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Brock Biology of Microorganisms

Twelfth Edition

Madigan / Martinko Dunlap / Clark

Principles of Bacterial Genetics

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Professor Bharat Patel

Brock Biology of Microorganisms

Principles of Bacterial Genetics

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

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

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 1 st generation sequencers  1970 – 2008: $1-2 million per microbial genome  2 nd generation sequencers (current)  Since 2009: $5,000 per microbial genome  3 rd 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. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

I. Genetics of Bacteria and Archaea

Lecture Content

 11.1 Genetic Map of the

Escherichia coli

**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

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

**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

Plasmid Plasmid

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

11.2 Plasmids: General Principles

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

11.2 Plasmids: General Principles

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

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

Examples of Phenotypes Conferred by Plasmids

11.3 Types of Plasmids and Their Biological Significance

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

11.3 Types of Plasmids and Their Biological Significance



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

11.3 Types of Plasmids and Their Biological Significance

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

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

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

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

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

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

Possible Effects of Base-Pair Substitution

11.5 Molecular Basis (consequences) of Mutation



Silent mutation

 Does not affect amino acid sequence 

Missense mutation

 Amino acid changed; polypeptide altered 

Nonsense mutation

11.5 Molecular Basis of Mutation

 Deletions and insertions cause more dramatic changes in DNA 

Frameshift mutations

Shifts in the Reading Frame of mRNA

11.5 Molecular Basis of Mutation

 Point mutations are typically reversible 

Reversion

11.5 Molecular Basis of 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

11.6 Mutation Rates

 F or most microorganisms, errors in DNA replication occur at a frequency of 10 -6 to10 -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 Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

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

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

Wavelengths of Radiation

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

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

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

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

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 2 nd & previously unbroken strand Two types of products of resolvase which differ in conformation can exist in E. coli – patch or splice Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Result of recombination events

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

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

S=smooth colonies, capsulated, virulent R = rough colonies, non capsulated, avirulent

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

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

11.10 Transformation

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

Animation: Transformation

Mechanisms of Transformation in Gram-Positive Bacteria

11.10 Transformation



Transfection

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 There are two types of transduction:

– generalized transduction:

a lytic bacteriophage A DNA fragment is that is now carrying maturation during the lytic life cycle.

Animation: Generalized Transduction A DNA fragment is carrying donor bacterial DNA due to an error 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.

http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/tran sduction/transduction.html

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

11.12 Conjugation: Essential Features



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

11.12 Conjugation: Essential Features

Formation of a Mating Pair

11.12 Conjugation: Essential Features

 DNA synthesis is necessary for DNA transfer by conjugation  DNA synthesized by

rolling circle replication

Transfer of Plasmid DNA by Conjugation

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

11.13 The Formation of Hfr Strains and Chromasome Mobilization

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

11.13 The Formation of Hfr Strains and Chromasome Mobilization

 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

Transfer of Chromosomal Genes by an Hfr Strain

11.13 The Formation of Hfr Strains and Chromosome Moblilization

Transfer of Chromosomal DNA by Conjugation

11.13 The Formation of Hfr Strains and Chromosome Moblilization

 Hfr strains that differ in the integration position of the F plasmid in the chromosome transfer genes in different orders Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Formation of Different Hfr Strains

11.13 The Formation of Hfr Strains and Chromosome Moblilization

 Identification of recombinant strains requires selective conditions in which the desired recombinants can grow but where neither of the parental strains can grow Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Example Experiment for the Detection of Conjugation

11.13 The Formation of Hfr Strains and Chromosome Moblilization

Time of Gene Entry in a Mating Culture

11.13 The Formation of Hfr Strains and Chromosome Mobilization



F ′ plasmids

11.14 Complementation



Merodiploid

(or partial diploid)  Bacterial strain that carries two copies of any particular chromosomal segment 

Complementation

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

Complementation Analysis

11.14 Complementation



Cistron

: gene defined by

cis-trans

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

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

11.16 Mobile DNA: Transposable Elements

 Two main types of transposable elements in

Bacteria

are

transposons

and

insertion sequences

Maps of Transposable Elements IS2 and Tn5

11.16 Mobile DNA: Transposable Elements



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

11.16 Mobile DNA: Transposable Elements



Transposons

are larger than insertion sequences  Transposase moves any DNA between inverted repeats  May include antibiotic resistance  Examples are the tn5 and tn10 Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

11.16 Mobile DNA: Transposable Elements

 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

Transposition

Two Mechanisms of Transposition

11.16 Mobile DNA: Transposable Elements

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