1. GN3502: Bacterial Genetics Ken Forbes Medical Microbiology

2. 1. Classical bacterial genetics 2. New approaches Physical mapping of genomes Whole genome sequencing Functional analysis 3. New perspectives on bacterial genetics Origin of species Bacterial lifestyles Lecture synopsis

3. Classical view of bacteria Single chromosome May have plasmids and phage Simple gene structure Genes have recognisable phenotype Can do genetics in lab –gene transfer transformation transduction Conjugation –molecular biology

4. Classical methods are not adequate Bacteria live in many diverse habitats Much diversity within a species Most genes in most species have not yet been identified

5. Have most of the genes in any species been identified? Traditional genetic and molecular methods have identified a function for only half of the genes in E. coli Constraints from –methodologies –many genes will not be expressed in the lab New approaches needed –genome oriented –sequence oriented

6. 1. Classical bacterial genetics 2. New approaches Physical mapping of genomes Whole genome sequencing Functional analysis 3. New perspectives on bacterial genetics Origin of species Bacterial lifestyles Lecture synopsis

7. Lecture synopsis: 2. New approaches Physical mapping of genomes Methods: PFGE clone libraries Discoveries: bacterial genomes size shape replicons Whole genome sequencing Methods: sequencing strategies Discoveries: gene organisation assigning function Functional analysis Discoveries: new genes Methods: for individual genes for whole genomes DNA arrays proteome

8. Physical mapping of genomes Low resolution restriction enzyme maps of whole genome Locate genes on the map using DNA-based techniques PHYSICAL map of chromosome not a GENETIC map

9. Restriction map whole chromosome with rare cutting REs –complete digests –partial –double digests Pulsed-Field Gel Electrophoresis E E E E E E E E HH H 1 Mb S S H S E HSHS(H)

10. Molten agarose Cultured cells Incubate with Proteinase K Trapped HMW DNA Embedded Cells Inactivate Proteinase K & wash to remove cell debris Pulsed-Field Gel Electrophoresis

11. Digest with Rare-cutting restriction enzyme + Periodic Switching (pulsing) between electrode pairs Net migration - + - Pulsed-Field Gel Electrophoresis

12. Mapping genes on whole genome RE maps E E E E E E E E HH H S S | geneA geneB | geneC | Hybridize cloned-gene DNA fragment to PFGE fragments –locate gene on map

13. Ordered clone libraries method Make clones of entire genome – Ø clones of whole genome Small (10s kb) size of inserts means 1000s clones required to cover whole chromosome –Bacterial Artificial Chromosomes (BAC) clone in E.coli F plasmid large (100s kb) size of inserts means fewer clones needed Order the clones into contigs –overlapping clones will cross hybridise

14. Ordered clone libraries Disadvantages –not all regions clonable –labour intensive and expensive Advantages –immortalised source of genomic DNA –minimally redundant –easy to find and sub-clone a gene of interest –identify adjacent genes –use in genome sequencing projects

15. Ordered clone libraries applications E. coli K12 –widely used lab strain Mycobacterium leprae –obligate human pathogen –not cultivable in vitro –genetic analysis impossible –ordered clone library allowed molecular genetic analysis

16. Physical mapping Pros –only need DNA of organism –standard molecular biology methods used Cons –low resolution –no phenotypic information about genes

17. Physical mapping of genomes Methods: PFGE clone libraries Discoveries: bacterial genomes size shape replicons Whole genome sequencing Methods: sequencing strategies Discoveries: gene organisation assigning function Functional analysis Discoveries: new genes Methods: for individual genes for whole genomes DNA arrays proteome Lecture synopsis: 2. New approaches

18. Bacterial genomes come in many different sizes Range 0.6Mb – 9Mb Bigger genomes encode more genes < 2Mb specialist species –restricted ecological niche (Mycoplasma) –fastidious growth (Haemophilus influenzae) –obligate intracellular parasites (Chlamidia) 3 – 5Mbgeneralist species –broad metabolic potential, few organic growth requirements (E. coli) > 5Mbspecies with developmental cycles –(Streptomyces: mycelial growth, spores, complex bioactive compounds)

19. Bacterial genomes come in different conformations Circular chromosomes –the traditional view: E. coli Linear chromosomes –Borrelia Plasmids –circular and linear forms

