Module 4
Microbial Genetics and Control
Microbial Genetics and Genetic Engineering
The genome is the sum total of genetic material of an organism. Although most of the
genome exists in the form of chromosomes, genetic material can appear as nonchromosomal
material as well (figure 8.1). For example, bacteria and some fungi contain tiny extra pieces of
DNA (plasmids). Also certain organelles of eukaryotes (the mitochondria and chloroplasts) are
equipped with their own DNA. Genomes of cells are composed exclusively of DNA, but the
viruses contain either DNA or RNA as the principal genetic material. Although the specific
genome of an individual organism is unique, the general pattern of nucleic acid structure and
function is similar among all organisms.
A chromosome is a distinct cellular structure composed of a neatly packaged DNA
molecule. The chromosomes of eukaryotes and bacterial cells differ in several respects. The
structure of eukaryotic chromosomes consists of a DNA molecule tightly wound around histone
proteins, whereas a bacterial chromosome is condensed into a packet by proteins that are histone-
like, but not actually histones. Eukaryotic chromosomes are located in the nucleus. They vary in
number from a few to hundreds. They can occur in pairs (diploid) or singles (haploid), and they
are doublestranded DNA in a more-or-less linear arrangement. In contrast, most bacterial
chromosomes are circular double strands of DNA. Bacteria generally have one, two, or
sometimes several chromosomes. The chromosomes of all cells are subdivided into basic
informational packets called genes. A gene can be defined from more than one perspective. In
classical genetics, the term refers to the fundamental unit of heredity responsible for a given trait
in an organism.
In the molecular and biochemical sense, it is a site on the chromosome that provides
information for a certain cell function. More specifically still, it has traditionally been
characterized as a certain segment of DNA that contains the necessary code to make a protein or
RNA molecule. Genes fall into three basic categories: (1) structural genes that code for proteins,
(2) genes that code for the RNA machinery used in protein production, and (3) regulatory genes
that control gene expression. The sum of all of these types of genes constitutes an organism’s
distinctive genetic makeup, or genotype (jee′-nohtyp). The expression of the genotype creates
traits (certain structures or functions) referred to as the phenotype (fee′-noh-typ). Just as a person
inherits a combination of genes (genotype) that gives a certain eye color or height (phenotypes),
a bacterium inherits genes that direct the formation of a flagellum, and a virus contains genes for
its capsid structure. All organisms contain more genes in their genotypes than are manifested as a
phenotype at any given time. In other words, the phenotype can change depending on which
genes are “turned on” (expressed).
Genomes vary greatly in size. The smallest viruses have four or five genes; the bacterium
Escherichia coli has a single chromosome containing 4,288 genes, and a human cell has about
19,000 to 20,000 genes on 23 chromosome pairs (46 chromosomes). The chromosome of E. coli
would measure about 1 mm if unwound and stretched out linearly, and yet this fits within a cell
that measures just over 1 μm across, making the stretched-out DNA 1,000 times longer than the
cell. This is possible because the DNA is very tightly wound around protein in the cytoplasm
(figure 8.2). Still, the bacterial chromosome takes up only about one-third to one-half of the
cell’s volume. Likewise, if the sum of all DNA making up the 46 human chromosomes in a cell
were unraveled and laid end to end, it would measure about 6 feet.
The general structure of DNA is universal, except in some viruses that contain single-
stranded DNA. The basic unit of DNA structure is a nucleotide, and a chromosome in a typical
bacterium consists of several million nucleotides linked end to end. Each nucleotide is composed
of a phosphate, a deoxyribose, and a nitrogenous base. The nucleotides covalently bond to each
other in a sugar-phosphate linkage that becomes the backbone of each strand. Each sugar
attaches in a repetitive pattern to two phosphates. One of the bonds is to the number 5′ (read
“five prime”) carbon on deoxyribose, and the other is to the 3′ carbon, which creates a certain
order and direction on each strand (figure 8.3). (In cyclical carbon molecules, like sugars, the
carbons are numbered so we can keep track of them. Deoxyribose has 5 carbons numbered 1—
5.)
The nitrogenous bases, purines and pyrimidines, attach along a strand by covalent bonds
at the 1′ position of the sugar (figure 8.3a). They join with complementary bases from the other
strand using hydrogen bonds. Such weak bonds are easily broken, allowing the molecule to be
“unzipped” into its complementary strands. This feature is of great importance in gaining access
to the information encoded in the nitrogenous base sequence. Pairing of purines and pyrimidines
is not random; it is dictated by the formation of hydrogen bonds between certain bases. So, in
DNA, the purine adenine (A) always pairs with the pyrimidine thymine (T), and the purine
guanine (G) always pairs with the pyrimidine cytosine (C). The bases are attracted to each other
in this pattern because each has a complementary three-dimensional shape that matches its pair.
Although the base-pairing partners generally do not vary, the sequence of base pairs along the
DNA molecule can assume any order, resulting in a nearly infinite number of possible nucleotide
sequences.
Other important considerations of DNA structure concern the nature of the double helix
itself. The halves are not oriented in the same direction. One side of the helix runs in the opposite
direction of the other, in what is called an antiparallel arrangement (figure 8.3b). The order of the
bond between the carbon on deoxyribose and the phosphates is used to keep track of the
direction of the two sides of the helix. This means that one helix runs from the 5′ to 3′ direction,
and the other runs from the 3′ to 5′ direction. This characteristic is a significant factor in DNA
synthesis and protein production.
DNA replication requires a careful orchestration of the actions of 30 different enzymes
(partial list in table 8.1), which separate the strands of the existing DNA molecule, copy one
strand, and produce two complete daughter molecules. A critical feature of DNA replication is
that each daughter molecule will be identical to the parent in composition, but neither one is
completely new; one of the strands in each double-stranded daughter molecule comes from the
parent DNA. The preservation of the parent molecule in this way, termed semiconservative
replication— semi- meaning “half,” as in semicircle—helps explain how the replication process
maintains accuracy and fidelity during successive cycles.
As with any language, DNA is occasionally “misspelled” when an incorrect base is added
to the growing chain. In bacteria such mistakes are made once in approximately 10 9 to 10 10
bases. Think of it this way: An overnight test tube culture of E. coli can be expected to have
around 10 9 to 10 10 bacteria per mL in it. That means a random mutation in every single base in
the E. coli genome will be represented in at least one cell in the culture. Because continued
cellular integrity is very dependent on accurate replication, cells have evolved their own
proofreading function for DNA. DNA polymerase III, the enzyme that elongates the molecule,
can detect incorrect, unmatching bases; excise them; and replace them with the correct base.
