CHAPTER 11
MICROBIAL GENETICS AND INFECTIOUS
DISEASE
© Dennis Kunkel
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
WHY IS THIS IMPORTANT?

Understanding genetic mechanisms lets us
study how microorganisms can mutate and
change in ways that allow them to defeat host
defenses.


These changes are one of the most important topics
in health care today.
To understand pathogenesis and virulence, we
must be familiar with microbial genetics.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
WHY IS THIS IMPORTANT?

Organisms become resistant through mutations

Mutations can be transferred to other bacteria.

Transfer of these mutations can make a harmless
bacterium dangerous and a dangerous bacterium
lethal.

One of the most difficult problems in medicine
today is antibiotic resistance.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
OVERVIEW
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA

DNA stands for deoxyribonucleic acid.

DNA is a blueprint for all components of the
cell.

The blueprint can be faithfully passed on from one
generation to the next.

The structure of DNA allows replication and
transcription to be a simple process.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA STRUCTURE

DNA is a double stranded helical structure.

It is composed of nucleotides.

Each nucleotide is a phosphate, a sugar
(deoxyribose), and a nucleotide base.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA STRUCTURE

The two strands are complementary and wind
around each other to form the double helix.

The components of DNA bind together in a
very specific way.


The bases project inward.
This permits a correct and precise orientation of
the nucleotide.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA STRUCTURE
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA STRUCTURE
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA STRUCTURE
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA STRUCTURE

Nucleotides join to each other to form a chain.

The 3’ hydroxyl group of a sugar joins to the 5’
hydroxyl of another nucleotide.

This makes the linkage inherently polarized

And gives structural orientation to the growing
chain.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA STRUCTURE
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA STRUCTURE

DNA has two types of base

Purines – adenine and guanine

Pyrimidines – thymine and cytosine


Purines are large double-ring structures.
Pyrimidines have smaller single ring structures.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA STRUCTURE
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA STRUCTURE

DNA has a helical geometry governed by how
the bases pair up.

Adenine always pairs with thymine.

Cytosine always pairs with guanine.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA STRUCTURE


The strands are anti-parallel.

One of the strands is oriented upside down relative
to the other.

The bases are stacked on top of each other.
DNA is a chemically stable molecule.

Any mismatched pairing is chemically unstable.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
RNA

RNA stands for ribose nucleic acid.

RNA differs from DNA in several ways.

It contains the sugar ribose (rather than
deoxyribose).

It contains uracil instead of thymine.


Uracil pairs up with adenine.
It is usually found in single-stranded form.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
RNA

There are three forms of RNA:



Messenger RNA – contains information derived
from DNA
Transfer RNA – carries amino acids to ribosomes
Ribosomal RNA – helps maintain the proper shape
of ribosomes.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA REPLICATION

This is the process by which DNA is copied.

It is carefully controlled and regulated.

It involves specific components and mechanisms.

It is remarkably accurate and amazingly fast.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA REPLICATION

DNA replication involves specific components
and mechanisms.

It is a critical cellular procedure accomplished
with remarkable accuracy and at astounding
speed.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA REPLICATION:
Separation and Supercoiling

Supercoiling is a characteristic of helical
structures.

Strands must be uncoiled, unwound, and
separated before replication.

This is accomplished by two enzymes:

Topoisomerase – unwinds the supercoils

Helicase – separates and unwinds the strands.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA REPLICATION:
Requirements

There are two requirements for replication:

An ample supply of each of the nucleotides
adenine, thymine, cytosine, and guanine

A primer:template junction

Each single strand of DNA is a template.

A portion of the DNA is paired with a short
piece of RNA called a primer.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA REPLICATION:
Requirements
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA REPLICATION:
Direction

DNA replication proceeds in only one
direction.

The primer:template junction gives the DNA
polymerase a place to add the next base

Binding is between the 3’end of one base and the
5’ end of the next base.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA REPLICATION:
Direction

Elongation of the bases is from the 3’ end


This is required for chemical stability.
The binding of a new base uses energy
released from pyrophosphate.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA REPLICATION:
Direction
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA POLYMERASE

DNA replication is performed by an enzyme
called DNA polymerase.

DNA polymerase forms new strands of DNA
using the primer:template junction as a guide.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA POLYMERASE

It works incredibly quickly.


