Bacterial Genetics 1 Genetic information Genetic elements Replication

1. Bacterial Genetics 1 • Genetic information • Genetic elements • Replication • Genetic information transfer • Regulation of gene expression • Mutation and recombination • Ch. 7, 8, 10

2. Sugar backbone (ribose or deoxiribose) Antiparallel – complementary (phosphate diester bond) purine – pyrimidine A T G C Deoxi-nucleotide triphosphate units: dATP, dTTP, dGTP, dCTP

3. The three key processes of macromolecular synthesis are: • DNA replication; • transcription (the synthesis of RNA from a DNA template); and • translation (the synthesis of proteins using messenger RNA as template).

4. Deoxyribose Thymine T-A G-C Double stranded Ribose Uracil U-A G-C Single but Loop-forming DNA vs. RNA

5. Types of RNA mRNA – messenger tRNA – transfer rRNA – ribosomal -------------------------------------------------- Roles: Genetic (informational) Functional: structure (ribosomes) function (ribozymes)

6. Bacteria: promoters are recognized by the sigma subunit of RNA polymerase. These promoters have very similar sequences. • Eukarya: the major classes of RNA are transcribed by three different RNA polymerases, with RNA polymerase II producing most mRNA. • Archaea: have a single RNAthat resembles in structure and function the RNA polymerase II.

7. Although the basic processes are the same in • both prokaryotes and eukaryotes, but more • complex in eukaryotes. • Eukaryotic genes have both: • Exons (coding regions and • Introns (noncoding regions). • 2. Both are transcribed to primary mRNA • 3. Introns are excised Exons ligated • 4. A cap is added at 3’ end • 5. A poly-A tail added at 5’ end is clipped and a • 6. Mature mRNA leaves the nucleus • 7. Translation takes place in cytoplasm

8. Transformation: Free DNA transfer; Requires competence factor Transduction: Virus mediated transfer Generalized: Accidental DNA fragments packed in the virion (at random; lytic cycle) Specialized: Genes next to prophage are transducted (lysogenic cycle) Conjugation:Plasmid-stimulated transfer F- Recipient w/o plasmid F+ Plasmid only is transferred Hfr Plasmid is integrated in the chromosome both transferred F’ Plasmid w. chromosomal genes

9. Bacterial Genetics 2 • Mutation • Recombination • In vivo – techniques, transformation • transduction, conjugation • Plasmids, Transposalbe elements • In vitro – techniques • Bacterial genomics • Genetic engineering • Ch. 10, 15, 31

10. Different types of mutations can occur at different frequencies. For a typical bacterium, mutation rates of 10–7–10–11 per base pair are generally seen. Although RNA and DNA polymerases make errors at about the same rate, RNA genomes typically accumulate mutations at much higher frequencies than DNA genomes.

11. Genetic Recombination General: RecA Site-specific: Transposase Eukaryotes: mating + crossing over Prokaryotes: transformation transduction conjungation

12. Transformation: Free DNA transfer; Requires competence factor Transduction: Virus mediated transfer Generalized: Accidental DNA fragments packed in the virion (at random; lytic cycle) Specialized: Genes next to prophage are transducted (lysogenic cycle) Conjugation:Plasmid-stimulated transfer F- Recipient w/o plasmid F+ Plasmid only is transferred Hfr Plasmid is integrated in the chromosome both transferred F’ Plasmid w. chromosomal genes

13. Recombination General: homologous: same sequence-different source --- complement partial heteroduplex (prokaryotes). --- crossing-over (eukaryotes) Plasmids: mobilize external receptors & pilus F- no plasmid (competent vs. incompetent) F+ complete transfer regular--- separate circular Hfr complete transfer rare --- integrated F' plasmid + chromosomal genes Interrupted mating Site-specific: Transposable elements: transposase, inverted sequences & repeats Conservative Replicative Integrons Inversions

14. PCR – Polymerase Chain Reaction • Target DNA (organismal) • Oligonucleotide primers flanking the target sequence, • DNA polymerase of a hyperthermophile (Taq) • Heat denaturation of the target dsDNA • Cooling – annealing of the Primers • Primer extension by polymerase in both directions • Repeat of the cycle • Accumulated sequence 10-6 – 10-9 • (Taq = Thermus aquaticus)

15. Microbial Evolution and Systematics

16. EARLY EARTH, THE ORIGIN OF LIFE, AND MICROBIAL DIVERSIFICATION Origin of Earth, Evidence for Microbial Life on Early Earth, Conditions on Early Earth: Hot and Anoxic. Origin of Life Catalysis and the Importance of Montmorillonite Primitive Life: The RNA World and Molecular Coding RNA Life The Modern Cell: DNA —> RNA —> Protein

17. Cambrian stromatolite, ca. 500 My. Old, South Australia Scale is in inches, Courtesy of Stanley M. Awramik

18. Great Slave Lake, Canada Ancient ca. 2 • 109y. Recent Hamelin Pool, Shark Bay, W. Australia Stromatolites dominated 5/6 of the etire history of Earth Cryptozoon Kalkowsky, V.H. 1908: Oolith und Stromatolith im Norddeutschen Buntsandstein

