BACTERIAL GENETICS

1. BACTERIAL GENETICS

2. Structure of DNA • Double stranded (double helix) • Chains of nucleotides • 5’ to 3’ (strands are anti-parallel) • Complimentary base pairing • A-T • G-C

3. DNA Structure Phosphate-P Sugar-blue Bases-ATGC

4. DNA Replication • Bacteria have closed, circular DNA • Genome: genetic material in an organism • E. coli • 4 million base pairs • 1 mm long (over 1000 times larger that actual bacterial cell) • DNA takes up around 10% of cell volume

5. DNA Replication-occurs at the replication fork • 5’ to 3 ‘ • DNA helicase-unzips + parental DNA strand that is used as a template • Leading stand (5’ to 3’-continuous) *DNA polymerase-joins growing DNA strand after nucleotides are aligned (complimentary) • Lagging strand (5’ to 3’-not continuous) *RNA polymerase (makes short RNA primer) *DNA polymerase (extends RNA primer then digests RNA primer and replaces it with DNA) *DNA ligase (seals Okazaki fragments-the newly formed DNA fragments)

6. Replication Fork

7. Protein Synthesis • DNA------- mRNA------ protein transcription translation Central Dogma of Molecular Genetics

8. Transcription • One strand of DNA used as a template to make a complimentary strand of mRNA • Promoter/RNA polymerase/termination site/5’ to 3’ • Ways in which RNA & DNA differ: • RNA is ss • RNA sugar is ribose • Base pairing-A-U

9. Transcription

10. Translation • Three parts: • Initiation-start codon (AUG) • Elongation-ribosome moves along mRNA • Termination: stop codon reached/polypeptide released and new protein forms • rRNA=subunits that form the 70 S ribosomes (protein synthesis occurs here) • tRNA=transfers amino acids to ribosomes for protein synthesis)

15. PHENOTYPIC VARIATION • Change in the colony characters , capsule or flagella

16. GENOTYPIC VARIATION • Change in the genes resulting in change in protein synthesis

17. Mutations – changes in the DNA • Point mutation – addition, deletion or substitution of a few bases • Missense mutation – causes change in a single amino acid • Nonsense mutation – changes a normal codon into a stop codon • Silent mutation – alters a base but does not change the amino acid

18. Mutations – changes in the DNA • Point mutation – addition, deletion or substitution of a few bases • Missense mutation – causes change in a single amino acid • Nonsense mutation – changes a normal codon into a stop codon • Silent mutation – alters a base but does not change the amino acid

19. Mutations • Changes in base sequence of DNA/lethal and inheritable • Can be: • Harmful • Lethal • Helpful • Silent

20. Normal DNA/Missense Mutation

21. Nonsense Mutation/Frameshift Mutation

22. Ames Test

23. General Features of Gene Transfer in Bacteria • Unidirectional • Donor to recipient • Donor does not give an entire chromosome • Merozygotes • Gene transfer can occur between species

24. Transformation • Definition: Gene transfer resulting from the uptake of DNA from a donor. • Factors affecting transformation • DNA size and state • Sensitive to nucleases • Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus) • Competence factor • Induced competence

25. Transformation • Steps • Uptake of DNA • Gram + • Gram - • Recombination • Legitimate, homologous or general • recA, recB and recC genes • Significance • Phase variation in Neiseseria • Recombinant DNA technology

26. Transduction • Definition: Gene transfer from a donor to a recipient by way of a bacteriophage

27. Head/Capsid Contractile Sheath Tail Tail Fibers Base Plate Phage Composition and Structure • Composition • Nucleic acid • Genome size • Modified bases • Protein • Protection • Infection • Structure (T4) • Size • Head or capsid • Tail

28. Generalized Transduction • Infection of Donor • Phage replication and degradation of host DNA • Release of phage • Assembly of phages particles • Infection of recipient • Legitimate recombination

29. Transduction • Types of transduction • Generalized - Transduction in which potentially any dornor bacterial gene can be transferred. • Specialized - Transduction in which only certain donor genes can be transferred