DNA polymerase I can also proofread the molecule and repair damaged DNA.
In addition to the RNA that is used to produce proteins, a wide variety of RNAs are used
to regulate gene function. This means that vast amounts of DNA sequences code for RNAs that
never get made into proteins (figure 8.5b). The DNA that codes for these very crucial RNA
molecules was called “junk” DNA until very recently because we didn’t understand its function,
and only knew that it did not produce proteins. While there is also a varying amount of junk
DNA in cells (whose function is not yet understood), much of the junk DNA has been found to
code for these very important regulatory RNAs. The action of these small RNAs is termed
epigenetic. This means that they are “on top of the DNA code” and don’t result in permanent
DNA changes (although sometimes epigenetic changes can be passed down to subsequent
generations).
Translation relies on a central principle: The mRNA nucleotides are read in groups of
three. Three nucleotides are called a codon, and it is the codon that dictates which amino acid is
added to the growing peptide chain. In figure 8.7, the mRNA codons and their corresponding
amino acid specificities are given. Because there are 64 different triplet codes and only 20
different amino acids, it is not surprising that some amino acids are represented by several
codons. For example, leucine and serine can each be represented by any of six different triplets,
and only tryptophan and methionine are represented by a single codon. This property is called
redundancy and allows for the insertion of correct amino acids (sometimes) even when mistakes
occur in the DNA sequence, as they do with regularity. Also, in codons such as those for leucine,
only the first two nucleotides are required to encode the correct amino acid. Any of the four
nucleotides can appear in the third position of the codon without changing the fact that leucine is
called for. This property, called wobble, is thought to permit some variation or mutation without
altering the message. Figure 8.8 shows the relationship between DNA sequence, RNA codons,
tRNA, and amino acids.
Eukaryotes and bacteria share many similarities in protein synthesis. The start codon in
eukaryotes is also AUG, but eukaryotes use a different form of methionine. Another difference is
that each eukaryotic mRNA codes for just one protein, unlike bacterial mRNAs, which often
contain information from several genes in series. As just mentioned, the presence of the DNA in
a separate compartment (the nucleus) means that eukaryotic transcription and translation cannot
be simultaneous. The mRNA transcript must pass through pores in the nuclear membrane and be
carried to the ribosomes in the cytoplasm for translation. We have given the simplified definition
of a gene that works well for bacteria, but most eukaryotic genes (and, surprisingly, archaeal
genes) do not exist as an uninterrupted series of triplets coding for a protein. A eukaryotic gene
contains the code for a protein, but located along the gene are one to several intervening
sequences of bases, called introns, that do not code for protein. Introns are interspersed between
coding regions, called exons, that will be translated into protein (figure 8.10). We can use words
as examples. A short section of colinear bacterial gene might read TOM SAW OUR DOG DIG
OUT; a eukaryotic gene that codes for the same portion would read TOM SAW XZKP FPL
OUR DOG QZWVP DIG OUT. The recognizable words are the exons, and the nonsense letters
represent the introns.
This unusual genetic architecture, sometimes called a split gene, requires further
processing for eukaryotes before translation. Transcription of the entire gene with both exons and
introns occurs first, producing a pre-mRNA. A series of adenosines is added to the mRNA
molecule. This protects the molecule and eventually directs it out of the nucleus for translation.
Next, a type of RNA and protein called a spliceosome recognizes the exon-intron junctions and
enzymatically cuts through them. The action of this splicer enzyme loops the introns into lariat-
shaped pieces, excises them, and joins the exons end to end. By this means, a strand of mRNA
with no intron material is produced. This completed mRNA strand can then proceed to the
cytoplasm to be translated.
Genetic Regulation of Protein Synthesis
In chapter 7, we surveyed the metabolic reactions in cells and the enzymes involved in
those reactions. At that time, we mentioned that some enzymes are regulated and that one form
of regulation occurs at the genetic level. Control mechanisms ensure that genes are active only
when their products are required. In this way, enzymes will be produced as they are needed and
prevent the waste of energy and materials in dead-end synthesis. Antisense RNAs, micro RNAs,
and riboswitches provide regulation in bacteria, archaea, and eukaryotes. Bacteria and archaea
have an additional strategy: They organize collections of genes into operons. Operons consist of
a coordinated set of genes, all of which are regulated as a single unit.
Operons are described as either inducible or repressible. Many catabolic operons, or
operons encoding enzymes that act in catabolism, are inducible, meaning that the operon is
turned on (induced) by the substrate of the enzyme(s) for which the structural genes code. In this
way, the enzymes needed to metabolize a nutrient (lactose, for example) are produced only when
that nutrient is present in the environment. Repressible operons are normally in the “on” position,
but are shut down in certain circumstances. Repressible operons often contain genes coding for
anabolic enzymes, such as those used to synthesize amino acids. In the case of these operons,
several genes in series are turned off (repressed) by the product synthesized by the enzyme.
When bacteria turn on or off a complement of genes that leads to obvious phenotypic
changes, it is sometimes called phase variation. Phase variation is a type of phenotypic variation,
but it has its own name because it has some special characteristics, the most important of which
is that the phenotype is heritable, meaning it is passed down to subsequent generations. The
process of turning on genes is often mediated by regulatory proteins, as described with operons.
The term phase variation is most often applied to traits affecting the bacterial cell surface and
was originally coined to describe the ability of bacteria to change components of their surface
that marked them for targeting by the host’s immune system. Because these surface molecules
also influenced the bacterium’s ability to attach to surfaces, the ability to undergo phase variation
allowed the microbes to adapt to—and stick in—different environments. Examples of phase
variation include the ability of Neisseria gonorrhoeae strains to produce attachment fimbriae and
the ability of Streptococcus pneumoniae to produce a capsule.
DNA Recombination Events
Genetic recombination through sexual reproduction is an important means of genetic
variation in eukaryotes. Although bacteria have no exact equivalent to sexual reproduction, they
exhibit a primitive means for sharing or recombining parts of their genome. An event in which
one bacterium donates DNA to another bacterium is a type of genetic transfer termed
recombination, the end result of which is a new strain different from both the donor and the
original recipient strain. Recombination in bacteria depends in part on the fact that bacteria
contain extrachromosomal DNA—that is, plasmids—and are adept at interchanging genes.