19. Early Proterozoic stromatolite ca. 3000 My. old, South Africa Scale bar is 10 cm long

20. Modern subtidal stromatolites Shark Bay, Australia

21. Modern Conophyton equivalent

24. Old Faithful Geyser Microbially guided silica deposition Yellowstone National Park

25. Microbial reefs with silica deposition Yellowstone National Park

26. Tom Brock in Yellowstone 1975

27. Modern subtidal stromatolites, Lee Stocking Island, Bahamas

28. Microbial mat (red) – Stromatolite (yellow) Sediment accumulation

29. Fossil2000 My old stromatolite Modern marine stromatolite Belcher Island Formation, Canada Shark bay, Western Australia Eoentophysalis belcherensis Entophysalis major Hofmann Ercegovic

30. Entophysalis major Baja California, Mexico Eoentophysalis belcherensis Mesoproterozoic, ca. 1400 Ma. Gauyuzhuang Formation, China

31. Planet Earth is approximately 4.6 billion years old. The first evidence for microbial life can be found in rocks about 3.86 billion years old. Early Earth was anoxic and much hotter than the present. The first biochemical compounds were made by abiotic syntheses that set the stage for the origin of life.

32. Condition on Early Earth: Hot and Anoxic Organic synthesis and stability Catalysis on clay and pyrite surfaces RNA and molecular coding

33. The Modern Cell: DNA —> RNA —> Protein

34. The first life forms may have been self-replicating RNAs. These were both catalytic and informational. Eventually, DNA became the genetic repository of cells and the three part system, DNA, RNA, and protein, became universal among cells.

35. Primitive Life: Energy and Carbon Metabolism

36. Molecular Oxygen:Banded Iron Formations

37. Oxygenation of the Atmosphere: New Metabolisms and the Ozone Shield

38. Primitive metabolism was anaerobic and likely chemolithotrophic, exploiting the abundant sources of FeS and H2S present. • Carbon metabolism may have included autotrophy. • Oxygenic photosynthesis led to development of banded iron formations, an oxic environment, and great bursts of biological evolution.

39. Phanerozoic 0-0.54 Neoproterozoic 0.54-1.0 Mesoproterozoic 1.0-1.6 Paleoproterozoic 1.6-2.5 Late Archaean 2.5-3.0 Early Archaean 3.0-3.5 Hadean 3.5-4.6

40. Origin of the Nucleus • Origin of organelles as organisms • Endosymbiosis • Lateral flow of genetic information • Reduction of redundancies

41. Evoluntionaryhistory of endosymbiosis: Mitochondria and Chloroplasts

42. 2 Evoluntionaryhistory of chloroplast endosymbiosis 3

43. Cyanobacteria 6. Euglenophyta • Glaucophyta 7. Chlorachniophyta • Cryptomonads 8. Dinoflagellata (green) . • Rhodophyta 9. Dinoflagellata (brown) • Chlorophyta 10. Chrysophyta, Heterocontae, Diatoms

44. Evoluntionaryhistory of chloroplast endosymbiosis: The Hosts

45. Evidence of Endosymbiosis • Size of ribosomes 80S vs. 70S • Organellar DNA present • Organellar DNA is circular • Multiple membranes • Sensitivity to antibiotics • Models of Symbioses

46. The eukaryotic nucleus and mitotic apparatus probably arose as a necessity for ensuring the orderly partitioning of DNA in large-genome organisms. • Mitochondria and chloroplasts, the principal energy-producing organelles of eukaryotes, arose from symbiotic association of prokaryotes of the domain Bacteria within eukaryotic cells, • The process is called endosymbiosis. • Assuming that an RNA world existed, self-replicating entities have populated Earth for over 4 billion years.

47. Microbial evolution, phylogeny and classification Fossil record vs. Molecular view of evolution DNA composition GC to AT ratio DNA-DNA-hybridization: melting – reanealing DNA-sequence similarity and interrelatedness DNA-sequencing: in vivo vs. in vitro Synthetic DNA – PCR – Molecular cloning Fossil record, and endosymbiotic events Phylogenetic classification Bacterial Classification by phenotypes

48. Universal distribution • Functional homology • Conserved sequences for alignment • Slow rates of evolutionary change • Lack of functional constraint • Ribosomal database Project (RDP) • >100,000 sequences. Criteria for Molecular Chronometers

49. Ribosomal RNAs as Evolutionary • Chronometers • Ribosomal RNA Sequences as a Tool of • Molecular Evolution • Sequencing Methodology • Generating Phylogenetic Trees from RNA • Sequences

50. Comparisons of sequences of ribosomal RNA can be used to determine the evolutionary relationships between organisms. • Phylogenetic trees based on ribosomal RNA have now been prepared for all the major prokaryotic and eukaryotic groups.