The addition of nucleotides is in the millisecond
range.
There are several types of DNA polymerase.

They perform specific functions and work at
different speeds.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA POLYMERASE:
Proofreading

DNA replication is extraordinarily accurate.

There are always some mistakes – mutations


Evolution relies on mutations.
During replication, an error occurs approximately
once in 1010 pairings.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
DNA POLYMERASE:
Proofreading

Proofreading takes place at the
primer:template junction active site.

Improperly paired bases are removed by an
exonuclease.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
DNA POLYMERASE:
Proofreading
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
THE REPLICATION FORK

In the replication fork, the double helix is
unwound and the strands separate.

DNA replication occurs at the replication fork.

The separated strands at the replication fork
are anti-parallel and are identified as:

Leading strand

Lagging strand
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
THE REPLICATION FORK
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
THE REPLICATION FORK

The leading strand is in the correct orientation
for bases added to the 3’ end of the
primer:template junction.

Replication moves towards the replication fork.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
THE REPLICATION FORK

The lagging strand is anti-parallel.



It moves away from the replication fork.
Bases are only added to the 3’ end of the
primer:template junction.
The lagging strand is replicated in pieces called
Okazaki fragments.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
THE REPLICATION FORK

Each Okazaki fragment has its own short RNA
primer.


It is created by an RNA polymerase called
primase.
When the fragment is finished, the enzyme
RNAase H removes the primer.

The gap is filled in by DNA polymerase.

Fragments are linked together by DNA ligase.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
INITIATION AND TERMINATION
OF REPLICATION

Initiation begins at a specific site on the
chromosome.

The origin of replication.

Termination occurs when the entire
chromosome has been copied.

Replicated chromosomes are separated by
topoisomerase.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
INITIATION AND TERMINATION
OF REPLICATION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
THE GENETIC CODE

Information in DNA is based on a four letter
alphabet (A, T, C, G).

The genetic code employs three letter
combinations called codons.
Microbiology: A Clinical Approach
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byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
THE GENETIC CODE

There are 64 possible 3 letter combinations


Only 20 amino acids are used to make
proteins.
The genetic code is degenerate.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
THE GENETIC CODE
Microbiology: A Clinical Approach
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byGarland
Tony Srelkauskas
Science © Garland Science
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THE GENETIC CODE

Three rules govern the arrangement and use of
codons:

Codons are always read in one direction.

The message is translated in a fixed reading frame.

There is no overlap or gap in the code.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
GENE EXPRESSION

A gene is a segment of DNA that codes for a
functional product.

Gene expression is the production of the
functional product.

Gene expression has two features:

It involves specific interactions between DNA and
RNA.

It is highly regulated.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
GENE EXPRESSION

There are two parts to gene expression:


Transcription – construction of RNA from a DNA
template
Translation – construction of the protein using
RNA instructions.
Microbiology: A Clinical Approach
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byGarland
Tony Srelkauskas
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TRANSCRIPTION

The process by which RNA is made from a
DNA template.

It does not require a primer:template junction.

RNA does not remain base-paired to DNA.

It is not as accurate as DNA synthesis.

RNA polymerase has no proofreading capability.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRANSCRIPTION

Transcription has three steps:

Initiation – a DNA sequence called the promoter
initially binds the RNA polymerase:




This produces a bubble in the DNA.
Elongation – RNA polymerase unwinds strands of
DNA and synthesizes the RNA:
It also re-anneals the strands.
Termination – a sequence of DNA signals the end of
transcription:

RNA polymerase detaches from DNA
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSCRIPTION
Microbiology: A Clinical Approach
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byGarland
Tony Srelkauskas
Science © Garland Science
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TRANSLATION

This is the process by which proteins are made

The sequence of nucleotides in messenger RNA is
translated into a sequence of amino acids.

It is directly affected by any errors in either DNA
or RNA.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRANSLATION

It is a highly conserved function seen in all
cells.

It requires high levels of energy.

Translation requires all three types of RNA –
messenger, transfer, and ribosomal.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
MESSENGER RNA (mRNA) IN
TRANSLATION

An open reading frame (ORF) indicates the
start of an amino acid sequence.

An ORF begins with a start codon.

Translation moves from the 5’end to the 3’ end.

