Microbial Genetics
Chapter 8:
Microbial Genetics
*
Plasmids Exist in Cells Separate from Chromosomes
Big Picture: Genetics
The science of heredity
Central dogma of molecular biology
Mutations
Gene expression controlled by operons
- Alteration of bacterial genes and/or gene expression
Cause of disease
Prevent disease treatment
Manipulated for human benefit
Big Picture: Genetics
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.
Structure and Function of the Genetic Material
Genetics: the study of genes, how they carry information, how information is expressed, and how genes are replicated
Chromosomes: structures containing DNA that physically carry hereditary information; the chromosomes contain genes
Genes: segments of DNA that encode functional products, usually proteins
Genome: all the genetic information in a cell
Structure and Function of the Genetic Material
- The genetic code is a set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein
- Central dogma:
Genotype and Phenotype
Genotype: the genetic makeup of an organism
Phenotype: expression of the genes
DNA and Chromosomes
Bacteria usually have a single circular chromosome made of DNA and associated proteins
Short tandem repeats (STRs): repeating sequences of noncoding DNA
Figure 8.1 A Prokaryotic Chromosome
Chromosome
The Flow of Genetic Information
Vertical gene transfer: flow of genetic information from one generation to the next
Horizontal gene transfer: flow of genetic information between individuals of the SAME generation (see the middle portion of the next slide!)
Figure 8.2 The Flow of Genetic Information
Parent cell
DNA
Genetic information is used
within a cell to produce the
proteins needed for the cell
to function.
Genetic information can be
transferred horizontally between
cells of the same generation.
Genetic information can be
transferred vertically to the
next generation of cells.
New combinations
of genes
Translation
Cell metabolizes and grows
Recombinant cell
Offspring cells
Transcription
DNA Replication
DNA forms a double helix
“Backbone” consists of deoxyribose-phosphate
Two strands of nucleotides are held together by hydrogen bonds between A-T and C-G
Strands are antiparallel
Order of the nitrogen-containing bases forms the genetic instructions of the organism
DNA Replication
One strand serves as a template for the production of a second strand
Topoisomerase and gyrase relax the strands
Helicase separates the strands
A replication fork is created
DNA Replication
DNA polymerase adds nucleotides to the growing DNA strand
In the 5‘ 3' direction
Initiated by an RNA primer
Leading strand is synthesized continuously
Lagging strand is synthesized discontinuously, creating Okazaki fragments
DNA polymerase removes RNA primers; Okazaki fragments are joined by the DNA polymerase and DNA ligase
Figure 8.5 A Summary of Events at the DNA Replication Fork
REPLICATION
Proteins stabilize the
unwound parental DNA.
The leading strand is
synthesized continuously
by DNA polymerase.
DNA polymerase
Enzymes unwind the
parental double
helix.
Primase
Parental
strand
The lagging strand is
synthesized discontinuously.
Primase, an RNA polymerase,
synthesizes a short RNA primer,
which is then extended by
DNA polymerase.
DNA polymerase
digests RNA primer
and replaces it with DNA.
DNA ligase joins
the discontinuous
fragments of the
lagging strand.
DNA
polymerase
DNA polymerase
Okazaki fragment
DNA ligase
RNA primer
Replication
fork
3'
5'
5'
3'
3'
5'
DNA Replication
Energy Needs
Energy for replication is supplied by nucleotides (remember, ATP is one example of a nucleotide!)
Hydrolysis of two phosphate groups on ATP provides energy
Figure 8.4 Adding a Nucleotide to DNA
New
Strand
Template
Strand
Sugar
Phosphate
When a nucleoside triphosphate bonds to the sugar, it loses
two phosphates.
Hydrolysis of the phosphate bonds
provides the energy for the reaction.
DNA Replication
Most bacterial DNA replication is bidirectional
Each offspring cell receives one copy of the DNA molecule
Replication is highly accurate due to the proofreading capability of DNA polymerase
Figure 8.6 Replication of Bacterial DNA
Check Your Understanding
Check Your Understanding
8-3 Describe DNA replication, including the functions of DNA gyrase, DNA ligase, and DNA polymerase.