Genetic exchanges have tremendous effects on the genetic diversity of bacteria. They provide
additional genes for resistance to drugs and metabolic poisons, new nutritional and metabolic
capabilities, and increased virulence and adaptation to the environment. In general, any organism
that contains genes that originated in another organism is called a recombinant.
Any transfer of DNA that results in organisms acquiring new genes that did not come
directly from parent organisms is called horizontal gene transfer. (Acquiring genes from parent
organisms during reproduction would be vertical gene transfer.) Bacteria have been known to
engage in horizontal gene transfer for decades. It is now becoming clear that eukaryotic
organisms—including humans—also engage in horizontal gene transfer, often aided and abetted
by microbes such as viruses. Earlier in this book we discussed the concept of “pangenomes,”
made necessary by the discovery that different representatives of the same species had dozens of
genes that the reference organisms did not. This comes about mainly from horizontal gene
transfer.
DNA transfer between bacterial cells typically involves small pieces of DNA in the form
of plasmids or chromosomal fragments. Plasmids are small, circular pieces of DNA that contain
their own origin of replication and therefore can replicate independently of the bacterial
chromosome. Plasmids are found in many bacteria (as well as some fungi) and typically contain,
at most, only a few dozen genes. Although plasmids are not necessary for bacterial survival, they
often carry useful traits, such as antibiotic resistance. Chromosomal fragments that have escaped
from a lysed bacterial cell are also commonly involved in the transfer of genetic information
between cells. An important difference between plasmids and fragments is that while a plasmid
has its own origin of replication and is stably replicated and inherited, chromosomal fragments
must integrate themselves into the bacterial chromosome in order to be replicated and eventually
passed to progeny cells.
Conjugation is a mode of genetic exchange in which a plasmid or other genetic material
is transferred by a donor to a recipient cell via a direct connection. Both gram-negative and
grampositive cells can conjugate. In gram-negative cells, the donor’s plasmid (called a fertility,
or F factor) has genes that direct the synthesis of a conjugative pilus. The recipient cell has a
recognition site on its surface. A cell’s role in conjugation is denoted by F + for the cell that has
the F plasmid and by F − for the cell that lacks it. Contact is made when a pilus grows out from
the F + cell, attaches to the surface of the F − cell, contracts, and draws the two cells together. In
gram-positive cells, an opening is created between two adjacent cells, and the replicated DNA
passes across from one cell to the other. Conjugation is a conservative process, in that the donor
bacterium generally retains (“conserves”) a copy of the genetic material being transferred.
Conjugation has great biomedical importance. Special resistance (R) plasmids, or factors,
that bear genes for resisting antibiotics and other drugs are commonly shared among bacteria
through conjugation. Transfer of R factors can pass on resistance to antibiotics such as
tetracycline, chloramphenicol, streptomycin, sulfonamides, and penicillin. Other types of R
factors carry genetic codes for resistance to heavy metals (nickel and mercury) or for
synthesizing virulence factors (toxins, enzymes, and adhesion molecules) that increase the
pathogenicity of the bacterial strain. One important example of this is E. coli strains that have
acquired the dangerous toxin from Shigella bacteria via conjugation, called shiga toxin.
We now know that a chromosome released by a lysed cell breaks into fragments small
enough to be accepted by a recipient cell. This nonspecific acceptance by a bacterial cell of small
fragments of soluble DNA from the surrounding environment is termed transformation.
Transformation is facilitated by special DNA-binding proteins on the cell wall that capture DNA
from the surrounding medium. Cells that are capable of accepting genetic material through this
means are termed competent. The new DNA is transported into the cytoplasm, where some of it
is inserted into the bacterial chromosome. Transformation is a natural event found in several
groups of gram-positive and gram-negative bacterial species.
Because transformation requires no special appendages, and the donor and recipient cells
do not have to be in direct contact, the process is useful for certain types of recombinant DNA
technology. With this technique, foreign genes from a completely unrelated organism are
inserted into a plasmid, which is then introduced into a competent bacterial cell through
transformation in the same way that small pieces are taken up naturally. These recombinations
can be carried out in a test tube, and human genes can be experimented upon and even expressed
outside the human body by placing them in a microbial cell. This same phenomenon in
eukaryotic cells, termed transfection, is an essential aspect of genetically engineered yeasts,
plants, and mice.
Earlier we described bacteriophages as bacterial parasites. Viruses can in fact serve as
genetic vectors (an entity that can bring foreign DNA into a cell). The process by which a
bacteriophage serves as the carrier of DNA from a donor cell to a recipient cell is transduction. It
occurs naturally in a broad spectrum of bacteria. The participating bacteria in a single
transduction event must generally be the same species because of the specificity of viruses for
host cells.
There are two versions of transduction. In generalized transduction (figure 8.12), random
fragments of disintegrating host DNA are taken up by the phage during assembly. Virtually any
gene from the bacterium can be transmitted through this means. In specialized transduction
(figure 8.13), a highly specific part of the host genome is regularly incorporated into the virus.
This specificity is explained by the prior existence of a temperate prophage inserted in a fixed
site on the bacterial chromosome. When activated, the prophage DNA separates from the
bacterial chromosome, carrying a small segment of host genes with it. During a lytic cycle, these
specific viral-host gene combinations are incorporated into the viral particles and carried to
another bacterial cell.
Several cases of specialized transduction have biomedical importance. The virulent
strains of bacteria such as Corynebacterium diphtheriae, Clostridium spp., and Streptococcus
pyogenes all produce toxins with profound physiological effects. The nonvirulent strains do not
produce toxins. It turns out that toxicity arises from the presence of bacteriophage genes that
have been introduced by transduction. Only those bacteria infected with a temperate phage are
toxin formers. (Details of toxin action are discussed in the organ system–specific disease
chapters.)
Another type of genetic transfer involves transposable elements, or transposons.
Transposons have the distinction of shifting from one part of the genome to another and so are
termed “jumping genes.” All transposons share the general characteristic of traveling from one
location to another on the genome—from one chromosomal site to another, from a chromosome
to a plasmid, or from a plasmid to a chromosome (figure 8.14). Because transposons can occur in
plasmids, they can also be transmitted from one cell to another in bacteria and a few eukaryotes.
