Chapter 11

You will be responsible for many of the items in this chapter


Principles of Bacterial Genetics
Chapter 11
I. Bacterial Chromosomes and Plasmids
11.1 Genetic Map of the Escherichia coli Chromosome

 Escherichia coli is a useful model organism for the study of biochemistry, genetics, and bacterial physiology
 The E. coli chromosome from strain MG1655 has been mapped using conjugation, transduction, molecular cloning, and sequencing
 Some Features of the E. coli Chromosome
 Many genes encoding enzymes of a single biochemical pathway are clustered into operons
 Operons equally distributed on both strands
 ~ 5 Mbp in size
 ~ 40% of predicted proteins are of unknown function
 Average protein contains ~ 300 amino acids
 Insertion sequences (IS elements)


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

 A cell can contain more than one plasmid, but it cannot be closely related
 Many Incompatibility (Inc) groups recognized
 Plasmids belonging to same group exclude each other from replicating in the same cell but can coexist with plasmids from other groups

 Some plasmids (episomes) can integrate into the cell chromosome; similar to situation seen with prophages
 Removal (curing) plasmids from host cells can result from various treatments
 Conjugative plasmids can be transferred between suitable organisms via cell-to-cell contact
 Conjugal transfer controlled by tra genes on plasmid


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

 R plasmids
 Resistance plasmids; confer resistance to antibiotics and other inhibitors
 Widespread and well-studied group of plasmids
 Many are conjugative

 In several pathogens, virulence characteristics are encoded by plasmid genes

 Bacteriocins
 Proteins produced by bacteria that inhibit or kill same/other species
 Genes encoding bacteriocins are often carried on plasmids

 Plasmids have been widely exploited in genetic engineering

II. Mutation

11.4 Mutations and Mutants
 Mutation
 Heritable change in DNA sequence that can lead to a change in phenotype  Mutant
 A strain of any cell or virus differing from parental strain in genotype ( Wild-type strain
 Typically refers to strain isolated from nature

 Selectable mutations
 give the mutant a growth advantage under certain environmental conditions
 Useful in genetic research
 Nonselectable mutations
 have neither an advantage nor a disadvantage over the parent
 requires examining a large number of colonies and looking for differences  Screening is always more tedious than selection
 Methods available to facilitate screening
 E.g., replica plating
 useful for identification of cells with a nutritional requirement for growth

11.5 Molecular Basis 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
 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


 Genetic engineering allows for the introduction of specific mutations
 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

11.7 Mutagenesis
 Mutagens: chemical, physical, or biological agents 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

 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
 Three Types of DNA Repair Systems
 Direct reversal: mutated base is still recognizable and can be repaired without referring to other strand
 Repair of single strand damage: damaged DNA is removed and repaired using opposite strand as template
 Repair of double strand damage: a break in the DNA
 Requires more error-prone repair mechanisms
 When DNA damage is large scale the cell may need to use a different type of repair system (i.e., damage interferes with DNA replication)
 This system is more error prone
 Allows replication to proceed and cell to replicate, but errors are likely
 Mechanism called the SOS regulatory system
 Translesion synthesis allows DNA to be synthesized with no template
 Perfection in organisms is counterproductive because it prevents evolution
 The mutation rate of an organism is subject to change
 Mutants are isolated that are hyperaccurate or have increased mutation rates
 Deinococcus radiodurans is 20–200 times more resistant to radiation

11.8 Mutagenesis and Carcinogenesis: The Ames Test
 The Ames test uses of mutations to detect for potentially hazardous chemicals
 Looks for an increase in mutation in the presence of suspected mutagen
 A wide variety of chemicals are screened for determining carcinogenicity

III. Genetic Exchange in Prokaryotes

11.9 Genetic Recombination
 Recombination
 Physical exchange of DNA between genetic elements
 Homologous recombination
 Process that results in genetic exchange between homologous DNA from two different sources
 Selective medium can be used to detect rare genetic recombinants

11.10 Transformation
 Genetic transfer process by which DNA is incorporated into a recipient cell and brings about genetic change
 Discovered by Fredrick Griffith in the late 1920s
 Worked with Streptococcus pneumococcus
 This process set the stage for the discovery of DNA

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

11.11 Transduction
 Transfer of DNA from one cell to another is mediated by a bacteriophage
 Two modes
 Generalized transduction: DNA derived from virtually any portion of the host genome is packaged inside the mature virion
 Specialized transduction: DNA from a specific region of the host chromosome is integrated directly in the virus genome
 Generalized transduction: DNA derived from virtually any portion of the host genome is packaged inside the mature virion
 Defective virus particle incorporates fragment of the cell’s chromosome randomly
 Can be temperate or virulent
 Low efficiency

 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
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

 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

 Sex pilus is essential for conjugation
 Only produced by donor cell
 DNA synthesis is necessary for DNA transfer by conjugation
 DNA synthesized by rolling circle replication; mechanism also used by some viruses

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

 Recipient cell does not become Hfr because only a portion of the integrated F plasmid is transferred by the donor

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

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

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

 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

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- -transposons and insertion sequences
 Both carry genes encoding transposase
 Both have inverted repeats at their ends

 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, Archaea , 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-inserted at second area
 Number of transposons present doubles

 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 not replicated
 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