Principles of Bacterial Genetics

Click to add TextCopyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Brock Biology of Microorganisms

Twelfth Edition

Madigan / Martinko

Dunlap / Clark

Principles of Bacterial Genetics

Cha

pter

11

Professor Bharat Patel

Click to add TextCopyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Brock Biology of Microorganisms

Twelfth Edition

Madigan / Martinko

Dunlap / Clark

Principles of Bacterial Genetics

Cha

pter

10

Professor Bharat Patel

Thirteenth Edition

Madigan / Martinko

Stahl / Clark

Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

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 genome2nd generation sequencers (current)

Since 2009: $5,000 per microbial genome3rd 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



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 MutationsAnimation: 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 PlatingAnimation: 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



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: MutagensAnimation: 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)



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: TransformationAnimation: Transformation


Mechanisms of Transformation in Gram-Positive BacteriaF

igu

re 1

1.1

6



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 TransductionAnimation: 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 TransductionAnimation: 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 TransductionAnimation: 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/transduction/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: ConjugationAnimation: 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 FAnimation: 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 HfrAnimation: 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



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



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

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