleus remove the intron-derived RNA and splice together the exon-derived RNA into mRNA.

mRNA

Test tube

Cytoplasm

Nucleus

3 mRNA is isolated from the cell, and reverse transcriptase is added.

5 The mRNA is digested by reverse transcriptase.

4 First strand of DNA is synthesized.

DNA strand being synthesized

6 DNA polymerase is added to synthesize second strand of DNA.

cDNA of gene without introns

Figure 9.9 Making complementary DNA (cDNA) for a eukaryotic gene. Reverse transcriptase catalyzes the synthesis of double-stranded DNA from an RNA template.

Q How does reverse transcriptase differ from DNA polymerase?

maintaining and retrieving DNA clones; they can even be pur- chased commercially.

Cloning genes from eukaryotic organisms presents a spe- cific problem. Genes of eukaryotic cells generally contain both exons, stretches of DNA that code for protein, and introns, intervening stretches of DNA that do not code for protein. When the RNA transcript of such a gene is converted to mRNA, the introns are removed (see Figure 8.11 on page 219). To clone genes from eukaryotic cells, it’s desirable to use a version of the gene that lacks introns because a gene that includes introns may be too large to work with easily. In addition, if such a gene is put into a bacterial cell, the bacterium usually won’t be able to remove the introns from the RNA transcript. Therefore, it won’t be able to make the correct protein product. However, an artificial gene that contains only exons can be produced by using an enzyme called reverse transcriptase to synthe- size complementary DNA (cDNA) from an mRNA template

Figure 9.10 A DNA synthesis machine. Short sequences of DNA can be synthesized by instruments such as this one.

Q What four reagents (in the brown bottles) are necessary to synthesize DNA?

252 PART ONE Fundamentals of Microbiology

The plasmid vector used contains a gene (amp) encoding resistance to the antibiotic ampicillin. The host bacterium won’t be able to grow on the test medium, which contains ampicillin, unless the vector has transferred the ampicillin-resistance gene. The plasmid vector also contains a second gene, this one for the enzyme β-galactosidase (lacZ). Notice in Figure 9.3 that there are several sites in lacZ that can be cut by restriction enzymes.

In the blue-white screening procedure shown in Figure 9.11, a library of bacteria is cultured in a medium called X-gal. X-gal contains two essential components other than those neces- sary to support normal bacterial growth. One is the antibiotic ampicillin, which prevents the growth of any bacterium that

CLINICAL CASE

The primer amplifies all eight samples and confirms that Dr. B. and seven of his former patients are all infected with HIV. The CDC then sequences the amplified DNA and compares the sequencing to an HIV isolate from Cleveland (local control) and an isolate from Haiti (outlier). A portion of the coding (5' to 3') is shown below.

Patient A GCTTG GGCTG GCGCT GAAGT GAGA

Patient B GCTAT TGCTG GCGCT GAATT GCAC

Patient C GCCAT AGCTG GCGCA GAAGT GCAC

Patient D GCTAT TGGCG TGGCT GACAG AGAA

Patient E GCACC TGCTG GCGCT GAAGT GAAA

Patient F CAGAT TGTGT TGATT GAACC TCAC

Patient G GCTAT TGCTG GCGCT GAAGT GAAA

Dentist GCTAT TGCTG GCGCT GAAGT GCAC

Local control CAGAC TACTG CTAGG AAAAA TATT

Outlier GAAGA CGAAA GGACT GCTAT TCAG

What is the percent similarity among the viruses?

243 249 252 254 257

way to predict the DNA sequence is by knowing the amino acid sequence of the gene’s protein product. If this amino acid sequence is known, in principle you can work backward through the genetic code to obtain the DNA sequence. Unfor- tunately, the degeneracy of the code prevents definitive deter- mination; thus, if the protein contains a leucine, for example, which of the six codons for leucine is the one in the gene?

For these reasons, it’s rare to clone a gene by synthesizing it directly, although some commercial products such as insu- lin, interferon, and somatostatin are produced from chemically synthesized genes. Desired restriction sites are added to the synthetic genes so the genes can be inserted into plasmid vec- tors and cloned in E. coli. Synthetic DNA plays a much more useful role in selection procedures, as we will see.

CHECK YOUR UNDERSTANDING

✓ 9-8 Contrast the five ways of putting DNA into a cell. ✓ 9-9 What is the purpose of a genomic library? ✓ 9-10 Why isn’t cDNA synthetic?

Selecting a Clone In cloning, it’s necessary to select the particular cell that con- tains the specific gene of interest. This is difficult to do because out of millions of cells, only a very few might contain the desired gene. Here we’ll examine a typical screening procedure known as blue-white screening, from the color of the bacterial colonies formed at the end of the screening process.

Bacterium

Colonies with foreign DNA

Plasmid

b-galactosidase gene (lacZ)

Ampicillin-resistance gene (amp)

Restriction site

Restriction sites

Foreign DNA

1 Plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The plasmid has the genes for lactose hydrolysis (the lacZ gene encodes the enzyme b-galactosidase) and ampicillin resistance.

2 Foreign DNA will insert into the lacZ gene. The bacterium receiving the plasmid vector will not produce the enzyme b-galactosidase if foreign DNA has been inserted into the plasmid.

3 The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant.

4 All treated bacteria are spread on a nutrient agar plate containing ampicillin and a b-galactosidase substrate and incubated. The b-galactosidase substrate is called X-gal.

5 Only bacteria that picked up the plasmid will grow in the presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and an indigo compound. The indigo turns the colonies blue. Bacteria that cannot hydrolyze X-gal produce white colonies.

Recombinant plasmid

Figure 9.11 Blue-white screening, one method of selecting recombinant bacteria.

Q Why are some colonies blue and others white?

CHAPTER 9 Biotechnology and DNA Technology 253

has not successfully received the ampicillin-resistance gene from the plasmid. The other, called X-gal, is a substrate for β-galactosidase.

Only bacteria that picked up the plasmid will grow, because they are now ampicillin resistant. Bacteria that picked up the recombinant plasmid—in which the new gene was inserted into the lacZ gene—will not hydrolyze lactose and will produce white colonies. If a bacterium received the original plasmid containing the intact lacZ gene, the cells will hydrolyze X-gal to produce a blue-colored compound; the colony will be blue.

What remains to be done can still be difficult. The above procedure has isolated white colonies known to contain for- eign DNA, but it is still not known whether it’s the desired frag- ment of foreign DNA. A second procedure is needed to identify these bacteria. If the foreign DNA in the plasmid encodes the production of an identifiable product, the bacterial isolate only needs to be grown in culture and tested. However, in some cases the gene itself must be identified in the host bacterium.

Colony hybridization is a common method of identifying cells that carry a specific cloned gene. DNA probes, short seg- ments of single-stranded DNA that are complementary to the desired gene, are synthesized. If the DNA probe finds a match, it will adhere to the target gene. The DNA probe is labeled with an enzyme or fluorescent dye so its presence can be detected. A typical colony hybridization experiment is shown in Figure 9.12. An array of DNA probes arranged in a DNA chip can be used to identify pathogens (see Figure 10.17, page 287).

Making a Gene Product We have just seen how to identify cells carrying a particular gene. Gene products are frequently the reason for genetic mod- ification. Most of the earliest work in genetic modification used E. coli to synthesize the gene products. E. coli is easily grown, and researchers are very familiar with the bacterium and its genetics. For example, some inducible promoters, such as that of the lac operon, have been cloned, and cloned genes can be attached to such promoters. The synthesis of great amounts of the cloned gene product can then be directed by the addi- tion of an inducer. Such a method has been used to produce gamma interferon in E. coli (Figure 9.13). However, E. coli also has several disadvantages. Like other gram-negative bacteria, it produces endotoxins as part of the outer layer of its cell wall. Because endotoxins cause fever and shock in mammals, their accidental presence in products intended for human use would be a serious problem.

Another disadvantage of E. coli is it doesn’t usually secrete protein products. To obtain a product, cells must usually be broken open and the product purified from the resulting “soup” of cell components. Recovering the product from such a mixture is expensive when done on an industrial scale. It’s more economical to have an organism secrete the product so it

Master plate with colonies of bacteria containing cloned segments of foreign genes

Fluorescence- labeled probes

Nitrocellulose filter

Strands of bacterial DNA

Bound DNA probe

Gene of interest

Single- stranded DNA

Colonies containing genes of interest

Replica plate

1 Make replica of master plate on nitrocellulose filter.

2 Treat filter with detergent (SDS) to lyse bacteria.

3 Treat filter with sodium hydroxide (NaOH) to separate DNA into single strands.

4 Add labeled probes.

5 Probe will hybridize with desired gene from bacterial cells.

6 Wash filter to remove unbound probe.

7 Compare filter with replica of master plate to identify colonies containing gene of interest.

Figure 9.12 Colony hybridization: using a DNA probe to identify a cloned gene of interest.

Q What is a DNA probe?

254 PART ONE Fundamentals of Microbiology

CLINICAL CASE

The sequences from Dr. B. and patients A, B, C, E, and G share 87.5% of the nucleotide sequence, which is comparable to reported similarities for known linked infections.

Identify the amino acids encoded by the viral DNA. Did this change the percent similarity? (Hint: Refer to Figure 8.8 on page 214).

243 249 252 254 257

TEM 0.25 mm

against infection. To produce huge amounts of CSF industri- ally, the gene is first inserted into a plasmid. Bacteria are used to make multiple copies of the plasmid (see Figure 9.1), and the resulting recombinant plasmids are then inserted into mam- malian cells that are grown in bottles.

Plant cells can also be grown in culture, altered by rDNA techniques, and then used to generate genetically modified plants. Such plants may prove useful as sources of valuable products, such as plant alkaloids (the painkiller codeine, for example), the isoprenoids that are the basis of synthetic rubber, and melanin (the animal skin pigment) for use in sunscreens. Genetically modified plants have many advantages for the pro- duction of human therapeutic agents, including vaccines and antibodies. The advantages include large-scale, low-cost agri- cultural production and a low risk of product contamination by mammalian pathogens or cancer-causing genes. Geneti- cally modifying plants often requires use of a bacterium. We’ll return to the topic of genetically modified plants later in the chapter (page 260).

