Microbiology- a clinical approach by Anthony
Strelkauskas et al. 2010
Chapter 11: Microbial genetics and infectious disease
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Understanding genetic mechanisms lets us
study how microorganisms can mutate in ways
that allows them to defeat host defenses and
antimicrobics.
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.
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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.
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DNA is a double stranded helical structure.
◦ It is composed of nucleotides.
◦ Each nucleotide is composed of a phosphate, a sugar
(deoxyribose), and a nucleotide base.
◦ Nucleotides are formed through the process of
dehydration (removal of water).
5’ end
3’ end
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DNA has two types of
base
◦ Purines – adenine and
guanine
 Purines are large doublering structures.
◦ Pyrimidines – thymine and
cytosine
Sugar
Hydrogen bonds
Sugar
 Pyrimidines have smaller
single ring structures
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DNA has a helical
geometry governed by
how the bases pair up.
◦ Adenine always pairs with
thymine (AT)
◦ Cytosine always pairs
with guanine (CG)
Sugar
Hydrogen bonds
Sugar
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The two strands are
complementary and wind
around each other to
form the double helix.
3’
◦ The bases project inward.
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The components of DNA
bind together in a very
specific way.
are stacked
◦ This permits a correct and
precise orientation of the
nucleotide.
5
’
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The strands are anti-parallel.
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DNA is a chemically stable molecule.
◦ One of the strands is oriented upside down relative to
the other.
◦ The bases are stacked on top of each other.
◦ Any mismatched pairing is chemically unstable.
No hydrogen bonds can be formed
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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 (UA).
◦ It is usually found in single-stranded form.
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There are three forms
of RNA:
◦ Messenger RNA (mRNA) –
contains information
derived from DNA; is
used as template for
protein synthesis whereby
3 bases form a codon,
coding for a specific
amino acid
◦ Transfer RNA (tRNA) –
carries amino acids to
ribosomes
◦ Ribosomal RNA (rRNA) –
helps maintain the proper
shape of ribosomes.
http://higheredbcs.wiley.co
m/legacy/college/boyer/047
1661791/structure/tRNA/tr
na.htm
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DNA stands for deoxyribonucleic acid
DNA is the informational molecule of the cell and is
a double-stranded molecule made of nucleotides.
A nucleotide is composed of a phosphate, a sugar,
and one of the four nucleotide bases (adenine,
thymine, guanine, and cytosine).
RNA stands for ribonucleic acid and is made by
copying one strand of DNA.
In RNA, thymine is replaced by uracil
There are three types of RNA: messenger,
transfer, and ribosomal RNA (mRNA, tRNA, rRNA).
View movie clip DNA structure: Movie clips\11.01_DNA_Structure.mov
a.
b.
c.
d.
e.
adenine: thymine
guanine: uracil
cytosine: guanine
adenine: uracil
All are correct.
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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.
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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
 The antibiotic ciprofloxacin
inhibits a bacteria-specific
topoisomerase.
 Helicase – separates and
unwinds the strands.
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There are two requirements for replication:
◦ An ample supply of each of the nucleotides adenine,
thymine, cytosine, and guanine
◦ A primer:template junction
◦ A DNA polymerase
Each single strand of DNA is a template.
A portion of the DNA is paired with a short
piece of RNA called a primer.
Grows in only one direction: from 5’ to 3’
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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.
It works incredibly quick.
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There are several types of DNA polymerase.
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◦ The addition of nucleotides is in the millisecond range.
◦ They perform specific functions and work at
different speeds.
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DNA replication is extraordinarily accurate.
But there are always some mistakes – mutations
◦ 1 per every 105 base pairing are likely to happen
◦ Evolution relies on mutations.
Improperly paired bases are removed by a
component of the polymerase with exonuclease
activity.
◦ During replication, an error is observed only
approximately once in 1010 base pairings.
DNA polymerase
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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: DNA strand is in perfect position for
adding bases to the 3’ end, continuous replication
◦ Lagging strand: DNA strand is in opposite direction;
discontinuous replication via Okazaki fragments that
need to be eventually ligated
View movie clip DNA replication: Movie clips\11.02_Replication.mov
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Initiation begins in bacteria at a specific site
on the chromosome.
