MICROBIOLOGY

AN EVOLVING SCIENCE
Fourth Edition
Joan L. Slonczewski and John W. Foster

9
Gene Transfer,
Mutations, and
Genome Evolution
PowerPoint® Lecture Outlines
Prepared by Johnny El-Rady, University of South Florida

Copyright © 2017 W. W. Norton & Company, Inc. Permission required for reproduction or display 1
 Chapter Overview
 The dynamic nature of genomes
 Gene transfer:
- Transformation, conjugation, and transduction
 Genetic recombination
 Mutations: types and causes
 Mechanisms of DNA repair
 Mobile genetic elements
- Insertion sequences and transposons
 How genomes evolve
2
 Introduction
 DNA sequences change over generations
through various mutations, rearrangements,
and inter- and intra-species gene transfer.

 But what are the consequences of DNA

plasticity?

 This chapter explores long-standing

evolutionary questions and shows how
microbial genomes continually change.
3
 9.1 Mosaic Genomes and
Gene Transfer
 A surprise arising from bioinformatic studies is the
mosaic nature of all microbial genomes.
- 37% of the proteins found in the archaeon
Methanocaldococcus jannaschii are found in all three
domains.
- Escherichia coli’s genome is rife with genomic islands,
inversions, deletions, and paralogs and orthologs.

 This is the result of heavy horizontal gene transfer,

recombinations, and a variety of mutagenic and
DNA repair strategies.
4
 The Discovery of Horizontal Gene Transfer
 Transformation is the process of importing free
DNA into bacterial cells.
- It was discovered by Frederick Griffith in 1928.
- The transforming principle, DNA, was identified in
1941.
 Transformation provided the first clue that gene
exchange can occur in microorganisms.
 Genome comparisons show that the fundamental
purpose of bacterial gene transfer is to acquire
genes that might be useful as the environment
changes.
5
 Transformation of Naked DNA – 1
 Many cells are capable of natural transformation.
- Requires specific protein complexes called
transformasomes
 Others require artificial manipulations.
- Perturbing the membrane by chemical (CaCl2) or
electrical (electroporation) methods
 Why do species undergo natural transformation?
- Use indiscriminate DNA as food
- Use specific DNA to repair damaged genomes
- Acquire new genes through horizontal gene transfer
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 Transformation of Naked DNA – 2
 Natural transformation in Gram-positive organisms
typically involves the growth phase–dependent
assembly of a transformasome complex across the
cell membrane.
- It is composed of a binding protein that captures
extracellular DNA floating in the environment, plus
proteins that form a transmembrane pore.
 Once the transformasome is assembled, the cell is
competent, meaning that it can import free DNA
fragments and incorporate them into its genome by
recombination.
7
Transformation of Naked DNA – 3

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 Transformation of Naked DNA – 4
 Gram-negative bacteria transform DNA without
the use of competence factors (CF).
- Either they are always competent or they become
competent when starved.
 Also, they do not use transformasomes.
 In another departure from the Gram-positives,
transformation in most Gram-negative species is
sequence specific.
- Thus limiting gene exchange between genera
9
 Gene Transfer by Conjugation – 1
 Conjugation is the transfer of DNA from one
bacterium to another, following cell-to-cell
contact.
- Sometimes called
“mating”
- It is typically initiated
by a special pilus
protruding from the
donor cell.

10
 Gene Transfer by Conjugation – 2
 Conjugation requires the presence of special
transferable plasmids.
- These usually contain all the genes needed for pilus
formation and DNA export.
 A well-studied example in Escherichia coli is the
fertility factor (F factor).
- Contains two replication origins:
- oriV: used in nonconjugating cells
- oriT: used during DNA transfer

 Conjugation begins with contact between the donor

cell, called the F+ cell, and a recipient F– cell.
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Gene Transfer by Conjugation – 3

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 Gene Transfer by Conjugation – 4
 The F-factor plasmid can integrate into the
chromosome.
- The cell is now designated Hfr, or high-frequency
recombination strain.
 An Hfr cell is capable of transferring chromosome
parts into a recipient cell.
- Genes are transferred in order.
- Entire chromosome takes about 100 minutes to
transfer.
 The process can be used to map genes.
13
Gene Transfer by Conjugation – 5

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 Gene Transfer by Conjugation – 6
 Another type of gene shuffling can occur when an
integrated F factor reverses course and excises from the
chromosome via host recombination mechanisms.
 Aberrant excision results in an F′ factor or
F′ plasmid, which carries chromosomal genes.
 Genes hitchhiking on an F′ plasmid do not have to
recombine into the recipient chromosome to be
maintained.
- The extra genes can be expressed as part of the F′
plasmid, and the resultant strain is called a “partial
diploid.”
15
Gene Transfer by Conjugation – 7