20. Bacterial genomes can have several chromosomes Chromosomes must harbour some essential genes –ribosomal RNA (rrn) Plasmids should not be required for viability –only encode supplementary functions –can be very large (1-2 Mb)

21. Bacterial genomes Most species have one chromosome –eg E. coli 1x circular chromosome with rrn, housekeeping genes Some species have 2 chromosomes (a few 3) –eg Agrobacterium tumefaciens 2x chromosomes each with rrn and housekeeping genes –1x circular 3Mb –1x linear 2Mb 2x plasmids, circular 200kb, 450kb

22. Physical mapping: conclusions Bacterial genomes are very variable –chromosome size, conformation, number –plasmids often very important, but not essential Genomes have a large coding capacity –this reflects bacterial biodiversity –there are many genes of unknown function –laboratory analysis imposes constraints on understanding of many genes How can you identify all of the genes in a species?

23. Physical mapping of genomes Methods: PFGE clone libraries Discoveries: bacterial genomes size shape replicons Whole genome sequencing Methods: sequencing strategies Discoveries: genome organisation identify genes Functional analysis Discoveries: new genes Methods: for individual genes for whole genomes DNA arrays proteome Lecture synopsis: 2. New approaches

24. Whole genome sequencing Whole genome sequences now available for –300 bacterial species/ strains –most pathogens –representatives of most bacterial lineages Haemophilus influenzae genome published 1995

25. Whole genome sequencing Advantages –inexpensive –all of genome seq available –all genes identified Requirements –automated DNA sequencing machines –massive computing power Factory sequencing

26. Fluorescent sequencing DNA sequencing reaction –Sanger terminator chemistry nt chain extension until blocked by terminator nt –terminator nt has fluorescent dye attached each nt has different colour

27. Phases of sequencing project Primary sequencing phase –random accumulation of seq into contigs Linking phase –contigs linked together using directed sequencing methods Polishing phase –removal of sequence ambiguities from the single contig Finished sequence –analyse, annotate

28. Genome sequencing strategies Total-genome shotgun sequencing Primer walking Mixed strategy

29. Total-genome shotgun sequencing Shotgun cloning –shear DNA into random fragments of 1-5kb –clone into vector Sequencing primers in vector vector cloned insert sequencing primers

30. Total-genome shotgun sequencing advantages Dont require map of genome Sequencing machines at continuous full capacity Sequence polishing only done once >er accuracy through multiple coverage –6-10 fold genome equivalents

31. Total-genome shotgun sequencing disadvantages Repeat coverage is wasteful Cant clone some genomic regions Repetitive regions in genome –cant map each to its correct genomic position –prevents contigs from being joined together other methods required to span across each repeat Sequence assembly and analysis can only be done at end of sequencing phase

32. Primer walking Require ordered clone library Primer walk along each cloned fragment –first primer in vector sequence into cloned DNA –next primer in new seq sequence further into cloned DNA –start at each end of cloned fragment –cycles of: sequencing polishing primer design primer synthesis

33. Primer walking Advantages –high quality, useable sequence obtained from start –sequence produced in large contigs –no repeat coverage –both strands sequenced Disadvantages –many expensive primers needed –time lag between walks –little automation, sequencing machines often idle

34. Mixed strategy Most popular strategy Combine advantages of both methods –initial random- sequencing phase on either whole genome or on set of ordered clones typically 3-6 fold coverage –final primer-walking over gaps

35. Ultrahigh throughput sequencing Sequencing by Synthesis – SBS –eg SOLEXA –generates short (18-35 base) reads video of chemistry

36. Ultrahigh throughput sequencing Sequencing by Synthesis – SBS –template of tens of millions of individual, clonally amplified DNA fragments –yields up to 1 gigabase sequence in total –avoids cloning steps –inexpensive: £500/ bacterial genome

37. Physical mapping of genomes Methods: PFGE clone libraries Discoveries: bacterial genomes size shape replicons Whole genome sequencing Methods: sequencing strategies Discoveries: genome organisation identify genes Functional analysis Discoveries: new genes Methods: for individual genes for whole genomes DNA arrays proteome Lecture synopsis: 2. New approaches

