Principles of Bacterial Genetics Chapter Summary

Click to add Text
Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Brock Biology of
Microorganisms
Twelfth Edition
Madigan / Martinko
Dunlap / Clark
Principles of Bacterial Genetics
Chapter
11
Professor Bharat Patel

Click to add Text
Brock Biology of
Microorganisms
Twelfth Edition
Madigan / Martinko
Dunlap / Clark
Principles of Bacterial Genetics
Chapter
10
Professor Bharat Patel
Thirteenth Edition
Madigan / Martinko
Stahl / Clark

NOTE
1. The following is a summary and are not full notes for the Lecture on
“Principles of Genetics”. This summary is a study guide only and it
is therefore recommended that students attend and take notes
during the lectures.
2. There are differences in the content of the chapters of the two
different editions of the recommended text book
3. The lecture & summary may not follow the same content as is in the
book chapter
4. There is extra content that has been sourced from other resources

CONTENT
The lecture content is divided into 3 parts:
I. Bacterial Chromosomes & Plasmids
• Physical location of the genes
II. Mutation
• Alterations in the genetic material
 Chemical, Physical
III. Genetic Transfer
• Gene transfer & exchange mechanisms
 Conjugation
 Transduction
 Transformation
• Gene exchange mechanisms

Note:
• Most of the techniques described here were used between 1950-80,
but advances in the past three decades in cloning and sequencing has
revolutionised studies on genomes & gene organisation:
• Developments in molecular biology:
Manual sequencing & Automated 1st generation sequencers
 1970 – 2008: $1-2 million per microbial genome
2nd generation sequencers (current)
 Since 2009: $5,000 per microbial genome
3rd generation sequencers
early next year,
semi-conductor real-time technology
 $1,000 per human genome
• Genomes OnLine Database (GOLD)- http://genomesonline.org – lists
all genome sequencing projects.

I. Genetics of Bacteria and Archaea
Lecture Content
 11.1 Genetic Map of the Escherichia coli Chromosome
 11.2 Plasmids: General Principles
 11.3 Types of Plasmids and Their Biological
Significance

11.1 Genetic Map of the Escherichia coli Chromosome
Escherichia coli a model organism for the study of
biochemistry, genetics, and bacterial physiology
The E. coli chromosome (strain MG1655, derivative of K-
12) was been mapped using
 Conjugation (initial mapping)
 Transduction (phage P1)
 Molecular cloning & sequencing
 Next Generation Sequencing (NGS) (most recent)
E. coli is (gram -ve) is inefficient at transformation unlike
Bacillus (gram +ve)

Circular Linkage Map of the Chromosome of E. coli K-12
Figure 11.1
Original map used
distance (centisomes)
0 – 100 mins, 0 = arbitrary
& set at thrABC (based on
transfer by conjugation)
Also shows kilobase pairs
(kb) from sequencing
studies
Replication starts at oriC
(84min)

11.1 Genetic Map of the Escherichia coli Chromosome
 Some Features of the E. coli Chromosome
 Many genes encoding enzymes of a single biochemical
pathway are clustered into operons
 Operons are equally distributed on both strands
 Transcription can occur clockwise or anticlockwise
 ~ 5 Mbp in size
 ~ 40% of predicted proteins are of unknown function
 Average protein size is ~ 300 amino acids
 Insertion sequences (IS elements) are present

Genomes of pathogenic E. coli contain PAIs.
Fig13.13
Genome size is indicated in the
centre. The outer ring shows
gene by gene comparison with
all 3 strains: common genes
(green), genes in pathogens only
(red), genes only in 536 (blue)

Pan Genome Versus Core Genome
Figure 13.14
Core genome is in black &
is present in all strains of
the same species.
The pan genome includes
elements (genes) that are
present in one or more
strains but not in all strains.
one coloured wedge =
single insertion
two coloured wedges =
alternative insertions
possible at the site but
only can be present

Plasmids

11.2 Plasmids: General Principles
Plasmids: Genetic elements
that replicate independently
of the host chromosome
 Small circular or linear DNA
molecules
 Range in size from 1 kbp to >
1 Mbp; typically less than 5%
of the size of the
chromosome
 Carry a variety of
nonessential, but often very
helpful, genes
 Abundance (copy number) is
variable
Plasmid
Plasmid