CHECK YOUR UNDERSTANDING

✓ 9-11 How are recombinant clones identified? ✓ 9-12 What types of cells are used for cloning rDNA?

Applications of DNA Technology LEARNING OBJECTIVES

9-13 List at least five applications of DNA technology.

9-14 Define RNAi.

9-15 Discuss the value of genome projects.

9-16 Define the following terms: random shotgun sequencing, bioinformatics, proteomics.

9-17 Diagram the Southern blotting procedure, and provide an example of its use.

9-18 Diagram DNA fingerprinting, and provide an example of its use.

9-19 Outline genetic engineering with Agrobacterium.

Figure 9.13 E. coli genetically modified to produce gamma interferon, a human protein that promotes an immune response. The product, visible here as a red mass in a violet ring, can be released by lysis of the cell.

Q What is one advantage of using E. coli for genetic engineering? One disadvantage?

can be recovered continuously from the growth medium. One approach has been to link the product to a natural E. coli pro- tein that the bacterium does secrete. However, gram-positive bacteria, such as Bacillus subtilis, are more likely to secrete their products and are often preferred industrially for that reason.

Another microbe being used as a vehicle for expressing rDNA is baker’s yeast, Saccharomyces cerevisiae. Its genome is only about four times larger than that of E. coli and is prob- ably the best understood eukaryotic genome. Yeasts may carry plasmids, which are easily transferred into yeast cells whose cell walls have been removed. As eukaryotic cells, yeasts may be more successful in expressing foreign eukaryotic genes than bacteria. Furthermore, yeasts are likely to continuously secrete the product. Because of all these factors, yeasts have become the eukaryotic workhorse of biotechnology.

Mammalian cells in culture, even human cells, can be genetically modified much like bacteria to produce various products. Scientists have developed effective methods of grow- ing certain mammalian cells in culture as hosts for growing viruses (see Chapter 13, page 371). Mammalian cells are often the best suited to making protein products for medical use because the cells secrete their products and there’s a low risk of toxins or allergens. Using mammalian cells to make foreign gene products on an industrial scale often requires a prelimi- nary step of cloning the gene in bacteria. Consider the exam- ple of colony-stimulating factor (CSF). A protein produced naturally in tiny amounts by white blood cells, CSF is valuable because it stimulates the growth of certain cells that protect

CHAPTER 9 Biotechnology and DNA Technology 255

DNA vaccines are usually circular plasmids that include a gene encoding a viral protein that’s under the transcriptional control of a promoter region active in human cells. The plas- mids are then cloned in bacteria. A DNA vaccine to protect against Zika virus disease is currently in clinical trials. Vac- cines are discussed in further detail in Chapter 18 (page 503). Table 9.2 lists some other important rDNA products used in medical therapy.

The importance of rDNA technology to medical research cannot be emphasized enough. Artificial blood for use in transfusions can now be prepared with human hemoglobin produced in genetically modified pigs. Sheep have also been genetically modified to produce a number of drugs in their milk. This procedure has no apparent effect upon the sheep, and they provide a ready source of raw material for the product that does not require sacrificing animals.

Gene therapy may eventually provide cures for some genetic diseases. It is possible to imagine removing some cells from a person and transforming them with a normal gene to replace a defective or mutated gene. When these cells are returned to the person, they should function normally. For example, gene therapy has been used to treat hemophilia B and severe combined immunodeficiency. Adenoviruses and retroviruses are used most often to deliver genes; how- ever, some researchers are working with plasmid vectors. An attenuated retrovirus was used as the vector when the first gene therapy to treat hemophilia in humans was performed in 1990. Glybera® is a gene therapy drug licensed in Europe to treat lipoprotein lipase deficiency. It uses an adenovirus to deliver the lipase gene to cells. Antisense DNA (page 262) introduced into cells is also being explored. Fomivirsen is an antisense DNA drug used in the treatment of cytomegalovirus retinitis.

Thus far, gene therapy results have not been impressive; there have even been a few deaths attributed to the viral vec- tors. A great deal of preliminary work remains to be done, but cures may not be possible for all genetic diseases.

Gene editing is a promising new technology to correct genetic mutations at precise locations. Gene editing uses CRISPR (pronounced “crisper”), which stands for clustered regularly interspaced short palindromic repeats. CRISPR enzymes are found in archaea and bacteria, where they destroy foreign DNA. A small RNA molecule, complementary to the desired target, binds DNA, and then the Cas9 enzyme cuts the DNA like molecular scissors. The cell’s DNA poly- merase and DNA ligase reattach the ends. A researcher can add template DNA for the correct gene, which can be attached by the DNA ligase. If may be possible to correct mutations in the human genome to treat genetic causes of disease. Gene editing was used to repair a defective muscle protein gene in mice with Duchenne muscular dystrophy. A parvovirus was used to deliver the gene-editing system into mice. In 2016, the

We have now described the entire sequence of events in cloning a gene. As indicated earlier, such cloned genes can be applied in a variety of ways. One is to produce useful substances more efficiently and less expensively. Another is to obtain infor- mation from the cloned DNA that is useful for either basic research, medicine, or forensics. A third is to use cloned genes to alter the characteristics of cells or organisms.

Therapeutic Applications An extremely valuable pharmaceutical product is the hormone insulin, a small protein produced by the pancreas that controls the body’s uptake of glucose from blood. For many years, peo- ple with insulin-dependent diabetes controlled their disease by injecting insulin obtained from the pancreases of slaughtered animals. Obtaining this insulin is an expensive process, and the insulin from animals is not as effective as human insulin.

Because of the value of human insulin and the protein’s small size, producing human insulin by rDNA techniques was an early goal for the pharmaceutical industry. To produce the hormone, synthetic genes were first constructed for each of the two short polypeptide chains that make up the insulin mol- ecule. The small size of these chains—only 21 and 30 amino acids long—made it possible to use synthetic genes. Follow- ing the procedure described earlier (page 249), each of the two synthetic genes was inserted into a plasmid vector and linked to the end of a gene coding for the bacterial enzyme β-galactosidase, so that the insulin polypeptide was copro- duced with the enzyme. Two different E. coli bacterial cultures were used, one to produce each of the insulin polypeptide chains. The polypeptides were then recovered from the bacte- ria, separated from the β-galactosidase, and chemically joined to make human insulin. This accomplishment was one of the early commercial successes of DNA technology, and it illus- trates a number of the principles and procedures discussed in this chapter.

Another human hormone that is now being produced com- mercially by genetic modification of E. coli is somatostatin. At one time 500,000 sheep brains were needed to produce 5 mg of animal somatostatin for experimental purposes. By contrast, only 8 liters of a genetically modified bacterial culture are now required to obtain the equivalent amount of the human hormone.

Subunit vaccines, consisting only of a protein portion of a pathogen, are being made by genetically modifying yeasts. Subunit vaccines have been produced for a number of diseases, notably hepatitis B. One of the advantages of a subunit vac- cine is that there is no chance that the vaccine will cause an infection. The protein is harvested from genetically modified cells and purified for use as a vaccine. Animal viruses such as vaccinia virus can be genetically modified to carry a gene for another microbe’s surface protein. When injected, the virus acts as a vaccine against the other microbe.

256 PART ONE Fundamentals of Microbiology

TABLE 9.2 Some Pharmaceutical Products of rDNA

Product Comments

Cervical Cancer Vaccine Consists of viral proteins; produced by Saccharomyces cerevisiae or by insect cells

Epidermal Growth Factor (EGF) Heals wounds, burns, ulcers; produced by E. coli

Erythropoietin (EPO) Treatment of anemia; produced by mammalian cell culture

Interferon

IFN–a Therapy for leukemia, melanoma, and hepatitis; produced by E. coli and S. cerevisiae (yeast)

IFN–b Treatment for multiple sclerosis; produced by mammalian cell culture

IFN–g Treatment of chronic granulomatous disease; produced by E. coli

Hepatitis B Vaccine Produced by S. cerevisiae that carries hepatitis-virus gene on a plasmid

Human Growth Hormone (hGH) Corrects growth deficiencies in children; produced by E. coli

Human Insulin Therapy for diabetes; better tolerated than insulin extracted from animals; produced by E. coli

Influenza Vaccine Vaccine made from E. coli or S. cerevisiae carrying virus genes

Interleukins Regulate the immune system; possible treatment for cancer; produced by E. coli

Orthoclone OKT3 Muromonab-CD3

Monoclonal antibody used in transplant patients to help suppress the immune system, reducing the chance of tissue rejection; produced by mouse cells

Pulmozyme (rhDNase) Enzyme used to break down mucous secretions in cystic fibrosis patients; produced by mammalian cell culture

Relaxin Used to ease childbirth; produced by E. coli

Superoxide Dismutase (SOD) Minimizes damage caused by oxygen free radicals when blood is resupplied to oxygen-deprived tissues; produced by S. cerevisiae and Komagataella pastoris (yeast)

Taxol Plant product used for treating ovarian cancer; produced in E. coli

Tissue Plasminogen Activator Dissolves the fibrin of blood clots; therapy for heart attacks; produced by mammalian cell culture

Tumor Necrosis Factor (TNF) Causes disintegration of tumor cells; produced by E. coli

Veterinary Use

Canine Distemper Vaccine Canarypox virus carrying canine distemper virus genes

Feline Leukemia Vaccine Canarypox virus carrying feline leukemia virus genes

first clinical trials were approved to modify a patient’s T cells (see page 476) to fight cancer.

Gene silencing is a natural process that occurs in a wide variety of eukaryotes and is apparently a defense against viruses and transposons. Gene silencing is similar to miRNA (page 219) in that a gene encoding a small piece of RNA is transcribed. Following transcription, RNAs called small interfering RNAs (siRNAs) are formed after processing by an enzyme called Dicer. The siRNA molecules bind to mRNA, which is then destroyed by proteins called the RNA-induced silencing complex (RISC), thus silencing the expression of a gene (Figure 9.14).