Some transposons replicate themsel
Mutations: Changes in the Genetic Code
As precise and predictable as the rules of genetic expression seem, permanent changes do
occur in the genetic code. Indeed, genetic change is the driving force of evolution. Any change to
the nucleotide sequence in the genome is called a mutation. Mutations are most noticeable when
the genotypic change leads to a change in phenotype. Mutations can involve the loss of base
pairs, the addition of base pairs, or a rearrangement in the order of base pairs. Do not confuse
this with genetic recombination, in which microbes transfer whole segments of genetic
information among themselves.
Mutations can be spontaneous or induced, depending upon their origin. A spontaneous
mutation is a random change in the DNA arising from errors in replication that occur randomly.
The frequency of spontaneous mutations has been measured for a number of organisms.
Mutation rates vary tremendously, from one mutation in 10 3 replications (a high rate) to one
mutation in 10 10 replications (a low rate). The rapid rate of bacterial reproduction allows these
mutations to be observed more readily in bacteria than in most eukaryotes. Induced mutations
result from exposure to known mutagens, which are physical or chemical agents that interact
with DNA in a disruptive manner. Examples of mutagens are some types of radiation (UV light,
X rays) and certain chemicals such as nitrous acid.
To understand how a change in DNA influences the cell, remember that the DNA code
appears in a particular order of triplets (three bases) that is transcribed into mRNA codons, each
of which specifies an amino acid. A permanent alteration in the DNA that is copied faithfully
into mRNA and translated can change the structure of the protein. A change in a protein can
likewise change the morphology and physiology of a cell. Some mutations have a harmful effect
on the cell, leading to cell dysfunction or death; these are called lethal mutations. Neutral
mutations produce neither adverse nor helpful changes. A small number of mutations are
beneficial in that they provide the cell with a useful change in structure or physiology.
A nonsense mutation, on the other hand, changes a normal codon into a stop codon that
does not code for an amino acid and stops the production of the protein wherever it occurs. A
nonsense mutation almost always results in a nonfunctional protein. (Table 8.8, row d, shows a
nonsense mutation resulting from a frameshift [described in next paragraph].) A silent mutation
(table 8.8, row c) alters a base but does not change the amino acid and thus has no effect. For
example, because of the redundancy of the code, ACU, ACC, ACG, and ACA all code for
threonine, so a mutation that changes only the last base will not alter the sense of the message in
any way. A back-mutation occurs when a gene that has undergone mutation reverses (mutates
back) to its original base composition.
Mutations also occur when one or more bases are inserted into or deleted from a newly
synthesized DNA strand. This type of mutation, known as a frameshift (table 8.8, rows d and e),
is so named because the reading frame of the mRNA has been changed. Frameshift mutations
nearly always result in a nonfunctional protein because most amino acids after the mutation are
different from what was coded for in the original DNA. Also note that insertion or deletion of
bases in multiples of three (3, 6, 9, etc.) results in the addition or deletion of amino acids but
does not disturb the reading frame.
One type of mutation recently found to be important in individual traits is called single
nucleotide polymorphism (SNP) because only a single nucleotide is altered. This is a result of a
point mutation at some point in the organism’s ancestry, and it is passed on genetically. Tens of
thousands of these differences at a single locus (when two different individuals are compared)
are known to exist throughout the genome.
The human genome contains 10 million SNPs. These variations are currently a hot area
of research and commerce. The ability to identify SNPs has proven critical to the new field of
personalized medicine, which is customized to a person’s genetic makeup. One example is when
patients’ genomes are examined for SNPs that have been found to be associated with a particular
disease, to determine their risk. For example, in a condition called thrombophilia (a blood-
clotting disorder), a point mutation in the gene for a clotting factor (factor V) causes an arginine
to become a glutamine (figure 8.15). This leads to increased clotting in the patient. Also, SNPs
can determine whether a patient will respond favorably to a particular treatment. The new field
of pharmacogenomics tailors drug treatments using this knowledge of SNPs.
DNA that has been damaged by ultraviolet radiation can be restored by photoactivation—
or light repair. This repair mechanism requires visible light and a light-sensitive enzyme, DNA
photolyase, an enzyme that can detect and attach to the damaged areas (sites of abnormal
pyrimidine binding). Ultraviolet repair mechanisms are successful only for a relatively small
number of UV mutations. Cells cannot repair severe, widespread damage and will die. Mutations
can be excised, or cut out, by a series of enzymes that remove the incorrect bases and add the
correct ones. This process is known as excision repair. First, enzymes break the bonds between
the bases and the sugar-phosphate strand at the site of the error. A different enzyme subsequently
removes the defective bases one at a time, leaving a gap that will be filled in by DNA
polymerase I and ligase. A repair system can also locate mismatched bases that were missed
during proofreading—for example, C mistakenly paired with A, or G with T.
Although most spontaneous mutations are not beneficial, a small number contribute to
the success of the individual and the population by creating variant strains with alternate ways of
expressing a trait. Microbes are not “aware” of this advantage and do not direct these changes;
they simply respond to the environment they encounter. A mutation is beneficial if it allows the
organism to more readily survive or reproduce. In the long-range view, mutations and the
variations they produce are the raw materials for change in the population and, thus, for
adaptation and evolution. Mutations that create variants occur frequently enough that any
population contains mutant strains for a number of characteristics, but as long as the environment
is stable, these mutants (for that particular trait) will never comprise more than a tiny percentage
of the population. When the environment changes, however, it can become hostile for the
survival of certain individuals, and only those microbes bearing protective mutations will be
equipped to survive in the new environment. In this way, the environment naturally “selects”
certain mutant strains that will reproduce, give rise to subsequent generations, and, in time, be
the dominant strain in the population. Through these means, any change that confers an
advantage due to this selection pressure will be retained by the population. One of the clearest
models for this sort of selection and adaptation is acquired drug resistance in bacteria.
Studying DNA in the Laboratory and Genetic Engineering
Knowing how DNA works within the cell to carry out the goals of a microbe allows
scientists to utilize these processes to accomplish goals more to the liking of human beings.
Since the 1970s, discoveries and advances have led to an explosion of new capabilities and, as a
result, an explosion of new knowledge about microbes and about biology in general. In this
section, we will highlight a few techniques that have relevance for microbiology and in particular
for infectious diseases.