A cell can contain more than one plasmid, but it
cannot be closely related genetically due to plasmid
incompatibility
 Many Incompatibility (Inc) groups recognized
 Plasmids belonging to same Inc group exclude each
other from replicating in the same cell but can coexist
with plasmids from other groups
 Borrellia burgdorferi (causes Lyme disease) - 17
different circular & liner plasmids

 Some plasmids (episomes) can integrate into the cell
chromosome; similar to prophage integration –
replication is under the control of the host cell
 Host cells can be cured of plasmids by agents that
interfere with plasmid (but not cell) replication
Acridine orange or can be spontaneous
 Conjugative plasmids can be transferred between
suitable organisms via cell-to-cell contact
Conjugal transfer controlled by tra genes on plasmid
 Plasmid replicate up to 10 times faster than host cell DNA
due to their small size
unidirectional (one fork) or bi-directional (two forks)

11.3 Types of Plasmids and Their Biological Significance
 Genetic information encoded on plasmids is not
essential for cell function under all conditions but may
confer a selective growth advantage under certain
conditions
 Plasmids are transferred by conjugation (refer to
Conjugation later) – provide cells with additional “coping
and fighting” strategies

Examples of Phenotypes Conferred by Plasmids

R plasmids
 Resistance plasmids; confer
resistance to antibiotics and
other growth inhibitors
 Widespread and well-studied
group of plasmids
 Many are conjugative
Outer ring: resistance genes (str streptomycin, tet tetracylcine, sul
sulfonamides, & other genes (tra transfer functions, IS insertion sequence,
Tn10 transposon). Inner ring: Plasmid size = 94.3 kb

 In several pathogenic bacteria, virulence characteristics
are encoded by plasmid genes

 Bacteriocins
 Proteins produced by bacteria that inhibit or kill closely
related species or even different strains of the same species
 Genes encoding bacteriocins are often carried on plasmids

 Plasmids have been widely exploited in genetic
engineering for biotechnology
 Plasmids are transferred by conjugation (refer to
Conjugation later) – provide cells with additional “coping
and fighting” strategies

Mutation

II. Mutation
 11.4 Mutations and Mutants - definitions
 11.5 Molecular Basis of Mutation
 11.6 Mutation Rates
 11.7 Mutagenesis
 11.8 Mutagenesis and Carcinogenesis: The Ames Test

11.4 Mutations and Mutants - definitions
 Mutation
 Heritable change in DNA sequence that can lead to a
change in phenotype (observable properties of an organism)
 Mutant
 A strain of any cell or virus differing from parental strain in
genotype (nucleotide sequence of genome)
 Wild-type strain
 Typically refers to strain isolated from nature
Animation: The Molecular Basis of Mutations

11.4 Mutations and Mutants – definitions (cont’d)
 Selectable mutations
 Those that give the mutant a growth advantage under certain
environmental conditions
 Useful in genetic research
 Nonselectable mutations
 Those that usually have neither an advantage nor a
disadvantage over the parent
 Detection of such mutations requires examining a large
number of colonies and looking for differences (screening)

Selectable and Nonselectable Mutations
Figure 11.4
Selectable mutants:
Antibiotic resistance
colonies can be detected
around a zone of clearance
created by the inhibition of
a sensitive bacterium
Nonselectable mutants:
Aspergilus nidulans produces
different interchangeable
spontaneously.

11.4 Mutations and Mutants
 Screening is always more tedious than selection
 Methods available to facilitate screening
 E.g., replica plating
 Replica plating is useful for identification of cells with a
nutritional requirement for growth (auxotroph)
Animation: Replica Plating

Screening for Nutritional Auxotrophs
Figure 11.5

11.5 Molecular Basis (Types ) of Mutation
 Induced mutations
 Those made deliberately
 Spontaneous mutations
 Those that occur without human intervention
 Can result from exposure to natural radiation or oxygen radicals
 Point mutations
 Mutations that change only one base pair
 Can lead to single amino acid change in a protein or no
change at all

