Principles of Bacterial Genetics

Principles of Bacterial Genetics
Chapter 11 Principles of Bacterial Genetics Professor Bharat Patel

Principles of Bacterial Genetics
Thirteenth Edition Madigan / Martinko Stahl / Clark Chapter 10 Principles of Bacterial Genetics Professor Bharat Patel

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 , 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)- – 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
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) Figure 11.1

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.
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) Fig13.13

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

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) 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
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
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. Figure 11.4

Animation: Replica Plating
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 DNA viruses have error rates 100 – 1,000 X greater
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

Disc, with added mutagen
The Ames Test to Assess the Mutagenicity of a Chemical Disc, no added mutagen Disc, with added mutagen 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) Figure 11.11

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
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
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
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 Figure 11.13

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 ( ) established many of the rules of heredity, now referred to as the laws of Mendelian inheritance.

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

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

11.10 Transformation 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)

Animation: Transformation
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

11.10 Transformation 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

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: Generalized Transduction 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

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.

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) Complementation
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

11.14 Complementation 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

11.14 Complementation 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

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