16
 Gene Transfer by Conjugation – 8
 Some bacteria can actually
transfer genes across
biological domains.
 Agrobacterium tumefaciens,
which causes crown gall
disease
- Contains a tumor-inducing
plasmid (Ti) that can be
transferred via conjugation
to plant cells
- Provides an indispensable
tool for plant breeding and
engineering
17
 Gene Transfer by Phage Transduction
 Transduction is the process in which
bacteriophages carry host DNA from one cell to
another.
- This occurs accidentally as an offshoot of the phage
life cycle.
 There are two basic types:
1. Generalized transduction: can transfer any gene
from a donor to a recipient cell
2. Specialized transduction: can transfer only a few
closely linked genes between cells
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Generalized Transduction

19
Specialized Transduction

20
 DNA Restriction and Modification – 1
 There are dangers to the cell associated with the
indiscriminate transfer of DNA between bacteria.
 Bacteria have developed a kind of “Halt! Who goes
there?” approach to gene exchange.
 This protection system, called restriction and
modification, involves:
- Enzymatic cleavage (restriction) of alien DNA, by
restriction endonucleases
- Protective methylation (modification) of host DNA

21
DNA Restriction and Modification – 2

22
 DNA Restriction and Modification – 3
 There are three main types of restriction
endonucleases.

23
 DNA Restriction and Modification – 4
 Types I and III have their restriction and modification
activities combined in one multifunctional protein.
- And cleave DNA some distance away from the
recognition site

 Type II possesses
only
endonuclease activity.
- Generally recognize
palindromic DNA
sequences and
cleave at those sites
24
 CRISPR Interference – 1
 CRISPR (clustered regularly interspaced short
palindromic repeats) is an adaptive system.
- An organism that manages to survive a phage attack
captures a piece of the invader’s genome and wields
it as defense against future attack.
 CRISPRs consist of repeats and spacers that do not
encode proteins, but near them lie CRISPR-
associated gene families that do encode proteins.
 CRISPR is a primitive microbial immune system.
- The function can be divided into three stages: spacer
acquisition, crRNA processing, and effector stage.
25
CRISPR Interference – 2

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CRISPR Interference – 3

27
 9.2 Recombination
 Two different DNA molecules in a cell can
recombine by one of two main mechanisms:
1. Generalized recombination requires that the two
recombining molecules have a considerable stretch
of homologous DNA sequences.
2. Site-specific recombination requires very little
sequence homology between the recombining DNA
molecules.
- But it does require a short sequence recognized
by the recombination enzyme.
28
 Generalized Recombination and RecA – 1
 There are three probable functions for generalized
recombination in the microbial cell:
- Recombination probably first evolved as an internal
method of DNA repair, useful to fix mutations or restart
stalled replication forks.
- Cells with damaged chromosomes use DNA donated by
others of the same species to repair their damaged
genes.
- Recombination is part of a “self-improvement” program
that samples genes from other organisms for an ability to
enhance the competitive fitness of the cell.
29
 Generalized Recombination and RecA – 2
 The central player in generalized recombination
is a protein called RecA.
- RecA molecules are able to scan DNA molecules for
homology and align the homologous regions, forming a
triplex DNA molecule, or synapse.

 The clearest model of RecA-mediated

recombination comes from E. coli.
 Homologs of RecA are found in many other species.
- Deinococcus radiodurans is thought to use RecA protein
to reconstruct the chromosome after extreme radiation
damage. 30
Mechanism of Generalized Recombination – 1

31
Mechanism of Generalized Recombination – 2

32
 Site-Specific Recombination
 In contrast to generalized recombination mechanisms,
site-specific recombination does not utilize RecA and
moves only a limited number of genes.
 It involves very short regions of homology between
donor and target DNA molecules.
- These are recognized specifically by dedicated
enzyme systems, which catalyze a crossover
between them to produce a cointegrate molecule.
 Examples:
- The integration of phage lambda
- Flagellar phase variation in Salmonella enterica
33
 9.3 Mutations – 1
 A mutation is a heritable change in the DNA.
 Mutations can come in several different forms:
- Point mutation: change in a single base
- Transition: purine → purine; pyrimidine → pyrimidine
- Transversion: purine ↔ pyrimidine
- Insertion (addition) and deletion (subtraction) of one
or more bases
- Inversion: DNA is flipped in orientation.
- Reversion: DNA mutates back to original sequence.