An ORF ends with a stop codon.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
mRNA IN TRANSLATION

mRNA contains a segment that recruits the
ribosomal subunits.

Ribosome and mRNA bind here through
complementary base pairing.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRANSFER RNA (tRNA) IN
TRANSLATION

Each tRNA attaches to a specific amino acid at
the acceptor arm.

It brings amino acids to the ribosome.

It binds to the ribosome at the anti-codon
region using complementary base pairing.
Microbiology: A Clinical Approach
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Tony Srelkauskas
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tRNA IN TRANSLATION
Microbiology: A Clinical Approach
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Tony Srelkauskas
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THE RIBOSOME IN
TRANSLATION

The ribosome is composed of three molecules
of rRNA and over 50 proteins.

It adds amino acids at a rate of 2-20 amino
acids per second.

More than one ribosome can move along the
same messenger RNA

This is called a polyribosome or polysome.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
THE RIBOSOME IN
TRANSLATION
Microbiology: A Clinical Approach
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Tony Srelkauskas
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FORMATION OF PEPTIDE
BONDS IN TRANSLATION

Peptide bonds form between amino acids while
on the ribosome.

The ribosome has 3 sites:

A site – tRNA brings in new amino acid

P site – tRNA holds the growing amino acid chain

E site – tRNA exits the ribosome.
Microbiology: A Clinical Approach
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FORMATION OF PEPTIDE
BONDS IN TRANSLATION
Microbiology: A Clinical Approach
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Tony Srelkauskas
Science © Garland Science
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Microbiology: A Clinical Approach [9780815365143] © Garland Science
FORMATION OF PEPTIDE
BONDS IN TRANSLATION

The ribosome is a honeycombed structure with
tunnels.

The components of protein synthesis enter
these tunnels and move through them.

mRNA

tRNA

Growing polypeptide chain
Microbiology: A Clinical Approach
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Tony Srelkauskas
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STAGES OF TRANSLATION

There are three stages of transcription:

Initiation

Elongation

Termination
Microbiology: A Clinical Approach
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Tony Srelkauskas
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INITIATION

Initiation requires:

Recruitment of the ribosome to the mRNA

Placement of a methionine tRNA complex at the P
site

Precise positioning of the ribosome over the start
codon of mRNA.
Microbiology: A Clinical Approach
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Tony Srelkauskas
Science © Garland Science
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Microbiology: A Clinical Approach [9780815365143] © Garland Science
INITIATION
Microbiology: A Clinical Approach
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ELONGATION

After initiation, three things must occur in order
for amino acids to be added to methionine.

A tRNA carrying the next amino acid is loaded into the
A site.

A peptide bond forms between the amino acids.


Each tRNA moves – the one at the A site to the P site,
the one at the P site to the E site.
The ribosome moves along the messenger RNA.
Microbiology: A Clinical Approach
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Tony Srelkauskas
Science © Garland Science
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TERMINATION


Translation continues until a stop codon enters
the A site.

Stop codons are recognized by specialized
proteins.

These specialized proteins cause the translation
complex to fall apart.
The peptide chain is released from the
ribosome and begins to form secondary and
tertiary structures.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
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Microbiology: A Clinical Approach [9780815365143] © Garland Science
REGULATION OF GENE
EXPRESSION

Protein synthesis is energetically expensive
and highly regulated.



Some genes are always turned on – constitutive
genes.
Some genes are on and can be turned off –
repressible genes.
Some genes are off and can be turned on –
inducible genes.
Microbiology: A Clinical Approach
Approach, ©
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Tony Srelkauskas
Science © Garland Science
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REGULATION OF GENE
EXPRESSION

Gene expression is controlled by regulatory
proteins:

Activators – involved in positive regulation

Repressors – involved in negative regulation

Both types are DNA binding proteins.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
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REGULATION OF GENE
EXPRESSION

Regulatory proteins recognize two sites on
DNA near the genes they control.

The promoter – where RNA polymerase binds

The operator – where regulatory proteins bind

The two sites are adjacent.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
INDUCTION

Induction turns on genes that are off
(repressed).

The best example is the lac operon:

An operon is a set of genes that is regulated.