Two very important techniques were developed in the 1970s and 1990s that made
massive advances in DNA study and manipulation possible. These are, first, the discovery of
restriction endonucleases, and, second, the invention of the polymerase chain reaction. The
groundbreaking discovery in 1971 of restriction endonucleases made almost everything we
discuss in this section possible. These enzymes come from bacterial cells. They recognize
foreign DNA and are capable of breaking the phosphodiester bonds between adjacent nucleotides
on both strands of DNA, leading to a break in the DNA strand. In the bacterial cell, this ability
protects against the incompatible DNA of bacteriophages or plasmids. In the biotechnologist’s
lab, the enzymes can be used to cleave DNA at desired sites and are necessary for the techniques
of recombinant DNA technology.
Thousands of restriction endonucleases have been discovered in bacteria. Each one has a
known sequence of 4 to 40 or more base pairs as its target, so sites of cutting can be finely
controlled. Many of these enzymes have the unique property of recognizing and clipping at base
sequences called palindromes (figure 8.16). Palindromes are sequences of DNA that are identical
when read from the 5′ to 3′ direction on one strand and the 5′ to 3′ direction on the other strand.
Endonucleases are usually named by combining the first letter of the bacterial genus, the
first two letters of the species, and the endonuclease number. For example, EcoRI is the first
endonuclease found in Escherichia coli, and HindIII is the third endonuclease discovered in
Haemophilus influenzae type d (figure 8.16). The pieces of DNA produced by restriction
endonucleases are termed restriction fragments. Because DNA sequences vary, even among
members of the same species, differences in the cutting pattern of specific restriction
endonucleases give rise to restriction fragments of differing lengths, known as restriction
fragment length polymorphisms (RFLPs).
RFLPs allow the direct comparison of the DNA of two different organisms at a specific
site. Another enzyme, called a ligase, is necessary to seal the sticky ends together by rejoining
the phosphate-sugar bonds cut by endonucleases. Its main application is in final splicing of genes
into plasmids and chromosomes. An enzyme called reverse transcriptase (RT) is best known for
its role in the replication of the AIDS virus and other retroviruses. It also provides geneticists
with a valuable tool for converting RNA into DNA. Copies called complementary DNA, or
cDNA, can be made from messenger, transfer, ribosomal, and other forms of RNA. The
technique provides a valuable means of synthesizing eukaryotic genes from mRNA transcripts.
The advantage is that the synthesized gene will be free of the intervening sequences (introns) that
can complicate the management of eukaryotic genes in genetic engineering.
Some of the techniques used to analyze DNA and RNA are limited by the small amounts
of test nucleic acid available. This problem was largely solved by the invention of a simple,
versatile way to amplify DNA called the polymerase chain reaction (PCR). This technique
rapidly increases the amount of DNA in a sample without the need for making cultures or
carrying out complex purification techniques. It is so sensitive that it holds the potential to detect
cancer from a single cell or to diagnose an infection from a single gene copy. It is comparable to
being able to pluck a single DNA “needle” out of a “haystack” of other molecules and make
unlimited copies of the DNA. The rapid rate of PCR makes it possible to replicate a target DNA
from a few copies to billions of copies in a few hours. To understand the idea behind PCR, it will
be helpful to review table 8.2, which describes synthesis of DNA as it occurs naturally in cells.
The PCR method uses essentially the same events, with the opening up of the double strand,
using the exposed strands as templates, the addition of primers, and the action of a DNA
polymerase.
Initiating the reaction requires a few specialized ingredients. As we saw earlier, the
primers are synthetic oligonucleotides (short DNA strands) of a known sequence of 15 to 30
bases that serve as landmarks to indicate where DNA amplification will begin. To keep the DNA
strands separated, processing must be carried out at a relatively high temperature. This
necessitates the use of special DNA polymerases isolated from thermophilic bacteria. The most
commonly used is Taq polymerase obtained from Thermus aquaticus. (Taq is an abbreviation of
the genus and species name of this microbe, from which the enzyme was isolated.) Enzymes
isolated from this thermophilic organism remain active at the elevated temperatures used in PCR.
Another vital component of PCR is a machine called a thermal cycler that automatically
performs the cyclic temperature changes.
There are many reasons scientists would want to know the characteristics of an
organism’s DNA. Knowing the entire genome sequence of an organism (such as a human) can
reveal genetic abnormalities, ancestry, and so forth. Sometimes the point is to determine if one
sample of DNA is the same as another sample. If you know the DNA patterns of a pathogen
causing an outbreak, for example, you can look at the DNA patterns of a microbe isolated from a
patient to see if they were made ill by that same pathogen. Also, you can look at the pattern of
human DNA left at a crime scene and match it against a suspect’s DNA. There are multiple ways
to analyze DNA, and we will highlight two of them: DNA profiling and DNA sequencing.
In modern sequencing, similar principles are used, but the whole process is scaled up in
what is called “high throughput” genome sequencing. High-throughput sequencing (also called
“deep sequencing” or “next generation sequencing”) requires four steps (figure 8.19): (1) A
library of DNA (or RNA) is prepared by fragmenting the DNA and fitting each fragment with
some common adaptors, or short sequences that are designed to match the probes in the next
step. (2) The collection of sequences is placed in a flow cell, whose surface is coated with the
probes that will bind with adaptor molecules on the DNA or RNA. Inside the flow cell, the
conditions and reagents are provided to use one or another form of PCR to amplify each
fragment many times. (3) Then all of the amplified fragments are sequenced so that each
nucleotide is “read” thousands of times, each time it is present on a sequence of any length. The
details of how this happens differ for each of the automated systems, but all rely on techniques
that originated in the Sanger method. (4) Finally, the millions of sequence lengths—also called
“reads”—are aligned using bioinformatics software. Putting all the reads together, the entire
sequence can be deduced.
The primary intent of recombinant DNA technology is to deliberately remove genetic
material from one organism and combine it with that of a different organism. Its origins can be
traced to 1970, when microbiologists first began to duplicate the clever tricks bacteria do
naturally with bits of extra DNA such as plasmids, transposons, and proviruses. As mentioned
earlier, humans have been trying to artificially influence genetic transmission of traits for
centuries. The discovery that bacteria can readily accept, replicate, and express foreign DNA
made them powerful agents for studying the genes of other organisms in isolation. The practical
applications of this work were soon realized by scientists. Bacteria could be genetically
engineered to mass-produce substances such as hormones, enzymes, and vaccines that were
difficult to synthesize by the usual industrial methods.