New technology called RNA interference (RNAi) holds promise for gene therapy for treating genetic diseases. A small DNA insert encoding siRNA against the gene of interest could be cloned into a plasmid. When transferred into a cell, the cell would produce the desired siRNA. Clinical trials are currently being conducted to test RNAi to prevent Ebola and respiratory syncytial virus infections.

CHECK YOUR UNDERSTANDING

✓ 9-13 Explain how DNA technology can be used to treat disease and to prevent disease.

✓ 9-14 What is gene silencing?

Genome Projects The first genome to be sequenced was from a bacteriophage in 1977. In 1995, the genome of a free-living cell—Haemophilus influ- enzae—was sequenced. Since then, 1000 prokaryotic genomes and over 400 eukaryotic genomes have been sequenced.

In shotgun sequencing, small pieces of a genome of a free- living cell are sequenced, and the sequences are then assem- bled using a computer. Any gaps between the pieces then have to be found and sequenced (Figure 9.15). This technique can be used on environmental samples to study the genomes of microorganisms that haven’t been cultured. The study of

CHAPTER 9 Biotechnology and DNA Technology 257

genetic material taken directly from environmental samples is called metagenomics.

The Human Genome Project was an international 13-year effort, formally begun in October 1990 and completed in 2003. The goal of the project was to sequence the entire human genome, approximately 3 billion nucleotide pairs, compris- ing 20,000 to 25,000 genes. Thousands of people in 18 coun- tries participated in this project. Researchers collected blood (female) or sperm (male) samples from a large number of donors. Only a few samples were processed as DNA resources, and the source names are protected so that neither donors nor scientists knew whose samples were used. Development of shotgun sequencing greatly speeded the process, and 99% of the genome has been sequenced.

One surprising finding was that less than 2% of the genome encodes a functional product—the other 98% includes miRNA genes, viral remnants, repetitive sequences (called short tandem repeats), introns, the chromosome ends (called telomeres), and transposons (page 231).

The next goal of researchers is the Human Proteome Proj- ect, which will map all the proteins expressed in human cells. Even before it is completed, however, it’s yielding data that are of immense value to our understanding of biology. It will also

CLINICAL CASE Resolved

The amino acid sequence reflects the nucleotide sequence. Analysis of the amino acid signature pattern confirms that the viruses from the dentist and patients are closely related. HIV has a high mutation rate, so HIVs from different individuals are genetically distinct. Dr. B.’s HIV is different from the local control and from the outlier. Dr. B.’s amino acid sequences and those of patients A, B, C, E, and G are distinct from those in the control and in the outlier and from two dental patients with known behavioral risks for HIV infection.

PCR and RFLP analyses have made it possible to track transmission of disease between individuals, communities, and countries. This tracking works best with pathogens that have enough genetic variation to identify different strains.

* * * Dr. B. died before the mode of transmission could be

established. But in the era when he practiced dentistry, it was not always the norm to wear gloves when performing procedures. Patient interviews indicated that Dr. B. didn’t like to wear gloves. It is likely that HIV was transmitted when a cut on the doctor’s bare hands allowed the virus to enter patients’ gums. Today the CDC and state health departments ask dental care providers to use universal precautions, including wearing gloves and masks and sterilizing equipment that is to be reused. Had Dr. B. used standard precautions, it is extremely unlikely he would have infected patients.

243 249 252 254 257

eventually be of great medical benefit, especially for the diag- nosis and treatment of genetic diseases.

Scientific Applications Recombinant DNA technology can be used to make products, but this isn’t its only important application. Because of its ability to produce many copies of DNA, it can serve as a sort of DNA “print- ing press.” Once a large amount of a particular piece of DNA is available, various analytic techniques, discussed in this section, can be used to “read” the information contained in the DNA.

In 2010, researchers synthesized the smallest known cellu- lar genome during the Minimal Genome Project. A copy of the Mycoplasma mycoides genome was synthesized and transplanted into an M. capricolum cell that had had its own DNA removed. The modified cell produced M. mycoides proteins. This experi- ment showed that large-scale changes to a genome can be made and that an existing cell will accept this DNA.

DNA sequencing has produced an enormous amount of information that has spawned the new field of bioinformatics, the science of understanding the function of genes through computer-assisted analysis. DNA sequences are stored in

1 An abnormal gene, cancer gene, or virus gene is transcribed in a host cell.

2 siRNA binds mRNA.

3 RISC breaks down the RNA complex.

4 No protein expression occurs.

mRNA

DNA

RNA transcript

Nucleus

Cytoplasm

siRNA

Figure 9.14 Gene silencing could provide treatments for a wide range of diseases.

Q Does RNAi act during or after transcription?

258 PART ONE Fundamentals of Microbiology

web-based databases referred to as GenBank. Genomic infor- mation can be searched with computer programs to find spe- cific sequences or to look for similar patterns in the genomes of different organisms. Microbial genes are now being searched to identify molecules that are the virulence factors of pathogens. By comparing genomes, researchers discovered that Chlamydia trachomatis (tra-KŌ-ma-tis) produces a toxin similar to that of Clostridium difficile (DIF-fi-sē-il).

The next goal is to identify the proteins encoded by these genes. Proteomics is the science of determining all of the pro- teins expressed in a cell.

Reverse genetics is an approach to discovering the function of a gene from a genetic sequence. Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism. For example, if you mutate or block a gene (see the earlier discussions of gene editing on page 255 and gene silenc- ing on page 256), you can then look for a characteristic the organism lost.

An example of the use of human DNA sequencing is the identification and cloning of the mutant gene that causes cystic fibrosis (CF). CF is characterized by the oversecretion of mucus, leading to blocked respiratory passageways. The sequence of the mutated gene can be used as a diagnostic tool in a hybridization technique called Southern blotting (Figure 9.16), named for Ed Southern, who developed the technique in 1975.

In this technique, subject DNA is digested with a restriction enzyme, yielding thousands of fragments of various sizes. The fragments are called RFLPs (pronounced “rif-lip”), for restric- tion fragment length polymorphisms. The different fragments are then separated by gel electrophoresis. The fragments are put

Isolate DNA.

(a) Constructing a gene library

Fragment DNA with restriction enzymes.

Clone DNA in a bacterial artificial chromosome (BAC).

Sequence DNA fragments.

BAC

(b) Random sequencing

Assemble sequences.

Edit sequences; fill in gaps.C T T TGA C

4 5

Figure 9.15 Shotgun sequencing. In this technique, a genome is cut into pieces, and each piece is sequenced. Then the pieces are fit together. There may be gaps if a specific DNA fragment was not sequenced.

Q Does this technique identify genes and their locations?

in a well at one end of a layer of agarose gel. Then an electrical current is passed through the gel. While the charge is applied, the different-sized RFLPs migrate through the gel at different rates. The RFLPs are transferred onto a filter by blotting and are exposed to a labeled probe made from the cloned gene of interest, in this case the CF gene. The probe will hybridize to this mutant gene but not to the normal gene. Fragments to which the probe binds are identified by a colored dye. With this method, any person’s DNA can be tested for the presence of the mutated gene.

Genetic testing can now be used to screen for several hun- dred genetic diseases. Such screening procedures can be per- formed on prospective parents and also on fetal tissue. Two of the more commonly screened genes are those associated with inherited forms of breast cancer and the gene responsible for Huntington’s disease. Genetic testing can help a physician pre- scribe the correct medication for a patient. The drug herceptin, for example, is effective only in breast cancer patients with a specific nucleotide sequence in the HER2 gene.

Forensic Microbiology For several years, microbiologists have used RFLPs in a method of identification known as DNA fingerprinting to identify

bacterial or viral pathogens (Figure 9.17). DNA chips (see Figure 10.18, page 288) or PCR microarrays

that can screen a sample for multiple pathogens at once are now being used. In a DNA chip, up to 22 primers from dif- ferent microorganisms can be used to initiate the PCR. A sus- pect microorganism is identified if DNA is copied from one of the primers. At the Centers for Disease Control and Prevention

CHAPTER 9 Biotechnology and DNA Technology 259

Gel

Human DNA fragments

Nitrocellulose filter

Gel DNA transferred to filter

Sealable plastic bag

Labeled probes

Sponge

Nitrocellulose filter

Salt solution

Gel

Paper towels

1 DNA containing the gene of interest is extracted from human cells and cut into fragments by restriction enzymes. Fragments are called restriction fragment length polymorphisms, or RFLPs (pronounced “rif-lips”).

2 The fragments are separated according to size by gel electrophoresis. Each band contains many copies of a particular DNA fragment. The bands are invisible but can be made visible by staining.

3 The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to the paper towels by capillary action.

4 This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel.

5 The filter is exposed to a labeled probe for a specific gene. The probe will base-pair (hybridize) with a short sequence present on the gene.

6 The fragment containing the gene of interest is identified by a band on the filter.

Restriction enzyme

Larger

Smaller

Gene of interest

Figure 9.16 Southern blotting.

Q What is the purpose of Southern blotting?

(CDC), PulseNet uses RFLPs to track outbreaks of foodborne disease. In some cases, PCR using specific primers can be used to track a bacterial strain to locate the source of an outbreak.

The genomics of pathogens has become a mainstay of mon- itoring, preventing, and controlling infectious disease. The use of genomics to trace a disease outbreak is described in the Clin- ical Focus box on page 264. The new field of forensic microbi- ology developed because hospitals, food manufacturers, and individuals can be sued in courts of law and because microor- ganisms can be used as weapons. In the 2001 anthrax attacks in the United States, DNA fingerprints of Bacillus anthracis were used to track the source and then the alleged attacker.

Northern Arizona University researchers determined that the B. anthracis endospores used in a 1993 attack by a cult in Japan were actually a nonpathogenic vaccine strain. No one was hurt when those endospores were released. Currently, a DNA data- base is being developed for microorganisms that could be used in biological crimes.

Microbial forensics has been used in court. In the 1990s, DNA fingerprints of HIV were used for the first time to obtain a rape conviction. Since then, a physician was convicted of injecting his former lover with HIV from one of his patients, based on the DNA fingerprint of the HIV.