38. Genome organisation Can identify –all protein and RNA coding genes –organisation of genes in genome wrt each other

39. E. coli genome Traditional genetic and molecular methods have identified 2220 genes in E. coli

40. E. coli genome Whole genome sequencing has identified 4288 protein coding genes in E. coli genome

41. E. coli genome genetic map = 100 min physical map = 4.6Mb 1min = 46Kb

42. Genome organisation >90% of genome codes for genes Genes –identified in genome sequence by Open Reading Frame (ORF) homology to known genes in other spp Regulation of gene expression –promoter and ribosome binding site sequences –operons and linked genes

43. Identifying genes: by phenotype Genes traditionally identified by genetic analysis –Robust identification of gene by its function

44. Identifying genes: by DNA homology Identify gene by sequence homology Need previously characterised gene in another species –high homology between them –robust identification of the previously characterised gene –But new gene may have different biological role

45. Identifying genes: by Open Reading Frame ORF: a DNA seq with no stop codons Only genes coding for proteins Ends of the gene not easily defined

46. Bacterial genomes have many genes with no known function 60% of genes have a recognisable function – but the specific role of many are unknown 40% of genes have no known function –10% found in other species conserved protein families important housekeeping genes? –30% unique to each sp determine pathogenicity, lifestyle

47. Physical mapping of genomes Methods: PFGE clone libraries Discoveries: bacterial genomes size shape replicons Whole genome sequencing Methods: sequencing strategies Discoveries: genome organisation identify genes Functional analysis Discoveries: new genes Methods: for individual genes for whole genomes DNA arrays proteome Lecture synopsis: 2. New approaches

48. Assigning function to novel genes How do you determine the function of genes identified by seq rather than by phenotype? For individual genes use an appropriate molecular genetic technique –gene knockouts –conditional lethal mutations –control region probes

49. Assigning function to new genes Individual genes gene knockouts conditional lethal mutations control region probes Whole genome DNA arrays proteome analysis

50. DNA arrays Macroarrays –DNA fragment probes (eg PCR product) –one per gene –array on membrane (10 3 s) Microarrays –oligonucleotide probes –several oligonucleotides per gene –array on glass (10 5 s)

51. DNA arrays Colour = relative ORF expression Intensity = extent ORF expression Sample A Sample B Expression in both samples

52. DNA arrays: applications Gene expression (mRNA) –transcriptome Presence/ absence genes (DNA) –genome polymorphisms

53. Proteomics 2D electrophoresis of cellular proteins –separate by charge then by size –AA sequence spot of interest –refer back to genome sequence Characterisation of all expressed proteins

54. 1. Classical bacterial genetics 2. New approaches Physical mapping of genomes Whole genome sequencing Functional analysis 3. New perspectives on bacterial genetics Origin of species Lifestyles Lecture synopsis

55. Why have bacteria so many genes? 60% have recognisable function – specific role of many genes unknown eg only to enzyme class 40% have no known function –10% common, conserved gene families –30% unique to each species

56. Some genes are common to many species Conserved gene families Presumably housekeeping genes Potential targets for novel antibacterials

57. Some genes are unique to one species These genes give a sp its unique characteristics Allow adaptation to a particular lifestyles Virulence genes

58. How many genes does a pathogen need? Mycobacterium tuberculosis –mechanism of pathogenesis unknown –4.4 Mb genome –3994 genes 1 / 3 known function 1 / 3 similar proteins 1 / 3 unknown in vivo 300 genes not required in vitro 3000 genes not required

59. Some species are apparently missing genes Many pathogens have complex growth requirements Some functions or pathways absent –genes for some pathways eliminated nutrients supplied by host –adaptation to niche H.pylori lives in acidic environment of stomach does not ferment sugars (acidic products) does ferment amino acids (alkaline products)

60. Community Genomics Among Stratified Microbial Assemblages in the Ocean's Interior (2006) DeLong, et al Science 311, pp. 496-503 Planktonic microbial communities in Pacific Ocean –sampled from ocean surface to sea floor –sequenced 64 million base pairs –thousands of new genes Variations in sequencs at different depths –near the ocean surface photosynthetic and mobile microorganisms more genes for iron uptake –deeps a predominance of "adhesive" microbes antibiotic synthesis genes

61. Organisms do not live in isolation Organisms interact with host/ environment Organisms often dependent on each other –nutrient flow through biological systems Use genomics to understand the interaction between spp at gene level Bacteria are diverse

62. Stereo micrograph of dental plaque. Nutrient flow from cocci to filamentous bacteria.