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Bacterial Genetics: Understanding DNA and Mutations

Explore the study of bacterial genetics, including DNA structure, mutations, genetic transfer mechanisms, and functional implications. Learn about plasmids, gene sequencing, and transformation processes.

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Bacterial Genetics: Understanding DNA and Mutations

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  1. Bacterial Genetics

  2. GENETICS-Study of genes their structure & function, heredity & variation • Genomics-Study & analysis of nucleotides of DNA • Nucleic acid-DNA and RNA

  3. Bacterial DNA • Single Haploid Chromosome • Super coiled circular dsDNA=1mm • Exception: • 2 chromosomes : Vibrocholerae

  4. Nucleotides-Structural units of Nucleic acids • Nitrogenous bases-Purines(A,G) and Pyrimidines(T,U,C) Nucleoside • Pentose sugar-Deoxyribose • Phosphate group

  5. Bacterial DNA • Ratio of A+T to G+C constant for each species • Genetic information is stored as a code • Codon-unit,triplet(3 bases) • 64 codon • 61 sense codon code for 20 AA • AGA/AGG/CGA-arginine— code is degenerate • 3 codon UAA/UAG/UGA- nonsense codons

  6. Gene or cistron • Segment of DNA carrying codons for a particular polypeptide synthesis • Locus • a large no of genes • Genome • large no of loci • Letter---------word-----sentence—paragraph--- • Nucleotide—Codon-----Gene--------Locus--- • 1000-3000 Gene • 580 -5200 kbp • length1-1.3mm

  7. Bidirectional replication Rolling circle mechanism • DNA Replication:

  8. RNA • mRNA • rRNA • tRNA

  9. Extra chromosomal elements Plasmids • Free Circular dsDNA-In Cytoplasm for several generations • Replicate independently • Episome-integrated form • Not essential for life of bacteria • Number: up to 40/cell • contain 50-100 genes

  10. Extra chromosomal elements Plasmids • Curing: process of eliminating plasmid from bacteria • Spontaneous • induced • Acridine • Radiation • Thymine starvation • High temp

  11. Classification On the basis of ability to perform conjugation: • Conjugative/self transmissible plasmid • Non conjugative plasmid Based on compatibility b/w plasmid: • Compatible • Incompatible

  12. Classification Based on function: • Fertility/F plasmid: contain tra gene: sex pili expression • Resistance/R plasmid • Col plasmid • Virulence plasmid • Metabolic plasmid

  13. Variation • Phenotypic • Genotypic

  14. MUTATIONRandom, heritable variation caused by alteration in nucleotide sequence of DNA Frequency 10-2 -10-10/bacterium/division CAUSES • Spontaneous • Induced (mutagen) – • Physical: UV • Chemical: alkylating agent, 5-FU, acridine dye

  15. Functionally affect: • Not able to produce Capsule/flagella • Antigenic structure alteration • Altered sensitivity to Bacteriophage • Drug resistance • Altered pigment production • Altered Biochemical reactions • Altered colony morphology

  16. Types: Forward mutation • Substitution • Transversion: • Transition:

  17. Types: Forward mutation • Substitution • Silent: New codon code for same AA • Neutral: New codon code for functional equivalent AA • Missense: Different AA • Non-sense: Stop

  18. Types: Forward mutation • Substitution • Silent: New codon codes for same AA • Neutral: New codon codes for functionally equivalent AA • Missense: Different AA • Non-sense: Stop

  19. Types: • Addition or deletion • Frame Shift • Reverse Mutation • True reversion

  20. Types: • Reverse Mutation • Equivalent reversion: 2nd Mutation different codon but same AA

  21. Types: • Reverse Mutation • Suppressor mutation: 2nd mutation in a different gene that revert the phenotypic effects of already existing mutation

  22. Demonstration of Mutation • Gene sequencing • Phenotypic changes • Fluctuation test • Replica plating

  23. Fluctuation test • Luria and Delbruck-Mutation is SPONTANEOUS and RANDOM- • Growth in presence of selective inhibitory pressure • Bacteriophage and E. coli

  24. Replica plating-Auxotrophic mutant

  25. Ames Test (carcinogenicity testing) • Mutational reversion assay Estimate Mutagenicity of mutant

  26. GENETIC TRANSFER • Vertical • Horizontal • Transformation • Transduction • Lysogenic conversion • Conjugation

  27. Transformation- Random uptake of free / naked DNA incorporation into chromosome • Natural – S. pneumoniae • express DNA-binding proteins on cell surface • natural competent state allows uptake of "naked DNA"

  28. Transformation- random uptake of free / naked DNA incorporation into chromosome

  29. 1928: Frederick Griffith (London): First demonstrated bacterial transformation

  30. An "S" or SMOOTH coat strain, which is lethal to mice.

  31. An “R" or rough coat strain, which is NOT lethal to mice.

  32. Griffith found that he could heat inactivate the smooth strain.

  33. heat-inactivated S strain,mixed with the R strain, the mouse would die.  Thus there was someMaterial in the heat-killed S strain that was responsible for "transforming" the R strain into a lethal form.

  34. GENETIC TRANSFER • Vertical • Horizontal • Transformation • Transduction • Lysogenic conversion • Conjugation

  35. Transduction-Transfer of genetic material through bacteriophage

  36. Transduction 2 types: • Generalized: • Packaging error • 3 outcome on transduction: • Abortive transduction: 70-90% • Stable gene transfer • Unstable gene transfer

  37. Transduction 2 types:

  38. Transduction 2 types: • Restricted/specialized • Defect in disintegration of lysogenic phage • 2 outcome when transduced to new bacteria • Cross over • Integrated as prophage

  39. Transduction 2 types:

  40. Importance of transduction • Drug resistance: Pn resistance in Staphylococci • Treatment: Genetic mapping, inborn error of metabolism • Phage vectors used in molecular transformation of bacteria

  41. Lysogenic Conversion • In Lysogenic bact prophage acts as additional segment of bact chromosome-new characters-lysogenic conversion eg. C.diphtheriae and its bacteriophage • Phage coded Toxins: • Diphtheria toxin • cholera toxin • Verocytotoxin of E. coli • Streptococcus pyrogenic exotoxin A & C • Botulism toxin C & D

  42. Lysogenic conversion: Phage DNA itself behave as new genetic element • Transduction: Phage act as vehicle carrying bacterial gene

  43. GENETIC TRANSFER • Vertical • Horizontal • Transformation • Transduction • Lysogenic conversion • Conjugation

  44. Bacterial Conjugation • Transfer of genetic information from one bacterium (donor or male) to another bacterium (recipient or female) bacterium by mating or contact with each other & forming conjunction tube

  45. F+ F- Mating • HFR conjugation • F’ Conjugation Col factor R factor-RTF + r determinants

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