260 PART ONE Fundamentals of Microbiology

SEM 1 mm

E. coli isolates from patients whose infections were not juice related

E. coli isolates from patients who drank contaminated juice

Apple juice isolates

that make nanoparticles from a variety of elements, includ- ing gold, silver, selenium, and cadmium. (Figure 9.18). Nano- technology research is growing, with researchers developing innovative ways of using bacteria to produce nanospheres for potential drug targeting and delivery. Researchers with the U.S. Department of Energy are using bacteria in nanoscale electrical circuits to make hydrogen gas. Swedish researchers are using Acetobacter xylinum to build cellulose nanofibers for artificial blood vessels.

CHECK YOUR UNDERSTANDING

✓ 9-15, 9-16 How are shotgun sequencing, bioinformatics, and proteomics related to genome projects?

✓ 9-17 What is Southern blotting? ✓ 9-18 Why do RFLPs result in a DNA fingerprint?

Agricultural Applications The process of selecting for genetically desirable plants has always been time-consuming. Conventional plant cross-breeding is laborious and involves waiting for the planted seed to germi- nate and the resulting plant to mature in order to learn whether the plant has the desired traits. Plant breeding has been revo- lutionized by the use of plant cells grown in culture. Clones of plant cells, including cells that have been genetically altered by rDNA techniques, can be grown in large numbers. These cells can then be induced to regenerate whole plants, from which seeds can be harvested.

Recombinant DNA can be introduced into plant cells in several ways. Previously we mentioned protoplast fusion and the use of DNA-coated “bullets.” The most elegant method, however, makes use of a plasmid called the Ti plasmid (Ti stands for tumor-inducing), which occurs naturally in the bacterium Agrobacterium tumefaciens (TOO-mah-fas9ē-enz). This bacterium infects certain plants, in which the Ti plasmid

Figure 9.17 DNA fingerprints used to track an infectious disease. This figure shows the RFLP patterns of bacterial isolates from an outbreak of Escherichia coli O157:H7. The isolates from apple juice are identical to the patterns of isolates from patients who drank the contaminated juice but different from those from patients whose infections were not juice related.

Q What is forensic microbiology?

The requirements to prove in a court of law the source of a microbe are stricter than for the medical community. For example, to prove intent to commit harm requires collecting evidence properly and establishing a chain of custody of that evidence. Microbial properties that are unimportant in pub- lic health may be important clues in forensic investigations. The genetic fingerprint of sexually transmitted pathogens, for instance, has been used as evidence in sexual abuse and rape cases. In the Clinic (page 242) offers another example of the use of bacterial genomics in a criminal investigation. These devel- opments suggest that the human microbiome may become an important law enforcement tool. The American Academy of Microbiology recently proposed professional certification in forensic microbiology.

Nanotechnology Nanotechnology deals with the design and manufacture of extremely small electronic circuits and mechanical devices built at the molecular level of matter. Molecule-sized robots or computers can be used to detect contamination in food, diseases in plants, or biological weapons. However, the small machines require small (a nanometer is 10−9 meters; 1000 nm fit in 1 mm) wires and components. Bacteria may provide the needed small metals without producing the toxic waste associ- ated with chemical manufacture. Bacteria have been isolated

Figure 9.18 Bacillus cells growing on selenium form chains of elemental selenium.

Q What might bacteria provide for nanotechnology?

Fingerprints, blood types, and DNA were once new to crime scene investigations (CSI). Each technique uses unique profiles from the human body to draw conclusions about a person’s actions or whereabouts. Now the microbiome might be the next CSI tool.

Even after we wash our hands, certain bacteria persist. These microbes can also be transferred to objects in the home or office or to other people we live with. But which microbes commonly live on the body also varies greatly throughout the population as a whole—meaning that the microbiome can become a unique identifier in certain situations.

A research project called The Home Microbiome Project followed seven families and their pets over 6 weeks. Researchers discovered distinct microbial

communities in each house. Couples and their young children shared most of their microbial community. When three of the families moved, it took less than a day for the new house to have the same microbial population as the old one.

In another study, it was shown that a person’s “microbiome fingerprint” remains fairly consistent over time. All this research suggests that microbiome composition may be the basis for a reliable forensic tool. Microbiome profiles could be used to track whether a person lived somewhere, used a particular cell phone, or walked over a surface. Humans also exchange microbes during intercourse, so microbes on pubic hair might also provide evidence of sexual assault.

Microbiota, like this skin biofilm, may one day be another crime scene “fingerprint.”

causes the formation of a tumorlike growth called a crown gall (Figure 9.19). A part of the Ti plasmid, called T-DNA, integrates into the genome of the infected plant. The T-DNA stimulates local cellular growth (the crown gall) and simultaneously causes the production of certain products used by the bacteria as a source of nutritional carbon and nitrogen.

For plant scientists, the attraction of the Ti plasmid is that it provides a vehicle for introducing rDNA into a plant (Figure 9.20). A scientist can insert foreign genes into the T-DNA, put the recombinant plasmid back into the Agrobacterium cell, and use the bacterium to insert the recombinant Ti plasmid into a plant cell. The plant cell with the foreign gene can then be used to generate a new plant. With luck, the new plant will express the foreign gene. Unfortunately, Agrobacterium does not naturally infect grasses, so it cannot be used to improve grains such as wheat, rice, or corn.

Noteworthy accomplishments of this approach are the introduction into plants of resistance to the herbicide glypho- sate. Normally, the herbicide kills both weeds and useful plants by inhibiting an enzyme necessary for making certain essential amino acids. Some Salmonella bacteria happen to have this enzyme, but are resistant to the herbicide. When the DNA for this enzyme is introduced into a crop plant, the crop

EXPLORING THE MICROBIOME Crime Scene Investigation and Your Microbiome

Crown gall

Figure 9.19 Crown gall disease on a rose plant. The tumorlike growth is stimulated by a gene on the Ti plasmid that Agrobacterium tumefaciens inserted into a plant cell.

Q What are some of the agricultural applications of rDNA technology? 261

262 PART ONE Fundamentals of Microbiology

becomes resistant to the herbicide, which then kills only the weeds. The Bt gene from Bacillus thuringiensis has been inserted into a variety of crop plants, including cotton and potatoes, so insects that eat the plants will be killed. Resistance to drought, viral infection, and several other environmental stresses has also been engineered into crop plants.

Another example involves FlavrSavr™ tomatoes, which stay firm after harvest because the gene for polygalacturonase (PG), the enzyme that breaks down pectin, is suppressed. The suppression was accomplished by antisense DNA technology. First, a length of DNA complementary to the PG mRNA is syn- thesized. This antisense DNA is taken up by the cell and binds to the mRNA to inhibit translation. The DNA-RNA hybrid is broken down by the cell’s enzymes, freeing the antisense DNA to disable another mRNA.

An example of a genetically modified bacterium now in agricultural use is Pseudomonas fluorescens that has been engi- neered to produce Bt toxin, normally produced by Bacillus thuringiensis. The genetically altered Pseudomonas, which pro- duces much more toxin than B. thuringiensis, can be added to plant seeds and in time will enter the vascular system of the growing plant. Its toxin is ingested by the feeding insect larvae and kills them (but is harmless to humans and other warm- blooded animals).

1 The plasmid is removed from the bacterium, and the T-DNA is cut by a restriction enzyme.

Ti plasmid

T-DNA

Restriction cleavage site

2 Foreign DNA is cut by the same enzyme.

3 The foreign DNA is inserted into the T-DNA of the plasmid.

Recombinant Ti plasmid

4 The plasmid is reinserted into a bacterium.

Agrobacterium tumefaciens bacterium

5 The bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of a plant cell.

6 The plant cells are grown in culture.

7 A plant is generated from a cell clone. All of its cells carry the foreign gene and may express it as a new trait.

Inserted T-DNA carrying foreign gene

Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants.

Q Why is the Ti plasmid important to biotechnology?

Animal husbandry has also benefited from rDNA technol- ogy to develop disease-resistant food animals. Techniques for making cattle resistant to bovine spongiform encephalopathy and chickens and pigs resistant to avian influenza are currently being researched.

Table 9.3 lists several rDNA products used in agriculture

and animal husbandry.

CHECK YOUR UNDERSTANDING

✓ 9-19 Of what value is the plant pathogen Agrobacterium?

Safety Issues and the Ethics of Using DNA Technology LEARNING OBJECTIVE

9-20 List the advantages of, and problems associated with, the use of genetic modification techniques.

There will always be concern about the safety of any new tech- nology, and genetic modification and biotechnology are cer- tainly no exceptions. One reason for this concern is it’s nearly impossible to prove that something is entirely safe under all conceivable conditions. People worry that the same tech- niques that can alter a microbe or plant to make them useful to humans could also inadvertently make them pathogenic to

CHAPTER 9 Biotechnology and DNA Technology 263

TABLE 9.3 Some Agriculturally Important Products of rDNA Technology Product Comments

AGRICULTURAL PRODUCTS

Button mushroom (Agaricus bisporus) Gene for polyphenyl oxidase, which causes browning, is deleted.

Bt cotton and Bt corn Plants have toxin-producing gene from Bacillus thuringiensis; toxin kills insects that eat plants.

Genetically modified tomatoes, raspberries Antisense gene blocks pectin degradation, so fruits have longer shelf life.

Pseudomonas syringae, ice-minus bacterium Lacks normal protein product that initiates undesirable ice formation on plants.

RoundUp (glyphosate)-resistant crops Plants have bacterial gene; allows use of herbicide on weeds without damaging crops.

ANIMAL PRODUCTS

Aedes aegypti Male mosquito with a gene that causes larvae to die; used to control spread of Zika virus.

Atlantic salmon Salmon grow faster with a gene from Chinook salmon and promoter from another fish (pout).