Possible Effects of Base-Pair Substitution
Figure 11.6

11.5 Molecular Basis (consequences) of Mutation
 Silent mutation
 Does not affect amino acid sequence
 Missense mutation
 Amino acid changed; polypeptide altered
 Nonsense mutation
 Codon becomes stop codon; polypeptide is incomplete

11.5 Molecular Basis of Mutation
 Deletions and insertions cause more dramatic
changes in DNA
 Frameshift mutations
 Deletions or insertions that result in a shift in the reading
frame
 Often result in complete loss of gene function

Shifts in the Reading Frame of mRNA
Figure 11.7

 Genetic engineering allows for the introduction of
specific mutations (site-directed mutagenesis)

 Point mutations are typically reversible
 Reversion
 Alteration in DNA that reverses the effects of a prior
mutation

 Revertant
 Strain in which original phenotype that was changed in
the mutant is restored
 Two types
 Same-site revertant: mutation restoration activity is at the
same site as original mutation
 Second-site revertant: mutation is at a different site in the
DNA
 suppressor mutation that compensates for the effect of the
original mutation

11.6 Mutation Rates
 For most microorganisms, errors in DNA replication
occur at a frequency of 10-6to10-7 per kilobase
 DNA viruses have error rates 100 – 1,000 X greater
 The mutation rate in RNA genomes is 1,000-fold higher
than in DNA genomes
 Some RNA polymerases have proofreading capabilities
 Comparable RNA repair mechanisms do not exist

11.7 Mutagenesis
 Mutagens: chemical, physical, or biological agents that
increase mutation rates
 Several classes of chemical mutagens exist
 Nucleotide base analogs: resemble nucleotides
 Chemical mutagens can induce chemical modifications
 I.e., alkylating agents like nitrosoguanidine
 Acridines: intercalating agents; typically cause frameshift
mutations
Animation: Mutagens

Nucleotide Base Analogs
Figure 11.8

Chemical and Physical Mutagens and their Modes of Action

11.7 Mutagenesis
 Several forms of radiation are highly mutagenic
 Two main categories of mutagenic electromagnetic
radiation
 Non-ionizing (i.e., UV radiation)
 Purines and pyrimidines strongly absorb UV
 Pyrimidine dimers is one effect of UV radiation
 Ionizing (i.e., X-rays, cosmic rays, and gamma rays)
 Ionize water and produce free radicals
 Free radicals damage macromolecules in the cell

Wavelengths of Radiation
Figure 11.9

11.7 Mutagenesis
 Perfect fidelity in organisms is counterproductive
because it prevents evolution
 The mutation rate of an organism is subject to change
 Mutants can be isolated that are hyperaccurate or have
increased mutation rates
 Deinococcus radiodurans is 20–200 times more
resistant to radiation than E. coli

11.8 Mutagenesis and Carcinogenesis: The Ames Test
 The Ames test makes practical use of bacterial
mutations to detect for potentially hazardous
chemicals
 Looks for an increase in the rate of back mutation
(reversion) of auxotrophic strains in the presence of
suspected mutagen
 A wide variety of chemicals have been screened for
determining carcinogenicity

The Ames Test to Assess the Mutagenicity of a Chemical
Figure 11.11
Auxotrophs with single point mutations will not grow in if the
required nutrient (eg an amino acid) is not included in the medium.
However, in the presence of an added mutagen, some of the cells will
revert to wild type an will grow. Eg Histidine-requiring mutants of
Salmonella entrica (above)- colonies grow on both plates due to
spontaneous mutation but colonies appear on the RHS plate which
contains a mutagen)
Disc, no added mutagen Disc, with added mutagen

DNA REPAIR

DNA Repair
 Three Types of DNA Repair Systems
 Direct reversal: mutated base is still recognizable and can
be repaired without referring to other strand eg by
photoreactivation fromUV damage in which T-T dimers are
formed
 Repair of single strand damage: damaged DNA is removed
and repaired using opposite strand as template eg Excision
repair
 Repair of double strand damage: a break in the DNA
Requires more error-prone repair mechanisms eg SOS repair

DNA Repair
Pyrimidine dimers form due to exposure to UV radiation (260 nm) –
an absorption maxima for DNA .
There are 4 mechanisms by which pyrimidine dimers can be
repaired – Refer to htp://trishul.ict.griffith.edu.au/courses/ss12bi/repair.html
Note: Some of the these mechanisms are also used for repairing
mutations caused by other mutagenic agents.