This process is called molecular cloning, or gene cloning. (Don’t confuse it with another
use of the word “clone,” namely, the cloning of whole organisms.) Gene cloning involves the
removal of a selected gene from an animal, plant, or microorganism (the genetic donor),
followed by its propagation in a different host organism. Cloning requires that the desired donor
gene first be selected, excised by restriction endonucleases, and isolated. The gene is next
inserted into a vector (usually a plasmid or a virus) that will insert the DNA into a cloning host.
The cloning host is usually a bacterium or a yeast that can replicate the gene and translate it into
the protein product for which it codes. In the next section, we examine the elements of gene
isolation, vectors, and cloning hosts and show how they participate in a complete recombinant
DNA procedure.
In recent years, researchers have staked out entirely new territory in genetic
manipulation: They are creating new biological molecules and organisms from scratch. This field
is called synthetic biology. A pioneer in the field is one of the same men who sequenced the
human genome, Craig Venter. In 2010, he successfully created a self-replicating bacterial cell
from four bottles of chemicals: the four nucleotides of DNA. This was a breakthrough of major
proportions, as it was the first time a living, replicating cell had been synthesized from
chemicals. Synthetic biology uses engineering-type methods to assemble molecules and cells.
Medical science is poised to be revolutionized when scientists can create precise chemicals to
replace those missing in disease, assemble customized immune components, or construct
biological molecules that can precisely target cancerous cells or pathogenic microbes. Synthetic
biology also holds promise for alternative energy production, for offering new and different
manufacturing processes, and for data storage. For example, researchers say that using DNA to
store information, they could fit the entire contents of the Internet in a shoebox. Of course, the
ability of scientists to “create” life, in a sense, makes many people nervous. The scientific
community, and those who monitor it, are engaged in intense conversations about the ethics—as
well as security issues—of synthetic biology.
Advances in genetic manipulation have allowed the development of several new
strategies to treating diseases such as cancer and other debilitating or life-threatening conditions.
Two of these are gene therapy and CRISPR. Gene therapy involves replacing a faulty gene that
is responsible for disease with a gene from a healthy organism. For example, a blood condition
called beta-thalassemia comes about when a gene for beta-globulin is defective. Patients require
monthly blood transfusions for survival. There are various strategies for this type of therapy. In
general, the normal gene is cloned in vectors such as retroviruses or adenoviruses that are
infectious but relatively harmless. In one technique, tissues can be removed from the patient and
incubated with these genetically modified viruses to transfect them with the normal gene. The
transfected cells are then reintroduced into the patient’s body by transfusion (figure 8.20).
Alternatively, naked DNA or a virus vector is directly introduced into the patient’s tissues. This
is the basis of a successful immunotherapy treatment for melanoma today
Experimentation with various types of gene therapy, or clinical testing, is performed on
human volunteers with the particular genetic condition. Thousands of these trials have been and
are being carried out in the United States and other countries. Most trials target cancer, single-
gene defects, and infections; and most gene deliveries are carried out by virus vectors. Early
therapeutic trials were hampered by several difficulties relating to effectiveness and safety. Some
of the safety issues were related to the use of (seemingly safe) viruses as delivery vehicles, which
then ended up causing malignancies. The strategies described so far are called somatic cell gene
therapy. This means that the changes are permanent in the individual who is treated, but they are
not passed on to offspring. The ultimate sort of gene therapy is germline therapy, in which genes
are inserted into an egg, sperm, or early embryo.
In this type of therapy, the new gene will be present in all cells of the individual. The
therapeutic gene is also heritable (that is, can be passed on to subsequent generations). There is
another system found in bacteria and archaea that can be exploited by scientists to alter genomes.
The system is called clustered regularly interspaced short palindromic repeats. You will see it in
the media as CRISPR. In bacteria and archaea, these are short lengths of DNA with repeating
nucleotides. After the repeats, short segments of spacer DNA are found. These turn out to be the
leftovers of DNA left behind by “invading” bacteriophages or plasmids. The CRISPR areas of
the genome are capable of recognizing and cutting this foreign DNA, keeping the bacterium or
archaea from being invaded. It is thought to be an adaptive immune system used by bacteria. In
other words, the bacteria “learn” the identity of an attacking phage by placing bits of its DNA in
its own genome, and in the future can cut it up before it causes trouble.
The system turns out to be highly adaptable for laboratory use, and scientists have started
using CRISPR in many genetic engineering applications. It is cheap, relatively easy to perform,
and very powerful. Researchers need only design a correct guide RNA that targets specific gene
sequences and mix it with nucleases associated with the CRISPR system, and they can cut DNA
in just about any organism exactly where they want to. It has already been used to make changes
in animals, and human trials began in 2019. Scientists hope to repair blood disorders and cancers
by removing malfunctioning cells and applying CRISPR to fix the DNA defects. They are also
injecting CRISPR molecules into the eyes of people with diseased retinas. The therapeutic and
research potential for this technology is huge. Some scientists warn that we don’t yet understand
the wide-reaching implications of changing DNA, especially in the germline, and that more
caution is needed. Another concern is that the CRISPR system can be used in a process called
gene drive. In this scenario, CRISPR is used to artificially cause an organism’s offspring to
accrue a particular mutation at a much accelerated rate. This might be a good thing, for instance,
if CRISPR can cause mosquitoes to no longer be susceptible to the malaria protozoan. The fear is
that this speeded-up evolution could have many unintended consequences.
Physical and Chemical Control of Microbes
Much of the time in the developed world, we take for granted tap water that is drinkable,
food that is not spoiled, shelves that are full of products to eradicate “germs,” and drugs that are
always available to treat infections. Controlling our degree of exposure to potentially harmful
microbes is a monumental concern in our lives. The ancient Greeks learned to burn corpses and
clothing during epidemics and the Egyptians embalmed the bodies of their dead, using strong
salts and pungent oils. These methods may seem rather archaic by modern measures, but these
examples illustrate that controlling microbes has been a concern for several centuries.
Actual comparative figures on the requirements for destroying various groups of
microorganisms are shown in table 9.2. Bacterial endospores have traditionally been considered
the most resistant microbial entities, being as much as 18 times harder to destroy than their
counterpart vegetative cells. Because of their resistance to microbial control, their destruction is
the goal of sterilization because any process that kills endospores will invariably kill all less-
resistant microbial forms. (Prions are a special case; see note in the margin.) Other methods of
control (disinfection, antisepsis) act primarily upon microbes that are less hardy than endospores.