GloFish® Brightly colored fluorescent aquarium fish with the color-protein genes from marine invertebrates.

humans or otherwise dangerous to living organisms or could create an ecological nightmare. Therefore, laboratories engaged in rDNA research must meet rigorous standards of control to avoid either accidental release of genetically modified organ- isms into the environment or exposure of humans to any risk of infection. To reduce risk further, microbiologists engaged in genetic modification often delete from the microbes’ genomes certain genes that are essential for growth in environments out- side the laboratory. Genetically modified organisms intended for use in the environment (in agriculture, for example) may be engineered to contain “suicide genes”—genes that eventually turn on to produce a toxin that kills the microbes, thus ensur- ing that they will not survive in the environment for very long after they have accomplished their task.

The safety issues in agricultural biotechnology are similar to those concerning chemical pesticides: toxicity to humans and to nonpest species. Although not shown to be harmful, geneti- cally modified foods have not been popular with consumers. In 1999, researchers in Ohio noticed that humans may develop allergies to Bacillus thuringiensis (Bt) toxin after working in fields sprayed with the insecticide. And an Iowa study showed that the caterpillar stage of Monarch butterflies could be killed by ingesting windblown Bt-carrying pollen that landed on milkweed, the caterpillars’ normal food. Crop plants can be genetically modified for herbicide resistance so that fields can be sprayed to eliminate weeds without killing the desired crop. However, if the modified plants pollinate related weed species,

weeds could become resistant to herbicides, making it more difficult to control unwanted plants. An unanswered question is whether releasing genetically modified organisms will alter evolution as genes move to wild species.

These developing technologies also raise a variety of ethi- cal issues. Genetic testing for diseases is becoming routine. Who should have access to this information? Should employers have the right to know the results of such tests? How can we be assured that such information will not be used to discriminate against certain groups? Should individuals be told they will get an incurable disease? If so, when?

Genetic counseling, which provides advice and counseling to prospective parents with family histories of genetic disease, is becoming more important in considerations about whether to have children.

There are probably just as many harmful applications of a new technology as there are helpful ones. It is particularly easy to imagine DNA technology being used to develop new and powerful biological weapons. In addition, because such research efforts are performed under top-secret conditions, it is virtually impossible for the general public to learn of them.

Perhaps more than most new technologies, molecular genetics holds the promise of affecting human life in previ- ously unimaginable ways. It is important that society and indi- viduals be given every opportunity to understand the potential impact of these new developments.

CLINICAL FOCUS Norovirus—Who Is Responsible for the Outbreak?

As you read through this box, you will encounter a series of questions that microbiologists ask themselves as they trace a disease outbreak. Whether the microbiologist is called as an expert witness in court will depend on whether a lawsuit is filed. Try to answer each question before going on to the next one.

1. On May 7, Nadia Koehler, a microbiologist at a county health department, is noti- fied of a gastroenteritis outbreak among 115 people. The case is defined as vomiting and diarrhea and fever, cramps, or nausea. What information does Nadia need?

2. Nadia needs to find out where the ill people have been in the past 48 hours. After several interviews, Nadia finds out that the ill people include 23 school employees, 55 publishing company employees, 9 employees of a social service organization, and 28 other people (see Figure A). Now what does Nadia need to know?

3. Next, Nadia finds out what these 115 people have in common. In her investigation, Nadia discovers that on May 2, the school staff had been served

N um

be r

of r

ep or

te d

ca se

School meal served

Initial reports of community cases

3 4 5 6 Date

5 Reported community cases

Social service group

School employees

Publishing company employees

KEY

a party-sized sandwich catered by a national franchise restaurant. On May 3, the publishing company and social service staff luncheons were catered by the same restaurant. The remaining 28 people ate sandwiches at the same restaurant, at varying times between these two days. What does Nadia do next?

4. Nadia analyzes exposures to 16 food items; the results show that eating lettuce is significantly associated with illness. What is Nadia’s next step?

5. Nadia then requests a reverse- transcription PCR (RT-PCR) using a norovirus primer to be done on stool samples (Figure B). What did Nadia conclude?

6. RT-PCR confirmed norovirus infection. Nadia’s next request is for a sequence analysis to be performed on 21 stool specimens. The results demonstrated 100% sequence homology for the 21 specimens. What should Nadia do next?

7. Nadia learns that a food handler employed by the restaurant had experienced vomiting and diarrhea on May 1. The food handler believes he had acquired the illness from his child. The child’s illness was traced to an ill cousin who had been exposed to norovirus at a child-care center. The food handler’s vomiting ended by the early morning of May 2, and he returned to work at the restaurant later that morning. What should Nadia look for now?

8. Now Nadia compares the virus strains from the food handler to the ones from the ill customers. She requests a sequence analysis on viruses from the

1 2 3 4 5 6 7 8

213

food handler and eight ill customers. They are identical to the strains identified in step 6. Where does Nadia look next?

9. Nadia looks for any areas in the restaurant that still may be contaminated by the norovirus. She finds out that the lettuce was sliced each morning by the food handler who had been sick. Nadia’s inspection reveals that the food preparation sink is also used for handwashing. The sink was not sanitized before and after the lettuce was washed. The health department closes the restaurant until it can be cleaned with the proper sanitizers.

Noroviruses are the most common cause of outbreaks of acute gastroenteritis worldwide. Annually, norovirus causes 20 million cases of gastroenteritis. During 2015, 316 norovirus outbreaks in the United States were reported.

Source: Adapted from CDC, Foodborne Outbreak Online Database (FOOD).

Figure B Results of PCR of patient samples. Lane 1, 123-bp size ladders. Lane 2, negative RT-PCR control; Lanes 3–8, patient samples. Norovirus is identified by the 213-bp band of DNA.

Figure A Number of cases reported.

264

CHAPTER 9 Biotechnology and DNA Technology 265

Like the invention of the microscope, the development of DNA techniques is causing profound changes in science, agri- culture, and human health care. With this technology not quite 50 years old, it is difficult to predict exactly what changes will occur. However, it is likely that within another 30 years, many of the treatments and diagnostic methods discussed in this book will have been replaced by far more powerful

Study Outline

techniques based on the unprecedented ability to manipulate DNA precisely.

CHECK YOUR UNDERSTANDING

✓ 9-20 Identify two advantages and two problems associated with genetically modified organisms.

Go to @MasteringMicrobiology for Interactive Microbiology, In the Clinic videos, MicroFlix, MicroBoosters, 3D animations, practice quizzes, and more.

Introduction to Biotechnology (pp. 243–245) 1. Biotechnology is the use of microorganisms, cells, or cell

components to make a product.

Recombinant DNA Technology (p. 243) 2. Closely related organisms can exchange genes in natural

recombination .

3. Genes can be transferred among unrelated species via laboratory manipulation, called rDNA technology.

4. Recombinant DNA is DNA that has been artificially manipulated to combine genes from two different sources.

An Overview of Recombinant DNA Procedures (pp. 243–245) 5. A desired gene is inserted into a DNA vector, such as a plasmid or a

viral genome.

6. The vector inserts the DNA into a new cell, which is grown to form a clone.

7. Large quantities of the gene product can be harvested from the clone.

Tools of Biotechnology (pp. 245–248)

Selection (p. 245) 1. Microbes with desirable traits are selected for culturing by artificial

selection .

Mutation (p. 245) 2. Mutagens are used to cause mutations that might result in a

microbe with desirable traits.

3. Site-directed mutagenesis is used to change a specific codon in a gene.

Restriction Enzymes (pp. 245–246) 4. Prepackaged kits are available for rDNA techniques.

5. A restriction enzyme recognizes and cuts only one particular nucleotide sequence in DNA .

6. Some restriction enzymes produce sticky ends, short stretches of single-stranded DNA at the ends of the DNA fragments.

7. Fragments of DNA produced by the same restriction enzyme will spontaneously join by base pairing. DNA ligase can covalently link the DNA backbones.

Vectors (pp. 246–247) 8. Vectors are DNA used to transfer other DNA between cells.

9. A plasmid containing a new gene can be inserted into a cell by transformation .

10. A virus containing a new gene can insert the gene into a cell.

Polymerase Chain Reaction (pp. 247–248) 11. The polymerase chain reaction (PCR) is used to make multiple

copies of a desired piece of DNA enzymatically.

12. PCR can be used to increase the amounts of DNA in samples to detectable levels. This may allow sequencing of genes, the diagnosis of genetic diseases, or the detection of viruses.

Techniques of Genetic Modification (pp. 248–254)

Inserting Foreign DNA into Cells (pp. 249–250) 1. Cells can take up naked DNA by transformation . Chemical

treatments are used to make cells that are not naturally competent take up DNA .

2. Pores made in protoplasts and animal cells by electric current in the process of electroporation can provide entrance for new pieces of DNA .

3. Protoplast fusion is the joining of cells whose cell walls have been removed.

4. Foreign DNA can be introduced into plant cells by shooting DNA- coated particles into the cells or by using a thin micropipette.

Obtaining DNA (pp. 250–252) 5. Genomic libraries can be made by cutting up an entire genome

with restriction enzymes and inserting the fragments into bacterial plasmids or phages.

6. Complementary DNA (cDNA) made from mRNA by reverse transcription can be cloned in genomic libraries.

7. Synthetic DNA can be made in vitro by a DNA synthesis machine.

Selecting a Clone (pp. 252–253) 8. Antibiotic-resistance markers on plasmid vectors are used to

identify cells containing the engineered vector by direct selection .

9. In blue-white screening, the vector contains the genes for amp and β-galactosidase.

10. The desired gene is inserted into the β-galactosidase gene site, destroying the gene.

11. Clones containing the recombinant vector will be resistant to ampicillin and unable to hydrolyze X-gal (white colonies).

12. Clones containing foreign DNA can be tested for the desired gene product.

13. A short piece of labeled DNA called a DNA probe can be used to identify clones carrying the desired gene.

266 PART ONE Fundamentals of Microbiology

Study Questions

11. Bioinformatics is the use of computer applications to study genetic data; proteomics is the study of a cell’s proteins.

12. Southern blotting can be used to locate a gene in a cell.

13. DNA probes can be used to quickly identify a pathogen in body tissue or food.

14. Forensic microbiologists use DNA fingerprinting to identify the source of bacterial or viral pathogens.

15. Bacteria may be used to make nano-sized materials for nanotechnology machines.

Agricultural Applications (pp. 260–262) 16. Cells from plants with desirable characteristics can be cloned

to produce many identical cells. These cells can then be used to produce whole plants from which seeds can be harvested.