RNA and Protein Synthesis
Ribonucleic acid
Single-stranded nucleotide
5-carbon ribose sugar
Contains uracil (U) instead of thymine (T)
Ribosomal RNA (rRNA): integral part of ribosomes
Transfer RNA (tRNA): transports amino acids during protein synthesis
Messenger RNA (mRNA): carries coded information from DNA to ribosomes
Transcription in Prokaryotes
Synthesis of a complementary mRNA strand from a DNA template
Transcription begins when RNA polymerase binds to the promoter sequence on DNA
Transcription proceeds in the 5‘ 3' direction; only one of the two DNA strands is transcribed
Transcription stops when it reaches the terminator sequence on DNA
Figure 8.7 The Process of Transcription
TRANSCRIPTION
DNA
mRNA
Protein
RNA
polymerase
DNA
RNA polymerase bound to DNA
RNA polymerase
RNA nucleotides
Template strand of DNA
RNA
Promoter
(gene begins)
RNA polymerase
RNA
RNA synthesis
Terminator
(gene ends)
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.
Complete
RNA strand
RNA and RNA
polymerase are
released, and the
DNA helix re-forms.
Promoter
Translation
mRNA is translated into the “language” of proteins
Codons are groups of three mRNA nucleotides that code for a particular amino acid (20 potential amino acids)
Each amino acid is coded by several codons… but each codon will code for just one amino acid (the chart on next slide shows you this better):
Translation
Translation of mRNA begins at the start codon: AUG
Translation ends at nonsense codons: UAA, UAG, UGA
Codons of mRNA are “read” sequentially
tRNA molecules transport the required amino acids to the ribosome
tRNA molecules also have an anticodon that base-pairs with the codon
Amino acids are joined by peptide bonds
Figure 8.9 The Process of Translation
Ribosome
P Site
Start
codon
Second
codon
mRNA
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.
Components needed to begin
translation come together.
mRNA
Anticodon
Ribosomal
subunit
Ribosomal
subunit
tRNA
Figure 8.9 The Process of Translation
Peptide bond forms
A site
mRNA
E site
mRNA
Ribosome moves
along 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.
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.
Figure 8.9 The Process of Translation
tRNA released
mRNA
The second amino acid joins to the third by another
peptide bond, and the first tRNA is released from the E
site.
The ribosome continues to move along the mRNA,
and new amino acids are added to the polypeptide.
mRNA
Growing
polypeptide
chain
Figure 8.9 The Process of Translation
mRNA
Polypeptide
released
Stop codon
When the ribosome reaches a stop
codon, the polypeptide is released.
Finally, the last tRNA is released, and the ribosome
comes apart. The released polypeptide forms a new
protein.
mRNA
New protein
Figure 8.10 Simultaneous Transcription and Translation in Bacteria
TRANSLATION
DNA
mRNA
Protein
DNA
RNA
polymerase
Direction of transcription
Peptide
Polyribosome
Ribosome
mRNA
Direction of translation
5'
In bacteria, translation can begin before transcription is complete
Transcription in Eukaryotes
In eukaryotes, transcription occurs in the nucleus, whereas translation occurs in the cytoplasm
Exons are regions of DNA that code for proteins
Introns are regions of DNA that do not code for proteins
Small nuclear ribonucleoproteins (snRNPs) remove introns and splice exons together
Check Your Understanding
8-5 How does mRNA production in eukaryotes differ from the process in prokaryotes?
Figure 8.11 RNA Processing in Eukaryotic Cells
The Regulation of Bacterial Gene Expression
Constitutive genes are expressed at a fixed rate
Other genes are expressed only as needed
Inducible genes
Repressible genes
Catabolite repression
Pre-Transcriptional Control
Repression inhibits gene expression and decreases enzyme synthesis
Mediated by repressors, proteins that block transcription
Default position of a repressible gene is on
Induction turns on gene expression
Initiated by an inducer
Default position of an inducible gene is off
The Operon Model of Gene Expression
Promoter: segment of DNA where RNA polymerase initiates transcription of structural genes
Operator: segment of DNA that controls transcription of structural genes
Operon: set of operator and promoter sites and the structural genes they control
The Operon Model of Gene Expression
In an inducible operon, structural genes are not transcribed unless an inducer is present
In the absence of lactose, the repressor binds to the operator, preventing transcription
In the presence of lactose, metabolite of lactose–allolactose (inducer)–binds to the repressor; the repressor cannot bind to the operator and transcription occurs
Figure 8.12 An Inducible Operon
Control region
Structural genes
Operon
I
P
O
Z
Y
A
DNA
Regulatory
gene
Promoter
Operator
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)
Figure 8.12 An Inducible Operon
RNA polymerase
I
P
Z
Y
A
Transcription
Translation
Repressor
mRNA
Active
repressor
protein
Repressor active, operon off. The repressor protein binds with the
operator, preventing transcription from the operon.