The terms sterilization, disinfection, and so on refer to processes. You will encounter
other terms that describe the agents used in the process. Two examples of these are the terms
bactericidal and bacteristatic. The root -cide, meaning “having the capacity to kill,” can be
combined with other terms to define an antimicrobial agent aimed at destroying a certain group
of microorganisms. For example, a bactericide is a chemical that destroys bacteria except for
those in the endospore stage. It may or may not be effective on other microbial groups. A
fungicide is a chemical that can kill fungal spores, hyphae, and yeasts. A virucide is any
chemical known to inactivate viruses, especially on living tissue. A sporicide is an agent capable
of destroying bacterial endospores. Germicide and microbicide are additional terms for chemical
agents that kill microorganisms. Sepsis is defined as the growth of microorganisms in the blood
and other tissues. The term asepsis refers to any practice that prevents the entry of infectious
agents into sterile tissues and therefore prevents infection. Aseptic techniques commonly
practiced in health care range from sterile methods that exclude all microbes to antisepsis. In
antisepsis, chemical agents called antiseptics are applied directly to exposed body surfaces (skin
and mucous membranes), wounds, and surgical incisions to destroy or inhibit pathogens.
Examples of antisepsis include preparing the skin before surgical incisions with iodine
compounds, swabbing an open root canal with hydrogen peroxide, and ordinary hand washing
with a germicidal soap.
The Greek words stasis and static mean “to stand still.” They can be used in combination
with various prefixes to indicate a state in which microbes are temporarily prevented from
multiplying but are not killed outright. Although killing or permanently inactivating
microorganisms is the usual goal of microbial control, microbistasis does have meaningful
applications. Bacteristatic agents prevent the growth of bacteria on tissues or on objects in the
environment, and fungistatic chemicals inhibit fungal growth. Materials used to control
microorganisms in the body (antiseptics and drugs) often have microbistatic effects because
many microbicidal compounds can be highly toxic to human cells. Note that even a -cidal agent
doesn’t necessarily result in sterilization, depending on how it is used.
Numerous factors should be taken into consideration when selecting a workable method
of microbial control. One useful framework for determining how devices that come in contact
with patients should be handled is whether they are considered critical, semicritical, or
noncritical. Critical medical devices are those that are expected to come into contact with sterile
tissues. A good example of this would be a syringe needle or an artificial hip. These must be
sterilized before use. Semicritical devices are those that come into contact with mucosal
membranes. An endoscopy tube is an example. These must receive at least high-level
disinfection and preferably should be sterilized. Noncritical items are those that do not touch the
patient or are only expected to touch intact skin, such as blood pressure cuffs or crutches. They
require only low-level disinfection unless they become contaminated with blood or body fluids.
A remarkable variety of substances can require sterilization. They range from durable
solids such as rubber to sensitive liquids such as serum. Hundreds of situations requiring
sterilization confront the network of persons involved in health care, whether technician, nurse,
doctor, or manufacturer, and no universal method works well in every case. Considerations such
as cost, effectiveness, and method of disposal are all important. For example, disposable plastic
items such as catheters and syringes that are used in invasive medical procedures have the
potential for infecting the tissues. These must be sterilized during manufacture by a nonheating
method (gas or radiation) because heat can damage plastics. After these items have been used, it
is often necessary to destroy or decontaminate them before they are discarded because of the
potential risk to the handler. Steam sterilization (autoclaving), which is quick and sure, is a
sensible choice at this point because it does not matter if the plastic is destroyed. As we become
more aware of the plastic pollution problem, it remains to be seen how these practices will
change.
Death is a phenomenon that involves the permanent termination of an organism’s vital
processes. Signs of life in complex organisms such as animals are self-evident, and death is made
clear by loss of nervous function, respiration, or heartbeat. In contrast, death in microscopic
organisms that are composed of just one or a few cells is often hard to detect because they reveal
no conspicuous vital signs to begin with. The permanent loss of reproductive capability, even
under optimum growth conditions, has become the accepted microbiological definition of death.
Methods of Physical Control
We can divide our methods of controlling microorganisms into two broad categories:
physical and chemical. We’ll start with physical methods. Microorganisms have adapted to the
tremendous diversity of habitats the earth provides, even severe conditions of temperature,
moisture, pressure, and light. For microbes that normally withstand such extreme physical
conditions, our attempts at control would probably have little effect. Fortunately for us, we are
most interested in controlling microbes that flourish in the same environment in which humans
live. The vast majority of these microbes are readily controlled by abrupt changes in their
environment. Most prominent among antimicrobial physical agents is heat. Other agents include
radiation, filtration, ultrasonic waves, and even cold. The following sections examine some of
these methods and explore their practical applications in medicine, commerce, and the home.
As a rule, elevated temperatures (exceeding the maximum growth temperature) are
microbicidal, whereas lower temperatures (below the minimum growth temperature) are
microbistatic. We’ll start with heat. Heat can be applied in either moist or dry forms. Moist heat
occurs in the form of hot water, boiling water, or steam (vaporized water). In practice, the
temperature of moist heat usually ranges from 60°C to 135°C. As we shall see, the temperature
of steam can be regulated by adjusting its pressure in a closed container. Dry heat refers to hot
air (such as in an oven) or an open flame. In practice, the temperature of dry heat ranges from
160°C to several thousand degrees Celsius.
Vegetative cells also vary in their sensitivity to heat. Among bacteria, the death times
with moist heat range from 50°C for 3 minutes (Neisseria gonorrhoeae) to 60°C for 60 minutes
(Staphylococcus aureus). It is worth noting that vegetative cells of endospore formers are just as
susceptible as vegetative cells of non-endospore-formers, and that pathogens are neither more
nor less susceptible than nonpathogens. Other microbes, including fungi, protozoa, and worms,
are rather similar in their sensitivity to heat. Viruses are surprisingly resistant to heat, with a
tolerance range extending from 55°C for 2 to 5 minutes (adenoviruses) to 60°C for 600 minutes
(hepatitis A virus).