30. gal gal bio bio gal bio gal bio bio gal Specialized TransductionLysogenic Phage • Excision of the prophage • Replication and release of phage • Infection of the recipient • Lysogenization of the recipient • Legitimate recombination also possible

31. Transduction • Definition • Types of transduction • Significance • Common in Gram+ bacteria • Lysogenic (phage) conversion

34. Donor Recipient Conjugation • Definition: Gene transfer from a donor to a recipient by direct physical contact between cells • Mating types in bacteria • Donor • F factor (Fertility factor) • F (sex) pilus • Recipient • Lacks an F factor

35. F+ Physiological States of F Factor • Autonomous (F+) • Characteristics of F+ x F- crosses • F- becomes F+ while F+ remains F+ • Low transfer of donor chromosomal genes

36. Hfr F+ PhysiologicalStates of F Factor • Integrated (Hfr) • Characteristics of Hfr x F- crosses • F- rarely becomes Hfr while Hfr remains Hfr • High transfer of certain donor chromosomal genes

37. F’ Hfr Physiological States of F Factor • Autonomous with donor genes (F’) • Characteristics of F’ x F- crosses • F- becomes F’ while F’ remains F’ • High transfer of donor genes on F’ and low transfer of other donor chromosomal genes

38. F+ F- F+ F- F+ F+ F+ F+ Mechanism of F+ x F- Crosses • DNA transfer • Origin of transfer • Rolling circle replication • Pair formation • Conjugation bridge

39. Hfr F- Hfr F- Hfr F- Hfr F- Mechanism of Hfr x F- Crosses • DNA transfer • Origin of transfer • Rolling circle replication • Homologous recombination • Pair formation • Conjugation bridge

40. F’ F- F’ F- F’ F’ F’ F’ Mechanism of F’ x F- Crosses • DNA transfer • Origin of transfer • Rolling circle replication • Pair formation • Conjugation bridge

41. Conjugation • Significance • Gram - bacteria • Antibiotic resistance • Rapid spread • Gram + bacteria • Production of adhesive material by donor cells

42. Transposable Genetic Elements • Definition: Segments of DNA that are able to move from one location to another • Properties • “Random” movement • Not capable of self replication • Transposition mediated by site-specific recombination • Transposase • Transposition may be accompanied by duplication

43. GFEDCBA ABCDEFG Transposase Types of Transposable Genetic Elements • Insertion sequences (IS) • Definition: Elements that carry no other genes except those involved in transposition • Nomenclature - IS1 • Structure • Importance • Mutation • Plasmid insertion • Phase variation

44. H2 gene H1 gene H1 flagella H2 flagella Phase Variation in Salmonella H Antigens IS

45. IS Resistance Gene(s) IS IS Resistance Gene(s) IS Types of Transposable Genetic Elements • Transposons (Tn) • Definition: Elements that carry other genes except those involved in transposition • Nomenclature - Tn10 • Structure • Composite Tns • Importance • Antibiotic resistance

46. Plasmids • Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) • Episome - a plasmid that can integrate into the chromosome

47. Classification of Plasmids • Transfer properties • Conjugative • Nonconjugative • Phenotypic effects • Fertility • Bacteriocinogenic plasmid • Resistance plasmid (R factors)

48. RTF Tn 10 Tn 8 Tn 9 Tn 21 R determinant Structure of R Factors • RTF • Conjugative plasmid • Transfer genes • R determinant • Resistance genes • Transposons

49. Genetic basis of drug resistance • Mutational drug resistance - Chromosomal • Transferable drug resistance – Plasmid mediated

50. Genetic engineering • Genetic engineering is the manipulation of genetic material to alter the characteristics of an organism. • Genetic fusion: allows transposition from one location on a chromosome to another, sometimes deleting a portion, thereby causing the joining of genes from two different operons. • Protoplast Fusion: combines protoplasts ( organisms without cell walls) and allows mixing of genetic information.