There are many operons in the chromosome.
Microbiology: A Clinical Approach
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lac Operon
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lac Proteins


The lac system has two regulatory proteins

The lac repressor

The lac activator - CAP (catabolite activator
protein).
Both proteins bind at the operator site on
DNA.
Microbiology: A Clinical Approach
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Tony Srelkauskas
Science © Garland Science
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Microbiology: A Clinical Approach [9780815365143] © Garland Science
lac Repressor

The lac repressor is always produced.

It binds at the operator site and overlaps part of
the promoter site

This blocks the RNA polymerase from attaching.

This prevents transcription of the lac gene.
Microbiology: A Clinical Approach
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OPERATION OF THE lac OPERON
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
lac ACTIVATOR

CAP also binds at the operator site

It recruits RNA polymerase to the site.

It then interacts with the polymerase so it binds
properly.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
EXPRESSION OF lac OPERON

For the genes of the lac operon to be turned
on, the repressor must first be inhibited.

This occurs through an allosteric control
mechanism.

It then interacts with the polymerase so it binds
properly.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
EXPRESSION OF lac OPERON

The expression of lac genes is leaky

A few transcripts are made and there is always a
low level of β-galactosidase.

This allows small amounts of lactose into the cell.

Lactose is converted to allolactose.

Allolactose binds the lac repressor.

This changes the shape of the lac repressor and it
can no longer bind the operator site.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
EXPRESSION OF lac OPERON

CAP acts in a similar fashion to allolactose.

Its activity is based on levels of cyclic AMP
(cAMP).
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
EXPRESSION OF lac OPERON

When cAMP levels rise, cAMP binds to CAP.

This causes a change in the three-dimensional
shape of CAP.

The CAP-cAMP complex binds to the promoter
site of the lac operon.

This helps the RNA polymerase bind to the
promoter site.

The lac genes are expressed.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
EXPRESSION OF lac OPERON

When cAMP levels fall, no complex is formed.

RNA polymerase does not bind to the promoter
site.

The lac genes are not expressed.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
REPRESSION

There are also cellular mechanisms that turn
off (repress) genes.


This is very important for the conservation of
energy.
Repression has similar mechanisms to
feedback inhibition.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
REPRESSION

A good example of repression is the synthesis
of tryptophan.

The tryptophan repressor is always produced but
cannot bind DNA in its normal form.

Excess tryptophan binds the repressor and changes
its shape so it can bind DNA and prevent gene
expression.

Tryptophan is a co-repressor of its own synthesis.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRYPTOPHAN OPERON
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRYPTOPHAN OPERON
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRYPTOPHAN OPERON
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
MUTATION & REPAIR OF DNA

Mutations are changes in the DNA sequence.

Change in DNA sequence can cause changes
in proteins.

Mutations must be kept to a minimum.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
MUTATION & REPAIR OF DNA

The simplest type of mutation is classified as a
point mutation.


In this instance, one base is switched for another.
More drastic mutations are classified as
frameshift mutations.

This is caused by insertion or deletion of bases.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
MUTATION & REPAIR OF DNA

Spontaneous mutation rates are low.

Certain sections of the chromosome have a
higher rate of spontaneous mutation.


These are called “hot spots”.
There are also suppressor mutations.

Suppressor mutations can reverse the primary
mutation.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
HOW DNA DAMAGE OCCURS

DNA can be damaged by:

Hydrolysis

Deamination

Chemical mutagens

Alkylation

Oxidation

Base analogs

Radiation
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
HOW DNA DAMAGE OCCURS

Gamma radiation and ionizing radiation cause
double-strand breaks in DNA.

Ultraviolet radiation causes DNA damage
through the formation of thymine dimers.

Radiation damage prevents replication.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
HOW DNA DAMAGE OCCURS

Base analogs look like DNA bases but aren’t.

They can be mistakenly used in replication.

This inhibits further replication.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
HOW DNA DAMAGE OCCURS
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
REPAIR OF DNA DAMAGE

Three principle mechanisms of DNA repair

Base excision

Nucleotide excision

Photoreactivation
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
REPAIR OF DNA DAMAGE

During base excision:

Repair enzymes look for damaged bases.

The damaged base is removed (excised) from the
double helix.

A DNA polymerase fills in the gap.

A DNA ligase repairs the break in the strand.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
REPAIR OF DNA DAMAGE

During nucleotide excision repair:

Repair enzymes look for distortions in the helix.

A short section of DNA surrounding the distortion
is removed.