◦ The origin of replication (ori)
◦ Contains multiple AT pairs which can be easier
separated
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Termination occurs when the entire
chromosome has been copied.
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DNA is faithfully replicated by the action of DNA
polymerase so that the same genetic information is
passed on from generation to generation.
The enzyme topoisomerase unwinds supercoils, the
enzyme helicase unwinds and separates the strands.
The replication fork is where DNA replication
begins.
DNA polymerase replicates only 5’ to 3’ and has
proof reading capability. The leading strand is
directly replicated 5’ to 3’. The lagging strand
requires discontinuous replication with Okazaki
fragments.
Replication in bacteria is initiated at the point of
origin (“ori “containing multiple AT pairs) and
proceeds until the entire circular genome has been
replicated.
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Information in DNA is based on a four letter
alphabet (A, T, C, G).
The genetic code employs three letter
combinations called codons.
There are 64 possible 3 letter combinations
Only 20 amino acids are used to make proteins.
◦ The genetic code is degenerate.
Typically, the last of the three letters of a codon can differ and
still yields the same amino acid (wobble hypothesis).
 This may minimize potentially devastating effects of
spontaneous mutations
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Three rules govern the arrangement and use of
codons:
◦ Codons are always read in one direction, from 5 to 3.
◦ The message is translated in a fixed reading frame
whereby the AUG codon (
methionine) always
indicates the start of a protein.
◦ There is no overlap or gap in the code.
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Mutations are changes in the DNA sequence.
Change in DNA sequence can cause changes in
proteins.
◦ Mutations must be kept to a minimum.
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The simplest type of mutation is classified as a
point mutation.
◦ In this instance, one base is switched for another.
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More drastic mutations are classified as frame
shift mutations.
◦ This is caused by insertion or deletion of bases.
Translation is aborted
A single amino acid is changed
A completely
different protein
made
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Spontaneous mutation rates are low.
Certain sections of the chromosome have a
higher rate of spontaneous mutation.
◦ These are called “hot spots”.
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There are also suppressor mutations.
◦ Suppressor mutations can reverse the primary
mutation.
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Mutagens increase the spontaneous mutation
rate.
DNA can be damaged by:
◦ Chemical modification of the bases
◦ Radiation
◦ Base analogs
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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.
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Base analogs look like DNA bases but aren’t.
◦ They can be mistakenly used in replication.
◦ This inhibits further replication.
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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.
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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.
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Mutation have an important role in the
infectious process because pathogens can
become resistant to antibiotics through
mutations.
Bacteria depend on a balance between mutation
and repair.
Mutations can result from point mutations and
frame shift mutations.
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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.
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There are two parts to gene expression:
◦ Transcription – construction of RNA from a DNA
template using an RNA polymerase (which has no
proof reading capability)
◦ Translation – construction of the protein using RNA
instructions.
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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
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Only certain portions of the DNA strand are
copied.
Initiation
Elongation
Termination
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View movie clips:
Movie clips\11.03_Transcription.mov
Movie clips\11.04_Transcription_II.mov
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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.
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.
It happens at the ribosomes.
tRNA brings aa
rRNA
tRNA brings aa
rRNA
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mRNA codes for the amino acid sequence of a
protein.
mRNA contains a segment that recruits the
ribosomal subunits.
◦ Ribosome and mRNA bind here through
complementary base pairing.
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An open reading frame (ORF) indicates the
start of an amino acid sequence.
◦ An ORF begins with a start codon (AUG)
◦ Translation moves from the 5’end to the 3’ end.
◦ An ORF ends with a stop codon (UAA, UAG, or UGA).
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Each tRNA
attaches to a
specific amino
acid at the
acceptor arm.
◦ Aminoacyl – tRNAsynthetase
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It brings amino
acids to the
ribosome.
It binds to the
mRNA at the
anti-codon region
using
complementary
base pairing.
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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.
S = Svedberg, sedimentation unit
AUG
UAA, UAG, or UGA
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View movie clips:
Movie clips\11.05_Translation.mov
Movie clips\11.06_Polyribosome.mov
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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.
