Chapter 10 Genetics of Bacteria and Archaea I. Mutation • 10.1 Mutations and Mutants • 10.2 Molecular Basis of Mutation • 10.3 Reversions and Mutation Rates • 10.4 Mutagenesis 10.1 Mutations and Mutants • 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 10.1 Mutations and Mutants • Selectable mutations (Figure 10.1) • • Those that give the mutant a growth advantage under certain conditions Useful in genetic research • Nonselectable mutations • Those that usually have neither an advantage nor a • disadvantage over the parent Detecting such mutations requires examining a large number of colonies and looking for differences (screening) 10.1 Mutations and Mutants • Screening is always more tedious than selection • • Methods are available to facilitate screening (Figure 10.2) • Example: replica plating Replica plating is useful for identifying cells with a nutritional requirement for growth (auxotroph) 10.2 Molecular Basis of Mutation • Induced mutations • • Those made environmentally or deliberately Can result from exposure to natural radiation or oxygen radicals • Spontaneous mutations • Those that occur without external intervention • Point mutations (Figure 10.3) • Mutations that change only one base pair • Can lead to single amino acid change in a protein, an incomplete protein, or no change at all 10.2 Molecular Basis 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 10.2 Molecular Basis of Mutation • Deletions and insertions cause more dramatic changes in DNA • Frameshift mutations (Figure 10.4) • Deletions or insertions that result in a shift in the reading • frame Often result in complete loss of gene function 10.3 Reversion and Mutation Rates • Point mutations are typically reversible • Reversion • Alteration in DNA that reverses the effects of a prior mutation 10.3 Reversion and Mutation Rates • Revertant • Strain in which original phenotype is restored • Two types • Same-site revertant: mutation 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 (Figure 10.5) 10.3 Reversion and Mutation Rates • For most microorganisms, errors in DNA replication occur at a frequency of 106 to107 per kilobase • • DNA viruses have error rates 100–1000 greater The mutation rate in RNA genomes is 1000-fold higher than in DNA genomes • Some RNA polymerases have proofreading capabilities • However, RNA repair mechanisms similar to DNA repair mechanisms do not exist 10.3 Reversion and Mutation Rates • The Ames test makes practical use of bacterial mutations to detect for potentially hazardous chemicals (Figure 10.6) • Looks for an increase in mutation of bacteria in the • presence of suspected mutagen A wide variety of chemicals have been screened for toxicity and carcinogenicity 10.4 Mutagenesis • Mutagens: chemical, physical, or biological agents that increase mutation rates • Several classes of chemical mutagens exist: • Nucleotide base analogs: resemble nucleotides (Figure • • 10.7) Chemical mutagens that induce chemical modifications • For example, alkylating agents such as nitrosoguanidine Chemical mutagens that cause frameshift mutations • For example, intercalating agents such as acridines 10.4 Mutagenesis • Several forms of radiation (Figure 10.8) • Two main categories of mutagenic electromagnetic radiation • Nonionizing (i.e., UV radiation) • • Purines and pyrimidines strongly absorb UV • Pyrimidine dimer 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 10.4 Mutagenesis • 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 10.4 Mutagenesis • When DNA damage is large scale, the cell may use a different type of repair system (i.e., damage interferes with DNA replication) • Mechanism called the SOS regulatory system (Figure 10.9) • This system is more error-prone • Allows replication to proceed and cell to replicate, but errors are more likely • Translesion synthesis allows DNA to be synthesized with no template 10.4 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 Mutator strains: bacteria that benefit from increased mutation rates II. Gene Transfer in Bacteria • 10.5 Genetic Recombination • 10.6 Transformation • 10.7 Transduction • • 10.8 Conjugation 10.9 The Formation of Hfr Strains and Chromosome Mobilization 10.5 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 (Figure 10.11) • Selective medium can be used to detect rare genetic recombinants (Figure 10.12) 10.6 Transformation • Transformation • Genetic transfer process by which DNA is incorporated into a recipient cell and brings about genetic change 10.6 Transformation • Competent: cells are 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 Electricity can be used to force cells to take up DNA (electroporation) 10.6 Transformation • During natural transformation, integration of transforming DNA is a highly regulated, multistep process (Figure 10.13) 10.6 Transformation • Transfection • Transformation of bacteria with DNA extracted from a bacterial virus 10.7 Transduction • Transduction • • Transfer of DNA from one cell to another by a bacteriophage Two modes • Generalized transduction: DNA from any portion of the host genome is packaged inside the virion • Specialized transduction: DNA from a specific region of the host chromosome is integrated directly into the virus genome 10.7 Transduction • Generalized transduction: DNA derived from virtually