Bacterial Genetics

1. Bacterial Genetics • Chapter 8

2. The Problem of Antibiotic Resistance • Staphylococcus aureus • Common Gram+ bacterium • Multi-drug resistant strains are prevalent in hospitals • These are described as methicillin-resistant Staphylococcus aureus (MRSA) • Antibiotic resistance is a genetic event

3. 8.1 Diversity in Bacteria • Bacterial diversity • Bacteria have haploid genomes • Mutations in bacterial genes alters genotype • Genotype is the DNA-level information encoded by the genome • Mutations also alter phenotype • Phenotype is principally controlled by proteins by alteration of amino acid composition

4. 8.1 Diversity in Bacteria • Organisms that acquire mutations are termed mutants • Mechanisms of mutagenesis • Spontaneous mutations of DNA • Horizontal gene transfer

5. 8.2 Spontaneous Mutations • Causes of spontaneous mutations • Chemicals and radiation can induce mutations of DNA • Mutases are enzymes that are expressed during times of stress that increase rates of mutations • Types of mutations • Nucleotide base substitution (aka, point mutation) • Deletion or insertion of nucleotides • Transposable elements (”jumping genes”)

6. 8.2 Spontaneous Mutations • Mutations are a numbers game • Rates of mutations vary from 10-4 to 10-12 • Cells have DNA repair mechanisms that repair most mutations

7. 8.2 Spontaneous Mutations • Base Substitution • Silent mutation has no effect on amino acid content • CCC, CCT, CCA, CCG all encode proline • Missense mutation results in an amino acid change • CCC➙CTC = proline to leucine • Can alter 3D shape of protein or compromise critical amino acid • Nonsense mutation results in a premature stop codon • TTG➙TAG = leucine to termination

8. 8.2 Spontaneous Mutations • Removal or addition of nucleotides • Deletions (removal) or insertion (addition) mutations alter the reading frame of DNA • These mutations are termed frameshift mutations • Frameshift mutations are usually intolerant for the bacterium • Spurious amino acid sequences • Premature stop codons

9. 8.2 Spontaneous Mutations • Transposable elements (aka, jumping genes, transposons) • Mobile genetic elements • Can “jump” from species to species • Often carry genes that alter the phenotype of recipient bacteria, including antibiotic resistance

10. 8.3 Induced Mutations • Mutagens are used to discover gene functions • Alterations in genes often result in changes in phenotype • Chemical mutagens • Base modifiers change bases that are misread during DNA replication • Nitrous acid converts NH2 groups to C=O groups

11. 8.3 Induced Mutations • Base analogs resemble normal bases, but have different H-bond characteristics and are mismatched during DNA replication • Intercalating agents, such as ethidium bromide, insert between adjacent bases on a strand, which can lead to the insertion of a base pair by DNA polymerases • This results in a frameshift mutation

12. 8.3 Induced Mutations • Radiation • Ultraviolet radiation induces thymine dimers • X-rays induce double strand DNA breaks

13. 8.4 Repair of Damaged DNA • Cells possess systems that can repair mutated DNA • Repair of errors in base incorporation • Proofreading: Some DNA polymerases can step backward and remove a misincorporated base • Mismatch repair: Endonucleases are enzymes that recognize inappropriate 3D structures of DNA and remove mutant bases, which are corrected by DNA polymerases

14. 8.4 Repair of Damaged DNA • Repair of thymine dimers • Photoreactivation • Recognizes bulges in DNA • Harness light energy to break covalent bond between adjacent thymines

15. 8.4 Repair of Damaged DNA • Repair of thymine dimers (cont.) • Excision repair removes several adjacent bases • DNA polymerases fill in the gap • DNA ligase forms phosphodiester bonds

16. 8.4 Repair of Damaged DNA • Repair of modified bases in DNA • Lesion-specific glycosylases recognize modified bases and remove them • Endonucleases then remove the deoxy-phosphate backbone (i.e., excision repair) • DNA polymerase adds the appropriate base • SOS repair • System of 30+ genes for repair of highly damaged DNA • System of desperation • Highly prone to error

17. 8.5 Mutations and Their Consequences • Mutations are a natural biological process • All DNA polymerases have inherent mutation rates • In times of stress, mutational rates increase (mutases) • Without mutations, evolution cannot occur and organisms will be ill-equipped to adjust to changes in their environment • Evolution requires three events • Genetic variation, which is mostly random (e.g., mutation) • The variations must be heritable • Natural selection of those traits most suitable for an environment

18. 8.6 Mutant Selection • Isolating a mutant is a statistically-unlikely event without selection • In nature, natural selection favors the outgrowth of mutant microbes • In the laboratory, artificial selection is employed to find these unlikely events • Many important strains of microbes have been developed using artificial selection • Oil-consuming bacteria • Heavy metal decomposition • Wines

19. Antibiotic -containing medium Inoculate entire surface with susceptible bacteria 2 weeks 1 week Normal medium Antibiotic gradient 8.6 Mutant Selection • A common method for producing an antibiotic-resistant bacterium

20. 8.7 DNA-Mediated Transformation • Competence • Cells that are receptive to DNA transfer are termed competent • The process of becoming competent is largely unknown, but requires protein synthesis • Competent cells permit DNA to pass through their cell walls and membranes • This process is termed transformation and can lead to the acquisition of new genes • Competent cells can be induced artificially and play an important role in biotechnology

21. 8.8 Transduction • Bacteriophages (aka. phages) are bacterial viruses • They frequently incorporate genes from previously-infected host cells • When the progeny viruses infect other bacteria, the new genetic information can be recombined with the host’s genome • This process is termed transduction

22. 8.9 Plasmids • Plasmids are circular molecules of DNA • They can be hundreds to thousands of nucleotides long • They frequently contain virulence factors that contribute to disease susceptibility • Antibiotic resistance • Toxins • They are considered promiscuous because they can disseminate between species of bacteria • Plasmids are routinely used in biotechnology for gene cloning and recombinant protein production

23. 8.10 Conjugation • Competent cells acquire plasmids by random chance • Conjugation is the direct transfer of plasmids (or chromosomes) between bacteria • It is a four-step process • Contact between a donor cell (F+) and recipient cell (F-) is mediated by a sex pilus, a tubular structure • The plasmid becomes mobilized by an enzyme that cleaves the plasmid • One strand of the plasmid is transferred to the recipient, presumably through the pilus • The copies of the plasmids are used as templates for DNA synthesis

24. 8.10 Conjugation

25. 8.11 Transposons • Transposons (transposable elements, jumping genes) are self-replicating DNA molecules • They occur in virtually all organisms • When they jump, they frequently take adjacent genes with them, such that integration in another cell leads to the introduction of novel genetic information • Trans-species transposition has been observed in nature • All transposons encode DNA and/or RNA polymerase, termed transposases, that mediate jumping

26. Integration TP TP TP D D D TP D C C C A X B Y C Z E Jump X Y Z 8.11 Transposons