17. Plant cells can be modified by using the Ti plasmid vector. The tumor-producing T genes are replaced with desired genes, and the rDNA is inserted into Agrobacterium. The bacterium naturally transforms its plant hosts.

18. Antisense DNA can prevent expression of unwanted proteins.

Safety Issues and the Ethics of Using DNA Technology (pp. 262–265) 1. Strict safety standards are used to avoid the accidental release of

genetically modified microorganisms.

2. Some microbes used in rDNA cloning have been altered so that they cannot survive outside the laboratory.

3. Microorganisms intended for use in the environment may be modified to contain suicide genes so that the organisms do not persist in the environment.

4. Genetic testing raises a number of ethical questions: Should employers have access to a person’s genetic records? Will genetic information be used to discriminate against people? Will genetic counseling be available to everyone?

5. Genetically modified crops must be safe for consumption and for release in the environment.

Making a Gene Product (pp. 253–254) 14. E. coli is used to produce proteins using rDNA because E. coli is

easily grown and its genomics are well understood.

15. Efforts must be made to ensure that E. coli’s endotoxin does not contaminate a product intended for human use.

16. To recover the product, E. coli must be lysed, or the gene must be linked to a gene that produces a naturally secreted protein.

17. Yeasts can be genetically modified and are likely to secrete a gene product continuously.

18. Genetically modified mammalian cells can be grown to produce proteins such as hormones for medical use.

19. Genetically modified plant cells can be grown and used to produce plants with new properties.

Applications of DNA Technology (pp. 254–262) 1. Cloned DNA is used to produce products, study the cloned DNA,

and alter the phenotype of an organism.

Therapeutic Applications (pp. 255–256) 2. Synthetic genes linked to the β-galactosidase gene (lacZ) in a

plasmid vector were inserted into E. coli, allowing E. coli to produce and secrete the two polypeptides used to make human insulin.

3. Cells and viruses can be modified to produce a pathogen’s surface protein, which can be used as a vaccine.

4. DNA vaccines consist of rDNA cloned in bacteria.

5. Gene therapy can be used to cure genetic diseases by replacing the defective or missing gene.

6. RNAi may be useful to prevent expression of abnormal proteins.

Genome Projects (pp. 256–257) 7. Nucleotide sequences of genomes from more than 1000 organisms,

including humans, have been completed.

8. This leads to determining the proteins produced in a cell.

Scientific Applications (pp. 257–260) 9. DNA can be used to increase understanding of DNA, for genetic

fingerprinting, and for gene therapy.

10. DNA sequencing machines are used to determine the nucleotide base sequence of restriction fragments in shotgun sequencing.

For answers to Knowledge and Comprehension questions, turn to the Answers tab at the back of the textbook.

Knowledge and Comprehension

Review 1. Compare and contrast the following terms:

a. cDNA and gene

b. RFLP and gene

c. DNA probe and gene

d. DNA polymerase and DNA ligase

e. rDNA and cDNA

f. genome and proteome

2. Differentiate the following terms. Which one is “hit and miss”— that is, does not add a specific gene to a cell? a. protoplast fusion b. gene gun c. microinjection d. electroporation

CHAPTER 9 Biotechnology and DNA Technology 267

3. Some commonly used restriction enzymes are listed in Table 9.1 on page 246. a. Indicate which enzymes produce sticky ends. b. Of what value are sticky ends in making rDNA?

4. Suppose you want multiple copies of a gene you have synthesized. How would you obtain the necessary copies by cloning? By PCR?

5. DRAW IT Using the following map of plasmid pMICRO, diagram the locations of the restriction fragments that result from digesting pMICRO with EcoRI, HindIII, and both enzymes together following electrophoresis. Which enzyme makes the smallest fragment containing the tetracycline resistance gene?

S iz

e la

dd er

E co

R I d

ig es

H in

dI II

di ge

D ou

bl e

di ge

Base pairs

2517

1517

1200

1000 900 800 700 600 500

400

300

200

100

tet

HindIII

100 bp 200 bp

650 bp

550 bp

1300 bp

HindIII

EcoRI

6. Describe an rDNA experiment in two or three sentences. Use the following terms: intron, exon, DNA, mRNA, cDNA, RNA polymerase, reverse transcriptase.

7. List at least two examples of the use of rDNA in medicine and in agriculture.

8. You are attempting to insert a gene for saltwater tolerance into a plant by using the Ti plasmid. In addition to the desired gene, you add a gene for tetracycline resistance (tet) to the plasmid. What is the purpose of the tet gene?

9. How does RNAi “silence” a gene?

10. NAME IT This virus family, normally associated with AIDS, may be useful for gene therapy.

Multiple Choice 1. Restriction enzymes were first discovered with the observation that

a. DNA is restricted to the nucleus. b. phage DNA is destroyed in a host cell. c. foreign DNA is kept out of a cell. d. foreign DNA is restricted to the cytoplasm. e. all of the above

2. The DNA probe, 3’-GGCTTA, will hybridize with which of the following? a. 5’-CCGUUA b. 5’-CCGAAT c. 5’-GGCTTA

d. 3’-CCGAAT e. 3’-GGCAAU

3. Which of the following is the fourth basic step to genetically modify a cell? a. transformation b. ligation c. plasmid cleavage d. restriction-enzyme digestion of gene e. isolation of gene

4. The following enzymes are used to make cDNA. What is the second enzyme used to make cDNA? a. reverse transcriptase b. ribozyme c. RNA polymerase d. DNA polymerase

5. If you put a gene in a virus, the next step in genetic modification would be a. insertion of a plasmid. b. transformation. c. transduction.

d. PCR. e. Southern blotting.

6. You have a small gene that you want replicated by PCR. You add radioactively labeled nucleotides to the PCR thermal cycler. After three replication cycles, what percentage of the DNA single strands are radioactively labeled? a. 0% b. 12.5% c. 50%

d. 87.5% e. 100%

Match the following choices to the statements in questions 7 through 10. a. antisense b. clone c. library

d. Southern blot e. vector

7. Pieces of human DNA stored in yeast cells.

8. A population of cells carrying a desired plasmid.

9. Self-replicating DNA for transmitting a gene from one organism to another.

10. DNA that hybridizes with mRNA.

268 PART ONE Fundamentals of Microbiology

Clinical Applications and Evaluation 1. PCR has been used to examine oysters for

the presence of Vibrio cholerae. Oysters from different areas were homogenized, and DNA was extracted from the homogenates. The DNA was digested by the restriction enzyme HincII. A primer for the hemolysin gene of V. cholerae was used for the PCR reaction. After PCR, each sample was electrophoresed and stained with a probe for the hemolysin gene. Which of the oyster samples were (was) positive for V. cholerae? How can you tell? Why look for V. cholerae in oysters? What is the advantage of PCR over conventional biochemical tests to identify the bacteria?

A B C

2. Using the restriction enzyme EcoRI, the following gel electrophoresis patterns were obtained from digests of various DNA molecules from a transformation experiment. Can you conclude from these data that transformation occurred? Explain why or why not.

Origin

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en e

N ew

g en

O rig

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c el

l’s D

N A

T ra

ns fo

rm ed

ce ll’

s D

N A

Analysis 1. Design an experiment using vaccinia virus to make a vaccine

against the AIDS virus (HIV).

2. Why did the use of DNA polymerase from the bacterium Thermus aquaticus allow researchers to add the necessary reagents to tubes in a preprogrammed heating block?

3. The following picture shows bacterial colonies growing on X-gal plus ampicillin in a blue-white screening test. Which colonies have the recombinant plasmid? The small satellite colonies do not have the plasmid. Why did they start growing on the medium 48 hours after the larger colonies?

Cover
Brief Contents
Title Page
Copyright Page
About the Authors
Digital Authors
Preface
Acknowledgments
Contents
Features
ASM Recommended Curriculum Guidelinesfor Undergraduate Microbiology
PART ONE Fundamentals of Microbiology

1 The Microbial World and You

Microbes in Our Lives

The Microbiome

Naming and Classifying Microorganisms

Nomenclature
Types of Microorganisms
Classification of Microorganisms

A Brief History of Microbiology

The First Observations
The Debate over Spontaneous Generation
The First Golden Age of Microbiology
The Second Golden Age of Microbiology
The Third Golden Age of Microbiology

Microbes and Human Welfare

Recycling Vital Elements
Sewage Treatment: Using Microbes to Recycle Water
Bioremediation: Using Microbes to Clean Up Pollutants
Insect Pest Control by Microorganisms
Biotechnology and Recombinant DNA Technology

Microbes and Human Disease

Biofilms
Infectious Diseases
Emerging Infectious Diseases

Study Outline
Study Questions

2 Chemical Principles

The Structure of Atoms

Chemical Elements
Electronic Configurations

How Atoms Form Molecules: Chemical Bonds

Ionic Bonds
Covalent Bonds
Hydrogen Bonds
Molecular Mass and Moles

Chemical Reactions

Energy in Chemical Reactions
Synthesis Reactions
Decomposition Reactions
Exchange Reactions
The Reversibility of Chemical Reactions

IMPORTANT BIOLOGICAL MOLECULES
Inorganic Compounds

Water
Acids, Bases, and Salts
Acid–Base Balance: The Concept of pH

Organic Compounds

Structure and Chemistry
Carbohydrates
Lipids
Proteins
Nucleic Acids
Adenosine Triphosphate (ATP)

Study Outline
Study Questions

3 Observing Microorganisms Through a Microscope

Units of Measurement
Microscopy: The Instruments

Light Microscopy
Two-Photon Microscopy
Super-Resolution Light Microscopy
Scanning Acoustic Microscopy
Electron Microscopy
Scanned-Probe Microscopy

Preparation of Specimens for Light Microscopy

Preparing Smears for Staining
Simple Stains
Differential Stains
Special Stains

Study Outline
Study Questions

4 Functional Anatomy of Prokaryotic and Eukaryotic Cells

Comparing Prokaryotic and Eukaryotic Cells: An Overview
THE PROKARYOTIC CELL
The Size, Shape, and Arrangement of Bacterial Cells
Structures External to the Cell Wall

