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

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

7. 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 8.2 Spontaneous Mutations

8. 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 8.2 Spontaneous Mutations

9. 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 8.2 Spontaneous Mutations

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 NH 2 groups to C=O groups

11. 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 8.3 Induced Mutations

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

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

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

16. 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 8.4 Repair of Damaged DNA

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

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. 8.11 Transposons ABC TPD EC D XYZ C D Jump XYZC TPD Integration