III. Genetic Exchange in Prokaryotes
 11.9 Genetic Recombination
 11.10 Transformation
 11.11 Transduction
 11.12 Conjugation: Essential Features
 11.13 The Formation of Hfr Strains and Chromosome
Mobilization
 11.14 Complementation
 11.15 Gene Transfer in Archaea
 11.16 Mobile DNA: Transposable Elements

11.9 Genetic Recombination –definition & mechanism
 Genetic Recombination
 Refers to physical exchange between two DNA
molecules – results in new combination of genes on
the chromosome
 Ex- fragment aligning, breaking at points, switching &
rejoining of alleles of the same gene on two different
chromosomes.
 Homologous recombination
 Process that results in genetic exchange between
homologous DNA from two different sources (alleles)
(next fig)
 Selective medium can be used to detect rare genetic
recombinants (fig, after next)

A Simplified Version of Homologous Recombination
Figure 11.13
Endonuclease nicks one strand of donor DNA,
is displaced (eg helicase), & ss binding protein
binds. RecBCD has both endonuclease & helicase activities
Strand invasion: RecA (error-prone repair)
binds to ss DNA to form a complex &
subsequently displaces the complimentary
sequence of the other strand to form a
heteroduplex (Holliday junction)
Holliday junctions are energised by several
proteins & can migrate along the DNA until
“resolved” by resolvase – cut & rejon the 2nd &
previously unbroken strand
Two types of products of resolvase which differ
in conformation can exist in E. coli – patch or
splice

Result of recombination events
 Recombination - a recombinant cell is formed
 Selective medium can be used to detect rare genetic
recombinants

Recombination and Gene Transfer
But one question still remains...how did the chromosome segment
get into the cell for recombination to occur:
The answer is Genetic Transfer!
The players in genetic recombination are:
 host cell (host DNA)
 donor cell (donor DNA)
 DNA is transferred from donor to host (gene transfer)
• Transformation (naked DNA)
• Conjugation (cell to cell contact)
• Transduction (phage mediated)

11.10 Transformation
 Transformation
 Genetic transfer process by which DNA is incorporated
into a recipient cell and brings about genetic change
 Discovered by Fredrick Griffith in 1928
 Worked with Streptococcus pneumoniae (see the next
slide to see how he deciphered this process)
 This process set the stage for the discovery of DNA
NOTE: Though farmers had known for centuries that crossbreeding of animals
and plants could favor certain desirable traits, Mendel's pea plant experiments
(1856 - 1863) established many of the rules of heredity, now referred to as the
laws of Mendelian inheritance.

Griffith’s Experiments with Pneumococcus
Figure 11.15
Death due to pneumonia
S=smooth colonies, capsulated,
virulent
R = rough colonies, non-
capsulated, avirulent

 Streptococcus pneumoniae, phylum Firmicutes causes pneumonia
in mammals. Colonies of the bacteria on petri plates are of two
types:
 Smooth due to presence of capsules (polysaccharide) are
virulent and rough (non-capsulated) are avirulent
 Cultures from blood samples from dead mice follow Koch's
postulates

 Competent: cells capable of taking up DNA and
being transformed
 In naturally transformable bacteria, competence is
regulated
 In other strains, specific procedures are necessary to
make cells competent and electricity can be used to
force cells to take up DNA (electroporation)

 During natural transformation, integration of transforming DNA is
a highly regulated, multi-step process
Animation: Transformation

Mechanisms of Transformation in Gram-Positive Bacteria
Figure
11.16

 Transfection
 Transformation of bacteria with DNA extracted from a
bacterial virus

11.11 Transduction
 Transduction
 Transfer of DNA from one cell to another is mediated by
a bacteriophage.
 Bacteriophage (phage) are obligate intracellular
parasites that multiply inside bacteria by making use
of some or all of the host biosynthetic machinery
(i.e., viruses that infect bacteria
Structure of T4 bacteriophage Contraction of the tail sheath of T4