any portion of the host genome is packaged inside the mature virion (Figure 10.14) • • • Defective virus particle incorporates fragment of the cell's chromosome randomly Virus can be temperate or virulent Low efficiency 10.7 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 (Figure 10.15) Transducing efficiency can be high 10.7 Transduction • Phage conversion: alteration of the phenotype of a host cell by a lysogenization • Nondefective temperate phage lysogenizes a cell and • • becomes a prophage Host cell becomes immune to further infection by same phage Other phenotypic changes can also occur • Salmonella enterica serovar Anatum and bacteriophage ε • Corynebacterium diphtheriae and bacteriophage β 10.8 Conjugation • 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 10.8 Conjugation • F (fertility) plasmid (Figure 10.16) • • • • 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 10.8 Conjugation • Sex pilus is essential for conjugation (Figure 10.17) • Produced only by donor cell 10.8 Conjugation • DNA synthesis is necessary for DNA transfer by conjugation • DNA synthesized by rolling circle replication (Figure 10.18) • Mechanism also used by some viruses 10.9 The Formation of Hfr Strains and Chromosome Mobilization • F plasmid is an episome; can integrate into host chromosome • • Cells possessing a nonintegrated 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 10.9 The Formation of Hfr Strains and Chromosome 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 10.9 The Formation of Hfr Strains and Chromosome Mobilization • Insertion sequences (mobile elements) are present in both the F plasmid and E. coli chromosome • Facilitate homologous recombination (Figure 10.19) • Plasmid is now part of chromosome • Chromosomal genes transferred with plasmid (Figure 10.20) 10.9 The Formation of Hfr Strains and Chromosome Mobilization • Recipient cell does not become Hfr because only a portion of the integrated F plasmid is transferred by the donor (Figure 10.21) 10.9 The Formation of Hfr Strains and Chromosome Mobilization • Hfr strains that differ in the integration position of the F plasmid in the chromosome transfer genes in different orders (Figure 10.22) 10.9 The Formation of Hfr Strains and Chromosome Mobilization • Genetic crosses with Hfr strains can be used to map the order of genes on the chromosome 10.9 The Formation of Hfr Strains and Chromosome Mobilization • F′ plasmids • Previously integrated F plasmids that have excised and captured some chromosomal genes III. Gene Transfer in Archaea and Other Genetic Elements • 10.10 Horizontal Gene Transfer in Archaea • 10.11 Mobile DNA: Transposable Elements • 10.12 Preserving Genome Integrity: CRISPR Interference 10.10 Horizontal Gene Transfer in Archaea • Archaea contain single circular chromosome (Figure 10.23) • Genetic manipulation of Archaea lags 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, transduction, and conjugation exist • Transformation works reasonably well in Archaea 10.11 Mobile DNA: Transposable Elements • Discrete segments of DNA that move as a unit from one location to another within other DNA molecules are 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 1000 to 1 in 10,000,000 • per generation First observed by Barbara McClintock 10.11 Mobile DNA: Transposable Elements • Two main types of transposable elements in Bacteria are transposons and insertion sequences (Figure 10.24) • Both carry genes encoding transposase • Both have inverted repeats at their ends 10.11 Mobile DNA: Transposable Elements • Insertion sequences are the simplest transposable element • ~1000 nucleotides long • Inverted repeats are 10–50 base pairs • Only gene is for the transposase • Found in plasmids and chromosomes of Bacteria and • Archaea Found in some bacteriophages 10.11 Mobile DNA: Transposable Elements • Transposons are larger than insertion sequences • • Transposase moves any DNA between inverted repeats Insertion of a transposable element generates a duplicate target sequence (Figure 10.25) • May include antibiotic resistance • Examples are Tn5 and Tn10 10.11 Mobile DNA: Transposable Elements • Mechanisms of transposition: two types • • Conservative: transposon is excised from one location and reinserted at a second location (e.g., Tn5; Figure 10.26) • Number of transposons stays constant Replicative: a new copy of transposon is produced and inserted at a second location • Number of transposons present doubles 10.11 Mobile DNA: Transposable Elements • Using transposons to make mutants (Figure 10.27) • • • • • Transposons with antibiotic resistance are used Transposon is on a plasmid that cannot be replicated in the cell Cells capable of growing on selective medium likely acquired transposon Most insertions will be in genes that encode proteins Next step: screen for loss of function to determine insertion site 10.12 Preserving Genome Integrity: CRISPR Interference • CRISPR: Clustered Regulatory Interspaced Short Palindromic Repeats • Type of prokaryotic "immune system" • Region of bacterial chromosome containing DNA • sequences similar to foreign DNA (spacers) alternating with identical repeated sequences (Figure 10.28) CRISPR-associated proteins (Cas proteins) • Obtain and store segments of foreign DNA as spacers • Recognize and destroy foreign DNA