Many perishable substances are processed with moist heat. Some of these products are
intended to remain on the shelf at room temperature for several months or even years. The
chosen heat treatment must render the product free of agents of spoilage or disease. At the same
time, the quality of the product and the speed and cost of processing must be considered. For
example, in the commercial preparation of canned green beans, one of the manufacturer’s
greatest concerns is to prevent growth of botulism bacteria. From several possible TDTs (i.e.,
combinations of time and temperature) for Clostridium botulinum endospores, the factory must
choose one that kills all endospores but does not turn the beans to mush. Out of these many
considerations emerges an optimal TDT for a given processing method. Commercial canneries
heat low-acid foods at 121°C for 30 minutes, a treatment that sterilizes these foods. Because of
such strict controls in canneries, cases of botulism due to commercially canned foods are rare.
The principal benefit of cold treatment is to slow down the growth of cultures and
microbes in food during processing and storage. Remember that cold can only be counted on to
be microbistatic, not microbicidal. Although it is true that some microbes are killed by cold
temperatures, most are not adversely affected by gradual cooling, long-term refrigeration, or
deep-freezing. In fact, freezing temperatures, ranging from −70°C to −135°C, are often used in
research labs to preserve cultures of bacteria, viruses, and fungi for long periods. Some
psychrophiles grow very slowly even at freezing temperatures and can continue to secrete toxic
products. Ignorance of these facts is probably responsible for numerous cases of food poisoning
from frozen foods that have been defrosted at room temperature and then inadequately cooked.
Pathogens able to survive several months in the refrigerator are Staphylococcus aureus,
Clostridium species (endospore formers), Streptococcus species, and several types of yeasts,
molds, and viruses. Outbreaks of Salmonella food infection traced back to refrigerated foods
such as ice cream, eggs, and tiramisu are testimony to the inability of cold temperatures to
reliably kill pathogens.
Vegetative cells directly exposed to normal room air gradually become dehydrated, or
desiccated. Delicate pathogens such as Streptococcus pneumoniae, the spirochete of syphilis, and
Neisseria gonorrhoeae can die after a few hours of air drying, but many others are not killed and
some are even preserved. Endospores of Bacillus and Clostridium are viable for thousands of
years under extremely dry conditions. Staphylococci and streptococci in dried secretions and the
microbe that causes tuberculosis surrounded by sputum can remain viable in air and dust for
lengthy periods. Many viruses (especially nonenveloped) and fungal spores can also withstand
long periods of desiccation. Desiccation can be a valuable way to preserve foods because it
greatly reduces the amount of water available to support microbial growth. It is interesting to
note that a combination of freezing and drying—called lyophilization (ly-off″- il-ih-za′-shun)—is
a common method of preserving microorganisms and other cells in a viable state for many years.
Pure cultures are frozen instantaneously and exposed to a vacuum that rapidly removes the water
(it goes straight from the frozen state into the vapor state, skipping the liquid state altogether).
This method avoids the formation of ice crystals that would damage the cells. Although not all
cells survive this process, enough of them do to permit future reconstitution of that culture. As a
general rule, chilling, freezing, and desiccation should not be construed as methods of
disinfection or sterilization because their antimicrobial effects are erratic and uncertain, and one
cannot be sure that pathogens subjected to them have been killed
Energy, in the form of radiation, is a useful source of antimicrobial activity. Radiation is
defined as energy emitted from atomic activities and dispersed at high velocity through matter or
space. Figure 9.5 illustrates the different wavelengths of radiation. In our discussion, we consider
only those types suitable for microbial control: gamma rays, X rays, and ultraviolet radiation.
These are the forms with shorter wavelengths than those of visible light. There are several
especially useful practices that take advantage of these forms of radiation (table 9.7). Hospitals
have begun using specialized UV-light machines to clean hospital rooms after patients have been
released and the rooms have been cleaned with traditional methods (figure 9.6). The UV
apparatus must be moved to more than one position so that the radiation reaches each area of the
room, but it can disinfect areas that are not usually reached by hand cleaning methods
Most modern microbiological filters are thin membranes of cellulose acetate,
polycarbonate, and a variety of plastic materials (Teflon, nylon) whose pore size can be carefully
controlled and standardized. Ordinary substances such as charcoal, diatomaceous earth, or
unglazed porcelain are also used in some applications. Viewed microscopically, most filters are
perforated by very precise, uniform pores (figure 9.8b). The pore diameters vary from coarse (8
μm) to ultrafine (0.02 μm), permitting selection of the minimum particle size to be trapped.
Those with even smaller pore diameters permit true sterilization by removing viruses, and some
will even remove large proteins. A sterile liquid filtrate is typically produced by suctioning the
liquid through a sterile filter into a presterilized container. These filters are also used to separate
mixtures of microorganisms and to count bacteria in water analysis. Filtration is used to prepare
liquids that cannot withstand heat, including serum and other blood products, vaccines, drugs, IV
fluids, enzymes, and media. Filtration has been employed as an alternative method for
decontaminating milk and beer without altering their flavor. It is also an important step in water
purification. Its use extends to filtering out particulate impurities (crystals, fibers, and so on) that
can cause severe reactions in the body. Filtration is also an efficient means of removing airborne
contaminants that are a common source of infection and spoilage. High-efficiency particulate air
(HEPA) filters are widely used to provide a flow of decontaminated air to hospital rooms and
sterile rooms.
Methods of Chemical Control
Antimicrobial chemicals occur in the liquid, gaseous, or even solid state, and they range
from disinfectants and antiseptics to sterilants and preservatives (chemicals that inhibit the
deterioration of substances). For the sake of convenience (and sometimes safety), many solid or
gaseous antimicrobial chemicals are dissolved in water, alcohol, or a mixture of the two to
produce a liquid solution. Solutions containing pure water as the solvent are termed aqueous,
whereas those dissolved in pure alcohol or alcohol-water mixtures are termed tinctures.
Germicides are evaluated in terms of their effectiveness in destroying microbes in
medical and dental settings. The three levels of chemical decontamination procedures are high,
intermediate, and low. High-level germicides kill endospores and, if properly used, are sterilants.
Materials that necessitate high-level control are medical devices—for example, catheters, heart-
lung equipment, and implants—that are not heat-sterilizable and are intended to enter body
tissues during medical procedures. Intermediate-level germicides kill fungal (but not bacterial)
spores, resistant pathogens such as the bacterium that causes tuberculosis, and viruses. They are
used to disinfect items (respiratory equipment, thermometers) that come into intimate contact
with the mucous membranes but are noninvasive. Low levels of disinfection eliminate only
vegetative bacteria, vegetative fungal cells, and some viruses. They are used to clean materials
such as electrodes, straps, and pieces of furniture that touch the skin surfaces but not the mucous
membranes.
