Chapter 8 The Genetics of Bacteria and Their Viruses Multi-Drug-Resistant Bacteria (MDRB) © John Wiley & Sons, Inc.

Chapter Outline Viruses and Bacteria in Genetics The Genetics of Viruses and Bacteria Mechanisms of Genetic Exchange in Bacteria The Evolutionary Significance of Genetic Exchange in Bacteria © John Wiley & Sons, Inc.

Bacteria and Viruses in Genetics Small size (~2 um) Rapid reproduction (~ hrs) Selective media (e.g., antibiotics) Relative Simple structures and physiology Complete genome sequences © John Wiley & Sons, Inc.

The Genetics of Viruses Viruses (DNA-RNA) can only reproduce by infecting living host cells. Bacteriophages are viruses that infect bacteria. Several important genetic concepts have been discovered through studies of bacteriophages. © John Wiley & Sons, Inc.

Bacteriophage T4 Double-stranded linear DNA genome Protein head Genome contains 168,800 base pairs and 150 characterized genes Lytic phage © John Wiley & Sons, Inc.

T4 DNAse: (Specific) Lysosyme: 5-hydroxymethylcytosine (HMC) Cell wall © John Wiley & Sons, Inc.

Bacteriophage Double-stranded linear DNA genome Genome contains, 48,502 base pairs and about 50 genes May be lytic or lysogenic (inserted in the bacterial chromosome) © John Wiley & Sons, Inc.

Differential genes expression © John Wiley & Sons, Inc.

Recombination process: ---site-specific attP (virus) and attB (bacteria) --- int gene (integrase) --- GCTTTTTTATACTAA --- CGAAAAAATATGATT --homologous recombination © John Wiley & Sons, Inc.

The Genetics of Bacteria Bacteria contain genes that mutate to produce altered phenotypes. Gene transfer in bacteria is unidirectional— from donor cells to recipient cells. S r Energy (Lac / lac); Synthesis Trp / trp; Resistant Amp / Tet Monopliod to monoploid? © John Wiley & Sons, Inc.

Bacteria One main chromosome with a few thousand genes. Variable number of plasmids and episomes. Asexual reproduction by simple fission. (Para) sexual transformation of DNA. © John Wiley & Sons, Inc.

Phenotypes in Bacteria Colony =one bacteria. Colony color and morphology. Nutritional mutants for energy sources. (any sugars-galactose-Lac+ and Lac-) Prototrophs-produce any metabolites- and auxotrophs- do note produce specific metabolites. Antibiotic resistance (Ampr, Tetr, Purr). © John Wiley & Sons, Inc.

Bacterial colonies: Serratia marcencens P. aeruginosa. © John Wiley & Sons, Inc.

Recombination in Bacteria --Small fragment to larger fragment (chromosome) --Monoploid (to partial diploid) -Donor to recipient cell © John Wiley & Sons, Inc.

Mechanisms of Genetic Exchange in Bacteria Bacteria exchange genetic material through three different (para)sexual processes. © John Wiley & Sons, Inc.

Bacteria exchange genetic material through three different (para)sexual processes. © John Wiley & Sons, Inc.

The U-tube Experiment Transformation Conjugation Transduction © John Wiley & Sons, Inc.

TRANSFORMATION Streptococcus pneumoniae---Phenotypes Polysaccharide capsule (S, type I,II, III, IV, V) Type II, agglutination Non- Polysaccharide capsule (R) © John Wiley & Sons, Inc.

Transformation Type IIIS to type IIR Transfer DNA Hydrophilic Non-membrane permeable Transporter (competence-Com-protein) Competent bacteria © John Wiley & Sons, Inc.

Transformation in Bacillus subtilis A heteroduplex is a double-stranded (duplex) molecule of DNA originated by recombination of single complementary strands derived from exogenous sources.

Conjugation in E. coli F+ DNA is transferred from a donor to acceptor cell, F: Fertility factor ; F pili © John Wiley & Sons, Inc.

The F Factor in E. coli F+ factor: --Autonomous and integrated states F’ factor: --Similar to F+ but with bacterial genes F: Fertility factor Hfr: high frequency recombination

Formation of Hfr Cells seq A -seq B seq A -seq B © John Wiley & Sons, Inc.

Conjugation MODEL Replication: Rolling-cycle © John Wiley & Sons, Inc.

F- Hfr strs strr gal+ gal- lac+ lac- leu+ leu- thr+ thr- azis azir tons strr gal- lac- leu- thr- azir tonr 25

Selected medium: -Thr, -Leu, +Streptomycin (str)

Interrupted Mating Experiments © John Wiley & Sons, Inc.

Plasmids A plasmid is a genetic element that can replicate independently of the main chromosome in an extrachromosomal state. Most plasmids are not required for the survival of the host cell. Plasmids in E. coli F Factor (Fertility Factor) R Plasmids (Resistance Plasmids) Col Plasmids (synthesize compounds that kill sensitive cells) © John Wiley & Sons, Inc.

Episomes An episome is a genetic element that is not essential to the host and that can either replicate autonomously or be integrated into the bacterial chromosome. Integration depends on the presence of IS elements. © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

Formation of an F’ Factor Homologous DNA recombination, but some bacterial DNA is translocated to the plasmid. © John Wiley & Sons, Inc.

Transduction In transduction, a bacteriophage transfers DNA from a donor cell to a recipient cell. In generalized transduction, a random fragment of bacterial DNA is packaged in the phage head in place of the phage DNA (normal Excision). In specialized transduction, recombination between the phage chromosome and the host chromosome produces a phage chromosome containing a piece of bacterial DNA (abnormal Excision). Auxotrophic Salmonella typhimurium: tyr, phe, trp and met, his. Infected with P22 bacteriophage © John Wiley & Sons, Inc.

Normal Excision of  Prophage © John Wiley & Sons, Inc.

Anomalous Excision of  Prophage © John Wiley & Sons, Inc.

Specialized Transduction © John Wiley & Sons, Inc.

Three parasexual processes—transformation, conjugation, and transduction—occur in bacteria. These processes can be distinguished by two criteria: whether the gene transfer is inhibited by deoxyribonuclease (DNAase) and whether it requires cell contact. © John Wiley & Sons, Inc.

The Evolutionary Significance of Genetic Exchange in Bacteria Genetic exchange is as important in bacteria as it is in other organisms. © John Wiley & Sons, Inc.

Genetic Exchange in Bacteria Mutation is the source of new genetic variation. (UV radiation) Recombination produces new combinations of allele. (normal and abnormal recombination) Transformation, conjugation, and transduction generate new combinations of genes in bacteria to allow bacteria to adapt to new environments. (soil, sewers and polluted waters) (animal/plants) © John Wiley & Sons, Inc.

Bacteria genetically engineer plants with the Ti plasmid to control their differentiation (tumorigenic- Crown gall disease).