34
 9.3 Mutations – 2
 Mutations can be categorized into several
information classes:
- Silent mutation: does not change the amino acid
sequence
- Missense mutation: changes the amino acid
sequence to another
- Nonsense mutation: changes the amino acid
sequence to a stop codon
- Frame-shift mutation: changes the open-reading
frame of the gene
35
Different Classes of Mutations – 1

36
Different Classes of Mutations – 2

37
 Mutations Arise in Diverse Ways – 1
 Spontaneous mutations are rare because of the
efficiency of DNA proofreading and repair.
 However, they can arise for many reasons:
1. Tautomeric shifts in DNA bases that alter base-
pairing properties
2. Oxidative deamination of bases
3. Formation of apurinic sites
4. Damage caused by reactive oxygen species
38
Mutations Arise in Diverse Ways – 2

39
Mutations Arise in Diverse Ways – 3

40
Mutations Arise in Diverse Ways – 4

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Mutations Arise in Diverse Ways – 5

42
 Mutations Arise in Diverse Ways – 6
 Mutations can be caused by mutagens:
 Chemical agents
- Base analogs
- Base modifiers
- Intercalators

 Electromagnetic radiation
- X-rays and gamma rays: break the DNA
- Ultraviolet rays: form pyrimidine dimers
43
Production of a Pyrimidine Dimer

44
Mutagenic Agents and Their Effects

45
 Mutation Rates and Frequencies
 The mutation rate for a given gene is often defined
as the number of mutations formed per cell division.
- It can be estimated according to the formula a =
m per number of cell divisions.
- a is the mutation rate.
- m is the number of mutations that occur as the
number of cells increases from N0 to N.
 A less complex calculation is mutation frequency.
- It is calculated simply as the ratio of mutants per
total cells in the population.
46
 Identifying Mutagens Using
Bacterial “Guinea Pigs”
 The Ames test relies on a mutant bacterial strain
that is defective in hisG.
- Cannot grow on a medium

lacking histidine
- A mutagen-containing disk
is placed on an agar plate
with the mutant.
- Mutagen causes reversion

mutations and colonies

start to appear around the 47
 The Modified Ames Assay

 The Ames assay

can be modified to
test for the
mutagenicity of
chemicals
processed in the
liver.

48
 9.4 DNA Repair
 DNA repair is divided into two types:
1. Error-proof repair pathways, which prevent
mutations
- Methyl mismatch repair, photoreactivation,
nucleotide excision repair, base excision repair,
and recombinational repair
2. Error-prone repair pathways, which risk
introducing mutations
- Operate only when damage is so severe that the
cell has no other choice but to die
49
 Error-Proof Repair Pathways – 1
 Methyl mismatch repair is based on recognition of
the methylation pattern in DNA bases.
- Uses methylation of the parental strand to
discriminate from newly replicated DNA
- The premise is that the parental strand will contain
the proper DNA sequence.
 The methyl-directed mismatch repair proteins (and
genes) are called Mut (and mut) because a high
mutation rate results in strains that are defective in
one of these proteins.
- A bacterial strain with a high mutation rate is called a
mutator strain. 50
Error-Proof Repair Pathways – 2

51
Error-Proof Repair Pathways – 3

52
 Error-Proof Repair Pathways – 4
 Photoreactivation
- The enzyme photolyase binds to the pyrimidine
dimer and cleaves the cyclobutane ring.

 Nucleotide excision repair

- An endonuclease removes a patch of single-
stranded DNA containing certain types of
damaged bases, including dimers.

53
Error-Proof Repair Pathways – 5

54
Error-Proof Repair Pathways – 6

55
 Error-Proof Repair Pathways – 7
 Base excision repair
- Specialized enzymes can recognize specific
damaged bases and remove them without
breaking the phosphodiester bonds.
- The result is an abasic site, or AP site (apurinic
or apyrimidinic).
- Can be recognized and cleaved by a specific AP
endonuclease
- This AP site allows DNA Pol I to synthesize a
replacement strand containing the proper base.
56
Error-Proof Repair Pathways – 8

57
 Error-Proof Repair Pathways – 9
 Recombinational repair
- Takes place at the replication fork
- A single-stranded segment of the undamaged
daughter strand can be used to replace a gap in
the damaged daughter strand.
- Carried out by RecA
- Recombinational repair is not limited to
pyrimidine dimers.
- It will work on any damage that causes gaps
during replication.
58
Error-Proof Repair Pathways – 10

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Error-Proof Repair Pathways – 11