Glycocalyx
Flagella and Archaella
Axial Filaments
Fimbriae and Pili

The Cell Wall

Composition and Characteristics
Cell Walls and the Gram Stain Mechanism
Atypical Cell Walls
Damage to the Cell Wall

Structures Internal to the Cell Wall

The Plasma (Cytoplasmic) Membrane
The Movement of Materials across Membranes
Cytoplasm
The Nucleoid
Ribosomes
Inclusions
Endospores

THE EUKARYOTIC CELL
Flagella and Cilia
The Cell Wall and Glycocalyx
The Plasma (Cytoplasmic) Membrane
Cytoplasm
Ribosomes
Organelles

The Nucleus
Endoplasmic Reticulum
Golgi Complex
Lysosomes
Vacuoles
Mitochondria
Chloroplasts
Peroxisomes
Centrosome

The Evolution of Eukaryotes
Study Outline
Study Questions

5 Microbial Metabolism

Catabolic and Anabolic Reactions
Enzymes

Collision Theory
Enzymes and Chemical Reactions
Enzyme Specificity and Efficiency
Naming Enzymes
Enzyme Components
Factors Influencing Enzymatic Activity
Feedback Inhibition
Ribozymes

Energy Production

Oxidation-Reduction Reactions
The Generation of ATP
Metabolic Pathways of Energy Production

Carbohydrate Catabolism

Glycolysis
Additional Pathways to Glycolysis
Cellular Respiration
Fermentation

Lipid and Protein Catabolism
Biochemical Tests and Bacterial Identification
Photosynthesis

The Light-Dependent Reactions: Photophosphorylation
The Light-Independent Reactions: The Calvin-Benson Cycle

A Summary of Energy Production Mechanisms
Metabolic Diversity among Organisms

Photoautotrophs
Photoheterotrophs
Chemoautotrophs
Chemoheterotrophs

Metabolic Pathways of Energy Use

Polysaccharide Biosynthesis
Lipid Biosynthesis
Amino Acid and Protein Biosynthesis
Purine and Pyrimidine Biosynthesis

The Integration of Metabolism
Study Outline
Study Questions

6 Microbial Growth

The Requirements for Growth

Physical Requirements
Chemical Requirements

Biofilms
Culture Media

Chemically Defined Media
Complex Media
Anaerobic Growth Media and Methods
Special Culture Techniques
Selective and Differential Media
Enrichment Culture

Obtaining Pure Cultures
Preserving Bacterial Cultures
The Growth of Bacterial Cultures

Bacterial Division
Generation Time
Logarithmic Representation of Bacterial Populations
Phases of Growth
Direct Measurement of Microbial Growth
Estimating Bacterial Numbers by Indirect Methods

Study Outline
Study Questions

7 The Control of Microbial Growth

The Terminology of Microbial Control
The Rate of Microbial Death
Actions of Microbial Control Agents

Alteration of Membrane Permeability
Damage to Proteins and Nucleic Acids

Physical Methods of Microbial Control

Heat
Filtration
Low Temperatures
High Pressure
Desiccation
Osmotic Pressure
Radiation

Chemical Methods of Microbial Control

Principles of Effective Disinfection
Evaluating a Disinfectant
Types of Disinfectants

Microbial Characteristics and Microbial Control
Study Outline
Study Questions

8 Microbial Genetics

Structure and Function of the Genetic Material

Genotype and Phenotype
DNA and Chromosomes
The Flow of Genetic Information
DNA Replication
RNA and Protein Synthesis

The Regulation of Bacterial Gene Expression

Pre-transcriptional Control
Post-transcriptional Control

Changes in Genetic Material

Mutation
Types of Mutations
Mutagens
The Frequency of Mutation
Identifying Mutants
Identifying Chemical Carcinogens

Genetic Transfer and Recombination

Plasmids and Transposons
Transformation in Bacteria
Conjugation in Bacteria
Transduction in Bacteria

Genes and Evolution
Study Outline
Study Questions

9 Biotechnology and DNA Technology

Introduction to Biotechnology

Recombinant DNA Technology
An Overview of Recombinant DNA Procedures

Tools of Biotechnology

Selection
Mutation
Restriction Enzymes
Vectors
Polymerase Chain Reaction

Techniques of Genetic Modification

Inserting Foreign DNA into Cells
Obtaining DNA
Selecting a Clone
Making a Gene Product

Applications of DNA Technology

Therapeutic Applications
Genome Projects
Scientific Applications
Agricultural Applications

Safety Issues and the Ethics of Using DNA Technology
Study Outline
Study Questions

PART TWO A Survey of the Microbial World

10 Classification of Microorganisms

The Study of Phylogenetic Relationships

The Three Domains
A Phylogenetic Tree

Classification of Organisms

Scientific Nomenclature
The Taxonomic Hierarchy
Classification of Prokaryotes
Classification of Eukaryotes
Classification of Viruses

Methods of Classifying and Identifying Microorganisms

Morphological Characteristics
Differential Staining
Biochemical Tests
Serology
Phage Typing
Fatty Acid Profiles
Flow Cytometry
DNA Sequencing
DNA Fingerprinting
Nucleic Acid Hybridization
Putting Classification Methods Together

Study Outline
Study Questions

11 The Prokaryotes: Domains Bacteria and Archaea

The Prokaryotic Groups
DOMAIN BACTERIA
Gram-Negative Bacteria

Proteobacteria
The Nonproteobacteria Gram-Negative Bacteria

The Gram-Positive Bacteria

Firmicutes (Low G + C Gram-Positive Bacteria)
Tenericutes
Actinobacteria (High G + C Gram-Positive Bacteria)

DOMAIN ARCHAEA
Diversity within the Archaea
MICROBIAL DIVERSITY
Discoveries Illustrating the Range of Diversity
Study Outline
Study Questions

12 The Eukaryotes: Fungi, Algae, Protozoa, and Helminths

Fungi

Characteristics of Fungi
Medically Important Fungi
Fungal Diseases
Economic Effects of Fungi

Lichens
Algae

Characteristics of Algae
Selected Phyla of Algae
Roles of Algae in Nature

Protozoa

Characteristics of Protozoa
Medically Important Protozoa

Slime Molds
Helminths

Characteristics of Helminths
Platyhelminths
Nematodes

Arthropods as Vectors
Study Outline
Study Questions

13 Viruses, Viroids, and Prions

General Characteristics of Viruses

Host Range
Viral Size

Viral Structure

Nucleic Acid
Capsid and Envelope
General Morphology

Taxonomy of Viruses
Isolation, Cultivation, and Identification of Viruses

Growing Bacteriophages in the Laboratory
Growing Animal Viruses in the Laboratory
Viral Identification

Viral Multiplication

Multiplication of Bacteriophages
Multiplication of Animal Viruses

Viruses and Cancer

The Transformation of Normal Cells into Tumor Cells
DNA Oncogenic Viruses
RNA Oncogenic Viruses
Viruses to Treat Cancer

Latent Viral Infections
Persistent Viral Infections
Plant Viruses and Viroids
Prions
Study Outline
Study Questions

PART THREE Interaction between Microbe and Host

14 Principles of Disease and Epidemiology

Pathology, Infection, and Disease
Human Microbiome

Relationships between the Normal Microbiota and the Host
Opportunistic Microorganisms
Cooperation among Microorganisms

The Etiology of Infectious Diseases

Koch’s Postulates
Exceptions to Koch’s Postulates

Classifying Infectious Diseases

Occurrence of a Disease
Severity or Duration of a Disease
Extent of Host Involvement

Patterns of Disease

Predisposing Factors
Development of Disease

The Spread of Infection

Reservoirs of Infection
Transmission of Disease

Healthcare-Associated Infections (HAIs)

Microorganisms in the Hospital
Compromised Host
Chain of Transmission
Control of Healthcare-Associated Infections

Emerging Infectious Diseases
Epidemiology

Descriptive Epidemiology
Analytical Epidemiology
Experimental Epidemiology
Case Reporting
The Centers for Disease Control and Prevention (CDC)

Study Outline
Study Questions

15 Microbial Mechanisms of Pathogenicity

How Microorganisms Enter a Host

Portals of Entry
The Preferred Portal of Entry
Numbers of Invading Microbes
Adherence

How Bacterial Pathogens Penetrate Host Defenses

Capsules
Cell Wall Components
Enzymes
Antigenic Variation
Penetration into the Host
Biofilms

How Bacterial Pathogens Damage Host Cells

Using the Host’s Nutrients: Siderophores
Direct Damage
Production of Toxins
Plasmids, Lysogeny, and Pathogenicity

Pathogenic Properties of Viruses

Viral Mechanisms for Evading Host Defenses
Cytopathic Effects of Viruses

Pathogenic Properties of Fungi, Protozoa, Helminths, and Algae

Fungi
Protozoa
Helminths
Algae

Portals of Exit
Study Outline
Study Questions

16 Innate Immunity: Nonspecific Defenses of the Host

The Concept of Immunity
FIRST LINE OF DEFENSE: SKIN AND MUCOUS MEMBRANES
Physical Factors
Chemical Factors
Normal Microbiota and Innate Immunity
SECOND LINE OF DEFENSE
Formed Elements in Blood
The Lymphatic System
Phagocytes

Actions of Phagocytic Cells
The Mechanism of Phagocytosis

Inflammation

Vasodilation and Increased Permeability of Blood Vessels
Phagocyte Migration and Phagocytosis
Tissue Repair

Fever
Antimicrobial Substances

The Complement System
Interferons
Iron-Binding Proteins
Antimicrobial Peptides
Other Factors

Study Outline
Study Questions

17 Adaptive Immunity: Specific Defenses of the Host

The Adaptive Immune System
Dual Nature of the Adaptive Immune System

Overview of Humoral Immunity
Overview of Cellular Immunity

Cytokines: Chemical Messengers of Immune Cells
Antigens and Antibodies

Antigens
Humoral Immunity: Antibodies

Humoral Immunity Response Process

Activation and Clonal Expansion of Antibody-Producing Cells
The Diversity of Antibodies

