2e lecture chapter_11 Streikauskas

ISBN: 978-0-8153-6514-3Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland ScienceMicrobiology: A Clinical Approach (2nd
Edition) © Garland Science
CHAPTER 11
MICROBIAL GENETICS AND INFECTIOUS
DISEASE

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

WHY IS THIS IMPORTANT?
 One of the most difficult problems in medicine
today is antibiotic resistance
 Organisms can become resistant to antibiotics
through mutations
 Mutations can be transferred to other bacteria
 Mutations can make a harmless bacterium
pathogenic and a pathogenic bacterium more
virulent and even lethal

OVERVIEW

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 maximizes the ease of
replication and makes gene expression, defined as the
process of transcription (DNA to RNA) and translation
(RNA to protein), efficient and almost error-free

DNA STRUCTURE
 DNA is a double-stranded helical structure
 It is composed of nucleotides
 Each nucleotide is a combination of a phosphate, a
sugar (which in DNA is deoxyribose), and a
nucleotide base

DNA STRUCTURE
 The two strands are complementary and wind
around each other to form the double helix
 The bases project inward
 The components of DNA bind together in a
very specific way
 This permits a correct and precise orientation of
the nucleotide

DNA STRUCTURE

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

DNA STRUCTURE
 DNA has two types of bases
 Purines – adenine and guanine

Purines are large double-ring structures
 Pyrimidines – thymine and cytosine

Pyrimidines have smaller single ring structures

DNA STRUCTURE
 DNA has a helical geometry governed by how
the bases pair up
 Adenine always pairs with thymine
 Cytosine always pairs with guanine

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

RNA
 RNA stands for ribonucleic 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

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

RNA

DNA REPLICATION
 This is the process by which DNA is copied
 DNA replication involves specific components
and mechanisms
 It is a critical cellular procedure accomplished
with remarkable accuracy and at astounding
speed

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

DNA REPLICATION:
Requirements
 There are two requirements for replication:
 An ample supply of each of the four nucleotides
 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

DNA REPLICATION:
Requirements

DNA REPLICATION:
Direction
 DNA replication proceeds in only one
direction
 The primer at 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

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

DNA REPLICATION:
Direction

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

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

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

DNA POLYMERASE:
Proofreading
 Proofreading takes place at the newly
synthesized strand
 DNA polymerase contains an exonuclease
component to remove improperly paired bases

DNA POLYMERASE:
Proofreading

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

 The leading strand is in a perfect position for
the addition of nucleotides to its 3 endʹ
 Replication moves towards the replication fork

 The lagging strand is anti-parallel
 It moves away from the replication fork
 For nucleotides to be added to a growing DNA
strand, there has to be a free 3 end that theʹ
polymerase can use
 The lagging strand is replicated in pieces called
Okazaki fragments

 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

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

INITIATION AND TERMINATION
OF REPLICATION

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

THE GENETIC CODE
 There are 64 possible three letter combinations
 Only 20 amino acids are used to make proteins
 The genetic code is degenerate

THE GENETIC CODE

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

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

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

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 is a poor proofreader

TRANSCRIPTION
 Transcription has three steps:
 Initiation – RNA polymerase binds to a DNA sequence
called the promoter:

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

TRANSCRIPTION

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

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

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

mRNA IN TRANSLATION
 mRNA contains a segment that recruits the
ribosomal subunits
 Ribosome and mRNA bind here through
complementary base pairing

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

tRNA IN TRANSLATION

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

THE RIBOSOME IN
TRANSLATION

FORMATION OF PEPTIDE
BONDS IN TRANSLATION
 Peptide bonds form between amino acids
while on the ribosome
 The ribosome has three sites:
 A site – tRNA brings in new amino acid
 P site – tRNA holds the growing amino acid chain
 E site – tRNA exits the ribosome

 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

STAGES OF TRANSLATION
 There are three stages of transcription:
 Initiation
 Elongation
 Termination

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

INITIATION

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

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 break down
 The peptide chain is released from the
ribosome and begins to form secondary and
tertiary structures

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

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

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

INDUCTION
 Induction turns on genes that are off
(repressed)
 The best example is the lac operon:
 An operon is a set of genes that are co-regulated
 There are many operons in the chromosome

lac Operon

lac Proteins
 The lac system has two regulatory proteins
 The lac repressor protein
 The lac activator - CAP (catabolite activator
protein).
 Both proteins bind at the operator site on DNA

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

OPERATION OF THE lac OPERON

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

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

 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

 CAP acts in a similar fashion to allolactose
 Its activity is based on levels of cyclic AMP
(cAMP)

 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 DNA
 This helps the RNA polymerase bind to the
promoter site
 The lac genes are expressed

 When cAMP levels fall, no complex is formed
 RNA polymerase does not bind to the promoter
site
 The lac genes are not expressed

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

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

TRYPTOPHAN OPERON

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

 The simplest type of mutation is classified as a
point mutation
 In this instance, one nucleotide is switched for
another
 More drastic mutations are classified as
frameshift mutations
 This is caused by insertion or deletion of
nucleotides

 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

HOW DNA DAMAGE OCCURS
 DNA can be damaged by:
 Hydrolysis
 Deamination
 Alkylation
 Oxidation
 Radiation

 Gamma radiation and ionizing radiation cause
double-strand breaks in DNA
 Ultraviolet radiation causes DNA damage
through the formation of thymine dimers
 Radiation damage impairs replication

 Base analogs look like DNA bases but are not
 They can be mistakenly used in replication
 This impairs further replication

REPAIR OF DNA DAMAGE
 Three principle mechanisms of DNA repair
 Base excision
 Nucleotide excision
 Photoreactivation

 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

 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

 Photoreactivation repairs thymine dimers
 It is accomplished by an enzyme called photolyase
 Photolyase binds to the dimer in the dark
 When the DNA is then exposed to light, the
photolyase becomes activated and breaks the
thymine–thymine bond

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

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

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

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

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

TRANSFORMATION

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

TRANSDUCTION
 There are two forms of transduction:
 Generalized – random
 Specialized – specific
 Transduction-related development of
pathogenicity or increase in virulence is
called phage conversion

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

TRANSDUCTION

TRANSDUCTION
 During specialized transduction:
 Phage DNA incorporates into the host
chromosome
 Phage DNA excises itself from the host
chromosome

Part of the host DNA is taken along
 Previous host DNA is incorporated into the next
host chromosome

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

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

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
 At the completion of this transfer, the recipient cell
becomes a donor and can conjugate with another
recipient cell

CONJUGATION

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

GENETICS AND
PATHOGENICITY
 Mutations that occur during DNA replication
and the various processes of gene expression
can lead to:
 a harmless bacterium becoming pathogenic
 increased virulence in a pathogen
 antibiotic resistance

GENETICS AND
PATHOGENICITY
 Genes found on plasmids can code for:
 toxins involved in infection and pathogenesis
 antibiotic resistance
 disinfectant resistance
 better adaptation to otherwise destructive
environments
 Plasmids are easily transported through
conjugation

2e lecture chapter_11 Streikauskas

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