11.11 Transduction
Animation: Generalized Transduction
There are two types of transduction:
– generalized transduction: A DNA fragment is
transferred from one bacterium to another by
a lytic bacteriophage that is now carrying
donor bacterial DNA due to an error in
maturation during the lytic life cycle.
– specialized transduction: A DNA fragment is
transferred from one bacterium to another by
a temperate bacteriophage that is now
carrying donor bacterial DNA due to an error
in spontaneous induction during the lysogenic
life cycle
Animation: Specialized Transduction

11.11 Transduction
 Specialized transduction: DNA from a specific
region of the host chromosome is integrated directly
in the virus genome
 DNA of temperate virus excises incorrectly and takes
adjacent host genes along with it
 Transducing efficiency can be high
Animation: Specialized Transduction

Seven steps in Generalised Transduction
1. A lytic bacteriophage adsorbs to a susceptible
bacterium.
2. The bacteriophage genome enters the bacterium. The
genome directs the bacterium's metabolic machinery to
manufacture bacteriophage components and enzymes
3. Occasionally, a bacteriophage head or capsid
assembles around a fragment of donor bacterium's
nucleoid or around a plasmid instead of a phage
genome by mistake.

4. The bacteriophages are released.
5. The bacteriophage carrying the donor
bacterium's DNA adsorbs to a recipient
bacterium

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/tran
sduction/transduction.html
6. The bacteriophage inserts the donor
bacterium's DNA it is carrying into the recipient
bacterium .
7. The donor bacterium's DNA is exchanged for
some of the recipient's DNA.

Six steps in Specialised Transduction
1. A temperate bacteriophage adsorbs to a
susceptible bacterium and injects its genome .
2. The bacteriophage inserts its genome into
the bacterium's nucleoid to become a
prophage.

3. Occasionally during spontaneous induction, a
small piece of the donor bacterium's DNA is
picked up as part of the phage's genome in
place of some of the phage DNA which remains
in the bacterium's nucleoid.
4. As the bacteriophage replicates, the
segment of bacterial DNA replicates as part
of the phage's genome. Every phage now
carries that segment of bacterial DNA.

5. The bacteriophage adsorbs to a recipient
bacterium and injects its genome.
6. The bacteriophage genome carrying the
donor bacterial DNA inserts into the recipient
bacterium's nucleoid.
http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/spectran.html

Summary – specialized transduction
DNA from a specific region of the host chromosome is
integrated directly in the virus genome
A of temperate virus excises incorrectly and takes adjacent
host genes along with it
Transducing efficiency can be high

11.12 Conjugation: Essential Features
 Bacterial conjugation (mating): mechanism of
genetic transfer that involves cell-to-cell contact
 Plasmid encoded mechanism
 Donor cell: contains conjugative plasmid
 Recipient cell: does not contain plasmid
Animation: Conjugation

 F (fertility) plasmid
 Circular DNA molecule; ~ 100 kbp
 Contains genes that regulate DNA replication
 Contains several transposable elements that allow the
plasmid to integrate into the host chromosome
 Contains tra genes that encode transfer functions
Animation: Conjugation F

Genetic Map of the F (Fertility) Plasmid of E. coli
Figure 11.19

 Sex pilus is essential for conjugation
 Only produced by donor cell

Formation of a Mating Pair
Figure 11.20

 DNA synthesis is necessary for DNA transfer by
conjugation
 DNA synthesized by rolling circle replication;
mechanism also used by some viruses

Transfer of Plasmid DNA by Conjugation
Figure 11.21a

Transfer of Plasmid DNA by Conjugation
Figure 11.21b

11.13 The Formation of Hfr Strains and Chromasome Mobilization
 F plasmid is an episome; can integrate into host
chromosome
 Cells possessing a non-integrated F plasmid are called
F+
 Cells possessing an integrated F plasmid are called Hfr
(high frequency of recombination)
 High rates of genetic recombination between genes on
the donor chromosome and those of the recipient

 Presence of the F plasmid results in alterations in
cell properties
 Ability to synthesize F pilus
 Mobilization of DNA for transfer to another cell
 Alteration of surface receptors so that cell can no longer
act as a recipient in conjugation

 Insertion sequences (mobile elements) are present
in both the F plasmid and E. coli chromosome
 Facilitate homologous recombination
Animation: Conjugation Hfr