DNA polymerase fills in removed sections.

DNA ligase repairs the break in the strand.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
REPAIR OF DNA DAMAGE
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
REPAIR OF DNA DAMAGE

Photoreactivation repairs thymine dimers.

It is accomplished by an enzyme called photolyase.

Photolyase binds to the dimer in the dark.

Photolyase is activated by light and breaks the
thymine-thymine bond.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
REPAIR OF DNA DAMAGE
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSFER OF GENETIC
INFORMATION

Bacteria can shuffle genes.


This is called genetic recombination.
There are four ways in which genetic
recombination can occur:

Transposition – within the same cell

Transformation – between cells

Conjugation – between cells

Transduction – between cells.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRANSPOSITION

Transposition is caused by transposons.

Transposons move from one place on the
chromosome to another.

They can move into or out of the chromosome.

They use cleavage and rejoining mechanisms.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSPOSITION

Transposition causes random rearrangements.

The results can be beneficial or detrimental.

Beneficial changes will be selected for and
maintained.

They may be the reason for several human
diseases.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSFORMATION

Transformation involves the transfer of genetic
material between cells.

It involves naked DNA.

This DNA is taken up by a bacterial cell and
recombines with genes of that cell.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRANSFORMATION

The recipient cell must be competent.

Must be able to take up large molecules such as
pieces of DNA.

Some bacteria are naturally competent, whereas
others can become competent after chemical
treatment.

Only a small amount of DNA is actually taken up.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSFORMATION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSDUCTION

Transduction involves the transfer of genetic
material between cells.

It is a common event in both Gram-positive
and Gram-negative bacteria.

It uses a bacterial virus (phage) for transfer.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRANSDUCTION

There are two forms of transduction:

Generalized – random

Specialized – specific
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSDUCTION

There are three phases to generalized
transduction.

The original infected cell chromosome is cleaved
into pieces.

Some of this bacterial DNA is incorporated into a
newly made phage.

When these phages infect the next cell, original
DNA recombines with host chromosome.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSDUCTION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
TRANSDUCTION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
TRANSDUCTION

During specialized transduction:

Phage DNA incorporates into the host chromosome.

Phage DNA excises itself from the host chromosome.

Original host DNA is incorporated into the next host
chromosome.


Part of the host DNA is taken along.
Specialized transduction is used in biotechnology.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
CONJUGATION

Conjugation involves the transfer of material
between cells.

Conjugation requires direct contact between
the donor and recipient cells.


Gram-positive cells stick to each other.

Gram-negative cells use pili as a conduit for DNA
transfer.
DNA moves from the donor to recipient cell.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
CONJUGATION
© Dennis Kunkel
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
CONJUGATION

There are several steps in conjugation:

The sex pilus of the donor cell recognizes specific
receptors on the cell wall of recipient cell.

An enzyme in the donor cell causes the plasmid
DNA to unwind.

One of the two single strands of plasmid DNA
stays in the donor cell.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
CONJUGATION

There are several steps in conjugation:

The other moves across the plasmid into the
recipient cell.

Both single strands are replicated.

After replication, the donor and the recipient
contain identical plasmids.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
CONJUGATION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
CONJUGATION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
CONJUGATION

Conjugation can have several outcomes for the
recipient cell:

The plasmid can remain as a plasmid.

The plasmid can become incorporated into the
recipient cell chromosome.


When this happens, the recipient cell is then referred to
as Hfr.
DNA from Hfr can be moved into a new recipient.

This replaces sections of the host chromosome.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
CONJUGATION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
CONJUGATION
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
GENETICS AND
PATHOGENICITY

Many genetic mechanisms are involved in
making pathogens more dangerous.

Mutations cause antibiotic resistance.

Genetic transfer is closely associated with
pathogenicity and virulence.

It transfers virulence genes into bacteria that were
previously harmless.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science
GENETICS AND
PATHOGENICITY

Genes for antibiotic resistance and toxins are
found on plasmids.

Genes for resistance to disinfectants and
environmental pressure are found on
dissimilation plasmids.
Microbiology: A Clinical Approach
Approach, ©
byGarland
Tony Srelkauskas
Science © Garland Science
ISBN: 978-0-8153-6514-3
Microbiology: A Clinical Approach [9780815365143] © Garland Science