Results of the Antigen–Antibody Interaction
Cellular Immunity Response Process

Antigen-Presenting Cells (APCs)
Classes of T Cells

Nonspecific Cells and Extracellular Killing by the Adaptive Immune System
Immunological Memory
Types of Adaptive Immunity
Study Outline
Study Questions

18 Practical Applications of Immunology

Vaccines

Principles and Effects of Vaccination
Types of Vaccines and Their Characteristics
Vaccine Production, Delivery Methods, and Formulations

Diagnostic Immunology

Use of Monoclonal Antibodies
Precipitation Reactions
Agglutination Reactions
Neutralization Reactions
Complement-Fixation Reactions
Fluorescent-Antibody Techniques
Enzyme-Linked Immunosorbent Assay (ELISA)
Western Blotting (Immunoblotting)
The Future of Diagnostic and Therapeutic Immunology

Study Outline
Study Questions

19 Disorders Associated with the Immune System

Hypersensitivity

Allergies and the Microbiome
Type I (Anaphylactic) Reactions
Type II (Cytotoxic) Reactions
Type III (Immune Complex) Reactions
Type IV (Delayed Cell-Mediated) Reactions

Autoimmune Diseases

Cytotoxic Autoimmune Reactions
Immune Complex Autoimmune Reactions
Cell-Mediated Autoimmune Reactions

Reactions to Transplantation

Immunosuppression to Prevent Transplant Rejection

The Immune System and Cancer

Immunotherapy for Cancer

Immunodeficiencies

Congenital Immunodeficiencies
Acquired Immunodeficiencies

Acquired Immunodeficiency Syndrome (AIDS)

The Origin of AIDS
HIV Infection
Diagnostic Methods
HIV Transmission
AIDS Worldwide
Preventing and Treating AIDS

Study Outline
Study Questions

20 Antimicrobial Drugs

The History of Chemotherapy

Antibiotic Use and Discovery Today

Spectrum of Antimicrobial Activity
The Action of Antimicrobial Drugs

Inhibiting Cell Wall Synthesis
Inhibiting Protein Synthesis
Injuring the Plasma Membrane
Inhibiting Nucleic Acid Synthesis
Inhibiting the Synthesis of Essential Metabolites

Common Antimicrobial Drugs

Antibacterial Antibiotics: Inhibitors of Cell Wall Synthesis
Inhibitors of Protein Synthesis
Injury to Membranes
Nucleic Acid Synthesis Inhibitors
Competitive Inhibition of Essential Metabolites
Antifungal Drugs
Antiviral Drugs
Antiprotozoan and Antihelminthic Drugs

Tests to Guide Chemotherapy

The Diffusion Methods
Broth Dilution Tests

Resistance to Antimicrobial Drugs

Mechanisms of Resistance
Antibiotic Misuse
Cost and Prevention of Resistance

Antibiotic Safety
Effects of Combinations of Drugs
Future of Chemotherapeutic Agents
Study Outline
Study Questions

PART FOUR Microorganisms and Human Disease

21 Microbial Diseases of the Skin and Eyes

Structure and Function of the Skin

Mucous Membranes

Normal Microbiota of the Skin
Microbial Diseases of the Skin

Bacterial Diseases of the Skin
Viral Diseases of the Skin
Fungal Diseases of the Skin and Nails
Parasitic Infestation of the Skin

Microbial Diseases of the Eye

Inflammation of the Eye Membranes: Conjunctivitis
Bacterial Diseases of the Eye
Other Infectious Diseases of the Eye

Study Outline
Study Questions

22 Microbial Diseases of the Nervous System

Structure and Function of the Nervous System
Bacterial Diseases of the Nervous System

Bacterial Meningitis
Tetanus
Botulism
Leprosy

Viral Diseases of the Nervous System

Poliomyelitis
Rabies
Arboviral Encephalitis

Fungal Disease of the Nervous System

Cryptococcus neoformans Meningitis (Cryptococcosis)

Protozoan Diseases of the Nervous System

African Trypanosomiasis
Amebic Meningoencephalitis

Nervous System Diseases Caused by Prions

Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease

Diseases Caused by Unidentified Agents
Study Outline
Study Questions

23 Microbial Diseases of the Cardiovascular and Lymphatic Systems

Structure and Function of the Cardiovascular and Lymphatic Systems
Bacterial Diseases of the Cardiovascular and Lymphatic Systems

Sepsis and Septic Shock
Bacterial Infections of the Heart
Rheumatic Fever
Tularemia
Brucellosis (Undulant Fever)
Anthrax
Gangrene
Systemic Diseases Caused by Bites and Scratches
Vector-Transmitted Diseases

Viral Diseases of the Cardiovascular and Lymphatic Systems

Burkitt’s Lymphoma
Infectious Mononucleosis
Other Diseases and Epstein-Barr Virus
Cytomegalovirus Infections
Chikungunya
Classic Viral Hemorrhagic Fevers
Emerging Viral Hemorrhagic Fevers

Protozoan Diseases of the Cardiovascular and Lymphatic Systems

Chagas Disease (American Trypanosomiasis)
Toxoplasmosis
Malaria
Leishmaniasis
Babesiosis

Helminthic Disease of the Cardiovascular and Lymphatic Systems

Schistosomiasis

Disease of Unknown Etiology

Kawasaki Syndrome

Study Outline
Study Questions

24 Microbial Diseases of the Respiratory System

Structure and Function of the Respiratory System
Normal Microbiota of the Respiratory System
MICROBIAL DISEASES OF THE UPPER RESPIRATORY SYSTEM
Bacterial Diseases of the Upper Respiratory System

Streptococcal Pharyngitis (Strep Throat)
Scarlet Fever
Diphtheria
Otitis Media

Viral Disease of the Upper Respiratory System

The Common Cold

MICROBIAL DISEASES OF THE LOWER RESPIRATORY SYSTEM
Bacterial Diseases of the Lower Respiratory System

Pertussis (Whooping Cough)
Tuberculosis
Bacterial Pneumonias
Melioidosis

Viral Diseases of the Lower Respiratory System

Viral Pneumonia
Respiratory Syncytial Virus (RSV)
Influenza (Flu)

Fungal Diseases of the Lower Respiratory System

Histoplasmosis
Coccidioidomycosis
Pneumocystis Pneumonia
Blastomycosis (North American Blastomycosis)
Other Fungi Involved in Respiratory Disease

Study Outline
Study Questions

25 Microbial Diseases of the Digestive System

Structure and Function of the Digestive System
Normal Microbiota of the Digestive System
Bacterial Diseases of the Mouth

Dental Caries (Tooth Decay)
Periodontal Disease

Bacterial Diseases of the Lower Digestive System

Staphylococcal Food Poisoning (Staphylococcal Enterotoxicosis)
Shigellosis (Bacillary Dysentery)
Salmonellosis (Salmonella Gastroenteritis)
Typhoid Fever
Cholera
Noncholera Vibrios
Escherichia coli Gastroenteritis
Campylobacteriosis (Campylobacter Gastroenteritis)
Helicobacter Peptic Ulcer Disease
Yersinia Gastroenteritis
Clostridium perfringens Gastroenteritis
Clostridium difficile–Associated Diarrhea
Bacillus cereus Gastroenteritis

Viral Diseases of the Digestive System

Mumps
Hepatitis
Viral Gastroenteritis

Fungal Diseases of the Digestive System
Protozoan Diseases of the Digestive System

Giardiasis
Cryptosporidiosis
Cyclosporiasis
Amebic Dysentery (Amebiasis)

Helminthic Diseases of the Digestive System

Tapeworms
Hydatid Disease
Nematodes

Study Outline
Study Questions

26 Microbial Diseases of the Urinary and Reproductive Systems

Structure and Function of the Urinary System
Structure and Function of the Reproductive Systems
Normal Microbiota of the Urinary and Reproductive Systems
DISEASES OF THE URINARY SYSTEM
Bacterial Diseases of the Urinary System

Cystitis
Pyelonephritis
Leptospirosis

DISEASES OF THE REPRODUCTIVE SYSTEMS
Bacterial Diseases of the Reproductive Systems

Gonorrhea
Nongonococcal Urethritis (NGU)
Pelvic Inflammatory Disease (PID)
Syphilis
Lymphogranuloma Venereum (LGV)
Chancroid (Soft Chancre)
Bacterial Vaginosis

Viral Diseases of the Reproductive Systems

Genital Herpes
Genital Warts
AIDS

Fungal Disease of the Reproductive Systems

Candidiasis

Protozoan Disease of the Reproductive Systems

Trichomoniasis

Study Outline
Study Questions

PART FIVE Environmental and Applied Microbiology

27 Environmental Microbiology

Microbial Diversity and Habitats

Symbiosis

Soil Microbiology and Biogeochemical Cycles

The Carbon Cycle
The Nitrogen Cycle
The Sulfur Cycle
Life without Sunshine
The Phosphorus Cycle
The Degradation of Synthetic Chemicals in Soil and Water

Aquatic Microbiology and Sewage Treatment

Aquatic Microorganisms
The Role of Microorganisms in Water Quality
Water Treatment
Sewage (Wastewater) Treatment

Study Outline
Study Questions

28 Applied and Industrial Microbiology

Food Microbiology

Foods and Disease
Industrial Food Canning
Aseptic Packaging
Radiation and Industrial Food Preservation
High-Pressure Food Preservation
The Role of Microorganisms in Food Production

Industrial Microbiology and Biotechnology

Fermentation Technology
Industrial Products
Alternative Energy Sources Using Microorganisms
Biofuels
Industrial Microbiology and the Future

Study Outline
Study Questions

Answers to Knowledge and Comprehension Study Questions
Appendix A Metabolic Pathways
Appendix B Exponents, Exponential Notation, Logarithms, and Generation Time
Appendix C Methods for Taking Clinical Samples
Appendix D Pronunciation Rules and Word Roots
Appendix E Classification of Prokaryotes According to Bergey’s Manual
Glossary

Credits
Trademark Attributions
Index