The Formation of an Hfr Strain
Figure 11.22

Transfer of Chromosomal Genes by an Hfr Strain
Figure 11.23

11.13 The Formation of Hfr Strains and Chromosome Moblilization
 Recipient cell does not become Hfr because only a
portion of the integrated F plasmid is transferred by the
donor

Transfer of Chromosomal DNA by Conjugation
Figure 11.24

 Hfr strains that differ in the integration position of the F
plasmid in the chromosome transfer genes in different
orders

Formation of Different Hfr Strains
Figure 11.25

 Identification of recombinant strains requires selective
conditions in which the desired recombinants can grow
but where neither of the parental strains can grow

Example Experiment for the Detection of Conjugation
Figure 11.26

 Genetic crosses with Hfr strains can be used to map
the order of genes on the chromosome

Time of Gene Entry in a Mating Culture
Figure 11.27

11.13 The Formation of Hfr Strains and Chromosome Mobilization
 F′ plasmids
 Previously integrated F plasmids that have excised and
captured some chromosomal genes

11.14 Complementation
 Merodiploid (or partial diploid)
 Bacterial strain that carries two copies of any particular
chromosomal segment
 Complementation
 Process by which a functional copy of a gene
compensates for a defective copy

 Complementation tests are used to determine if two
mutations are in the same or different genes
 Necessary when mutations in different genes in the same
pathway yield the same phenotype
 Two copies of region of DNA under investigation must be
present and carried on two different molecules of DNA (trans
configuration)
 Placing two regions on a single DNA molecule (cis
configuration) serves as a positive control for these tests

Complementation Analysis
Figure 11.28

 Cistron: gene defined by cis-trans test
 Equivalent to defining a structural gene as a segment
of DNA that encodes a single polypeptide chain

11.15 Gene Transfer in Archaea
 Development of gene transfer systems for genetic
manipulation lag far behind Bacteria
 Archaea need to be grown in extreme conditions
 Most antibiotics do not affect Archaea
 No single species is a model organism for Archaea
 Examples of transformation, viral transduction, and
conjugation exist
 Transformation works reasonably well in Archaea

An Archaeal Chromosome Viewed by Electron Microscope
Figure 11.29

11.16 Mobile DNA: Transposable Elements
 Discrete segments of DNA that move as a unit from one
location to another within other DNA molecules (i.e.,
transposable elements)
 Transposable elements can be found in all three domains
of life
 Move by a process called transposition
 Frequency of transposition is 1 in 1,000 to 1 in 10,000,000
per generation
 First observed by Barbara McClintock

 Two main types of transposable elements in
Bacteria are transposons and insertion sequences
 Both carry genes encoding transposase
 Both have inverted repeats at their ends

Maps of Transposable Elements IS2 and Tn5
Figure 11.30

 Insertion sequences are the simplest transposable
element
 ~1,000 nucleotides long
 Inverted repeats are 10–50 base pairs
 Only gene is for the transposase
 Found in plasmids and chromosomes of Bacteria and
Archaea and some bacteriophages

 Transposons are larger than insertion sequences
 Transposase moves any DNA between inverted repeats
 May include antibiotic resistance
 Examples are the tn5 and tn10

 Mechanisms of Transposition: Two Types
 Conservative: transposon is excised from one location
and reinserted at a second location (i.e., Tn5)
 Number of transposons stays constant
 Replicative: a new copy of transposon is produced and
inserted at a second location
 Number of transposons present doubles

Transposition
Figure 11.31

Two Mechanisms of Transposition
Figure 11.32

 Using transposons is a convenient way to make mutants
 Transposons with antibiotic resistance are often used
 Transposon is introduced to the target cells on a plasmid that
can’t be replicated in the cell
 Cells capable of growing on selective medium likely acquired
transposon
 Most insertions will be in genes that encode proteins
 You can then screen for loss of function and determine
insertion site

Transposon Mutagenesis
Figure 11.33

Principles of Bacterial Genetics Chapter Summary

Recommended

Recommended

More Related Content

Similar to Principles of Bacterial Genetics Chapter Summary

Similar to Principles of Bacterial Genetics Chapter Summary (20)

More from dawitg2

More from dawitg2 (20)

Recently uploaded

Recently uploaded (20)

Principles of Bacterial Genetics Chapter Summary