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Gene expression is controlled by regulatory
proteins:
◦ Activators – involved in positive regulation
◦ Repressors – involved in negative regulation
◦ Both types are DNA binding proteins.
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The expression of a gene is carefully
regulated.
Genes can be constitutive (always on),
inducible (off and can be turned on), and
repressible (on and can be turned off).
Regulatory proteins control induction and
repression through binding on the DNA at
the site known as the operator site.
A.
B.
C.
D.
E.
inducible
repressible
constitutive
suppressible
high frequency of
recombination
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Bacteria can shuffle genes.
◦ This is called genetic recombination.
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There are four ways in which genetic
recombination can occur:
◦ Within the same cell
 Transposition
◦ Between cells
 Transformation –uptake of free DNA
 Transduction – DNA delivered via a viral vector, a
bacteriophage
 Conjugation –cell to cell contact, in gram negative bacteria
DNA delivered via sex pili
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Transposition is caused by transposons.
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Transposition causes random rearrangements.
◦ Transposons move from one place on the chromosome
to another.
◦ They can move into or out of the chromosome in a
random fashion.
◦ They use cleavage and rejoining mechanisms.
◦ The results can be beneficial or detrimental.
◦ Changes beneficial to the bacterium will be selected
for and maintained.
◦ They may be the reason for several human diseases.
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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.
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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.
In 1944 Avery, MacLeod & MacCarty proved DNA is the transforming element.
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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.
There are two forms of transduction:
◦ Generalized : only host DNA is transferred
◦ Specialized: viral DNA along with host DNA is
transferred
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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.
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DNA moves from the donor to recipient cell.
View movie clip: Movie clips\11.07_Conjugation.mov
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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.
◦ 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.
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Genetic recombination occurs in bacteria through
transposition, transformation, transduction, and
conjugation.
Transposition is a specific form of recombination in
which genetic elements call transposons randomly move
from one place in the chromosome to the another
within the same cell.
Transformation involves the uptake of naked DNA
from one cell to another.
Transduction is caused by a virus transferring pieces
of DNA from one cell to another.
Conjugation occurs when DNA is moved from a donor
cell (designated F+) to a recipient cell (designated F-)
by direct cell to cell contact.
Each of the transfer mechanisms causes genetic
recombination in the recipient cell and thus can be
important in making a pathogen more dangerous.
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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.
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DNA is the informational molecule of the cell.
DNA is a double-stranded molecule made up of
nucleotides (which consist of a phosphate, a
deoxyribose sugar and one of the four bases adenine,
thymine, guanine, or cytosine).
Nucleotides are bound together through complementary
base pairing.
RNA is a single-stranded molecule and contains uracil
instead of thymine. It can be found in the form of
messenger RNA, transfer RNA, or ribosomal RNA.
DNA is faithfully replicated by the enzyme DNA
polymerase.
DNA polymerase has a proofreading capability that
prevents errors in replication.
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Replication occurs at the replication fork (a separation
of the DNA strands) and is continuous on one strand
and discontinuous (made in pieces) on the other.
DNA is transcribed into RNA by the enzyme RNA
polymerase.
The genetic code is based on codons, which are
combinations of three nucleotides.
Gene expression is the process of making a functional
product by transcription and translation; it is highly
regulated.
Mutations are changes in the DNA and are important in
infectious disease because they can lead to antibiotic
resistance.
Genetic recombination can occur in bacteria through
transposition, transformation, transduction, or
conjugation.
A.
B.
C.
D.
E.
Transcribes DNA
to RNA
Transfers DNA
vertically to a new
generation of cells.
Replicates DNA
Transfers DNA
horizontally, to
cells in the same
generation.
None of the above.
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11:40am – 1:20pm
Chapters 1 thru 13: Lecture, Reading, Chapter
End Self Study Questions
Fifty Multiple Choice Questions = 100 points
Please bring:
◦ Scantron (form No. 882-E for the Quiz – available at
no cost at the Student Bookstore)
◦ No. 2 pencil only