Figure 8.12 An Inducible Operon (3 of 3)
Allolactose
(inducer)
I
P
O
Z
Y
A
Transcription
Translation
Transacetylase
Permease
β-Galactosidase
Inactive
repressor
protein
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.
Operon
mRNA
The Operon Model of Gene Expression
In repressible operons, structural genes are transcribed until they are turned off
Excess tryptophan is a corepressor that binds and activates the repressor to bind to the operator, stopping tryptophan synthesis
Figure 8.13 A Repressible Operon
Control region
Structural genes
Operon
I
P
O
E
C
A
DNA
Regulatory
gene
Promoter
Operator
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)
D
B
Figure 8.13 A Repressible Operon (2 of 3)
RNA polymerase
I
P
O
E
D
C
B
A
Transcription
Repressor
mRNA
Translation
Inactive
repressor
protein
Polypeptides
comprising the
enzymes for
tryptophan
synthesis
Operon
mRNA
Repressor inactive, operon on. The repressor is inactive, and
transcription and translation proceed, leading to the synthesis
of tryptophan.
Figure 8.13 A Repressible Operon (3 of 3)
I
P
E
D
C
B
A
Active
repressor
protein
Tryptophan
(corepressor)
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.
Positive Regulation
Catabolite repression inhibits cells from using carbon sources other than glucose
Cyclic AMP (cAMP) builds up in a cell when glucose is not available
cAMP binds to the catabolic activator protein (CAP) that in turn binds the lac promoter, initiating transcription and allowing the cell to use lactose
Figure 8.14 The Growth Rate of E. Coli on Glucose and Lactose
Bacteria growing on
glucose as the sole carbon
source grow faster than on
lactose.
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, more lactose is transported into the cell, and β-galacto-sidase is synthesized to break down lactose.
Glucose
Lactose
All glucose
consumed
Glucose
used
Lag
time
Lactose used
Figure 8.15 Positive Regulation of the Lac Operon
Promoter
lacZ
lacl
DNA
Operator
RNA
polymerase
can bind
and transcribe
cAMP
Inactive
CAP
Active
CAP
Inactive lac
repressor
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.
CAP-binding site
CAP-binding site
DNA
lacl
Promoter
lacZ
Operator
RNA
polymerase
can't bind
Inactive
CAP
Inactive lac
repressor
Lactose present, glucose present (cAMP level low). When glucose is
present, cAMP is scarce, and CAP is unable to stimulate transcription.
Epigenetic Control
Methylating nucleotides turn genes off
Methylated (off) genes can be passed to offspring cells
Not permanent
Changes in Genetic Material
Mutation: a permanent change in the base sequence of DNA
Mutations may be neutral, beneficial, or harmful
Mutagens: agents that cause mutations
Spontaneous mutations: occur in the absence of a mutagen
Types of Mutations
Base substitution (point mutation)
Change in one base in DNA
Missense mutation
Base substitution results in change in an amino acid
Nonsense mutation
Base substitution results in a nonsense (stop) codon
Frameshift mutation
Insertion or deletion of one or more nucleotide pairs
Shifts the translational “reading frame”
Chemical Mutagens & radiation
Nitrous acid: causes adenine to bind with cytosine instead of thymine
Nucleoside analog: incorporates into DNA in place of a normal base; causes mistakes in base pairing
Glyphosate / RoundUp has also been seen to be directly mutagenic (again, see paper in supplemental folder)
Ionizing radiation (X-rays and gamma rays) causes the formation of ions that can oxidize nucleotides and break the deoxyribose-phosphate backbone
UV radiation causes thymine dimers
Repair of mutations can happen:
Photolyases separate thymine dimers
Nucleotide excision repair: Enzymes cut out incorrect bases and fill in correct bases
Ultraviolet light
Exposure to ultraviolet light
causes adjacent thymines to
become cross-linked, forming
a thymine dimer and disrupting
their normal base pairing.
Thymine dimer
An endonuclease cuts the
DNA, and an exonuclease
removes the damaged DNA.
New DNA
DNA polymerase fills the gap
by synthesizing new DNA,
using the intact strand as
a template.