60
 Error-Prone DNA Repair – 1
 SOS (“Save Our Ship”) repair
- Induced by extensive DNA damage
- RecA coprotease activity stimulates
autodigestion of the LexA repressor.
- Expression of many DNA repair enzymes
- Among them, two “sloppy” DNA polymerases
that lack proofreading activity
- However, the cell has no other option but
to “mutate or die.”
61
Error-Prone DNA Repair – 2

62
 Error-Prone DNA Repair – 3
 Nonhomologous end joining (NHEJ)
- Common in eukaryotes
- Found in slow-growing bacteria such as
Mycobacterium tuberculosis, and in Bacillus
- Carried out by the proteins Ku and LigD
- Does not require homology, and so is error-prone
- May cause loss or addition of a few nucleotides or
even the joining of two previously unlinked DNA
molecules
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Error-Prone DNA Repair – 4

64
Summary of Types of DNA Repair

65
 9.5 Mobile Genetic Elements
 Transposable elements move from one DNA
molecule to another.
- Exist in virtually all life-forms
- Can move within and between chromosomes
- Are incapable of existing outside a larger DNA molecule

 Insertion sequence (IS)

- Simple transposable elements containing a transposase
gene, flanked by short inverted repeat sequences
- Are targets for the transposase enzyme
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Basic Transposition

67
 Mechanisms of Transposition
 Transposable elements transpose by one of two
mechanisms:
- Replicative or nonreplicative transposition

68
Nonreplicative Transposition – 1

69
Nonreplicative Transposition – 2

70
 Transposons – 1
 Transposons are complex transposable elements
that carry other genes in addition to those required
for transposition.
- Composite transposons
- Typically consist of two insertion sequences that flank
a drug resistance gene, or one or more catabolic
genes
- Carry out nonreplicative transposition

71
 Transposons – 2
- Complex transposons
- Example 1: Tn3 family of transposons
- Possess transposase and antibiotic resistance
genes, but also includes a gene whose product is
called resolvase
- When present in a plasmid can bring about
cointegration of the plasmid into a target DNA
molecule during replicative transposition
- Example 2: Conjugative transposons
- Are able to transfer from one cell to another by
conjugation.
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 Transposition-Mediated Cointegrate
Formation between Two DNA Elements

73
 9.6 Genome Evolution
 Microbial genomes are dynamic entities that
continually evolve.
 Evolution of a genome is a random process driven
by natural selection.
 Two basic processes are thought to contribute to
genome restructuring:
1. Horizontal gene transfer
2. Duplication followed by functional divergence
through mutation
74
 Horizontal Gene Transfer – 1
 Microbial genomes evolve by randomly assembling
an eclectic array of genes from many sources.
 Genomic islands provide evidence for horizontal
gene transfer.
- These include:
- Pathogenicity islands
- Symbiosis islands
- Fitness islands

75
Horizontal Gene Transfer – 2

76
 Horizontal Gene Transfer – 3
 Interdomain transfer of DNA over the course of evolution
may be more common than previously realized.
- DNA sequences from many eukaryotes, including
humans, appear to include a variety of genes horizontally
transferred from bacteria.

77
 Genome Reduction
 Genome reduction is the large-scale loss of
genes through evolution.
- Pathogenic shigellae exhibit chromosomal
“black holes,” regions lacking genes that occur in
the closely related Escherichia coli.
- Over half of the Mycobacterium leprae genome is
composed of nonfunctional pseudogenes.
- Presumably formed from genes it no longer needs
after becoming an obligate intracellular pathogen

78
 Duplications and Divergence
 Gene duplication is the most important mechanism
for generating new genes and biochemical
processes.
 Duplication frees a gene from its previous functional
constraints and allows divergent evolution through
mutation.
 Superfamilies of proteins arising from divergent
evolution share structural and functional features but
may catalyze different reactions.
- Example: ABC superfamily of ABC transporters
79
 Chapter Summary – 1
 All microbial genomes have a mosaic nature.
 There are three types of gene transfer in bacteria:
- Transformation: free DNA from environment
- Conjugation: DNA transfer after cellular contact
- Transduction: DNA transfer via bacteriophages

 A restriction-modification system protects bacteria

from invasion by foreign DNA.
 Recombination is the exchange of DNA sequences
between DNA molecules.
80
 Chapter Summary – 2
 Mutations can be classified based on their effects on
DNA or the protein.
 Mutations can be spontaneous or induced (by
mutagens).
 DNA repair pathways can be divided into two types:
- Error-proof pathways and error-prone pathways
 Transposable elements include insertion sequences and
transposons.
 Genomes evolve primarily by horizontal gene transfer
and by duplications followed by functional divergence
through mutation.
81