DNA ligase seals the
remaining gap by joining the
old and new DNA.
The Frequency of Mutation
Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes
Mutagens increase the mutation rate to per 10-5 or 10-3 replicated gene
Identifying Mutants
Positive (direct) selection detects mutant cells because they grow or appear different than unmutated cells
Negative (indirect) selection detects mutant cells that cannot grow or perform a certain function
Auxtotroph: mutant that has a nutritional requirement absent in the parent
Identifying Chemical Carcinogens
The Ames test exposes mutant bacteria to mutagenic substances to measure the rate of reversal of the mutation
Indicates degree to which a substance is mutagenic
IMPORTANT: If the Ames test suggests no mutagenicity, this is NOT a “for-sure” negative– sometimes chemicals react with a human protein to yield a carcinogen !
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 Transfer and Recombination
Genetic recombination: exchange of genes between two DNA molecules…in particular, between 2 same-aged individuals instead of from parent to offspring; creates genetic diversity especially among microbes (which do not normally sexually reproduce…sexual reproduction / meiosis and fertilization is the norm for most multicellular creatures, but unicellular organisms cannot do that)
Vertical gene transfer: transfer of genes from an organism to its offspring
Horizontal gene transfer: transfer of genes between cells of the same generation
Plasmids and Transposons
Transposons = Mobile genetic elements
Move from one chromosome to another or from one cell to another
Occur in prokaryotic and eukaryotic organisms
Plasmids are self-replicating circular pieces of DNA
1 to 5% the size of a bacterial chromosome
Often code for proteins that enhance the pathogenicity of a bacterium
Plasmids
Conjugative plasmid: carries genes for sex pili and transfer of the plasmid
Dissimilation plasmids: encode enzymes for the catabolism of unusual compounds
Resistance factors (R factors): encode antibiotic resistance
Transposons
Transposons are segments of DNA that can move from one region of DNA to another
Contain insertion sequences (IS) that code for transposase that cuts and reseals DNA
Complex transposons carry other genes (e.g., in antibiotic resistance)
Transformation in Bacteria
Transformation: genes transferred from one bacterium to another as “naked” DNA
Figure 8.28 The Mechanism of Genetic Transformation in Bacteria
a
b
c
d
DNA fragments
from donor cells
Recipient cell
A
D
B
C
Chromosomal DNA
Recipient cell takes
up donor DNA.
Donor DNA aligns
with complementary
bases.
Recombination occurs
between donor DNA
and recipient DNA.
A
D
B
C
A
D
B
C
Degraded
unrecombined DNA
Genetically transformed cell
a
b
c
d
b
c
d
B
C
D
a
5'
3'
5'
3'
Conjugation in Bacteria
Conjugation: plasmids transferred from one bacterium to another
Requires cell-to-cell contact via sex pili
Figure 8.30a Conjugation in E. coli
RECOMBINATION
Bacterial
chromosome
Mating bridge
Replication
and transfer
of F factor
F factor
F+ cell
F– cell
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.
F+ cell
F+ cell
Transduction in Bacteria
DNA is transferred from a donor cell to a recipient via a bacteriophage
Generalized transduction: Random bacterial DNA is packaged inside a phage (virus that infects bacteria) and transferred to a recipient cell
Specialized transduction: Specific bacterial genes are packaged inside a phage and transferred to a recipient cell
Figure 8.32 Transduction by a Bacteriophage
RECOMBINATION
Phage protein coat
Phage DNA
Bacterial
chromosome
A phage infects the
donor bacterial cell.
Phage DNA and proteins are made,
and the bacterial chromosome is
broken into pieces.
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
DNA
Bacterial
DNA
A phage carrying
bacterial DNA infects
a new host cell, the
recipient cell.
Recipient
cell
Donor
bacterial
DNA
Recipient
bacterial
DNA
Recombinant
cell reproduces
normally
Recombination can
occur, producing a
recombinant cell with
a genotype different
from both the donor
and recipient cells.
Many cell
divisions
Donor
cell
Genes and Evolution
Mutations and recombination create cell diversity
Diversity is the raw material for evolution
Natural selection acts on populations of organisms to ensure the survival of organisms fit for a particular environment
Check Your Understanding
Check Your Understanding
8-17 Natural selection means that the environment favors survival of some genotypes. From where does diversity in genotypes come?