Vol 6 | June 2008

1. Vol 6 | June 2008 extra information in the notepad! Presenter: Constantin Bode

2. Classifing bacteria • in the 1970s: DNA-DNA hybridization was introduced • Isolates that showed >70% DNA homology were considered to belong to the same species • 16S ribosomal RNA • ubiquitous in bacterial and archeal genomes • matches to 98% of the 70% cut-off method • High-throughput method • with a few drawbacks: • you cannot distinguish between some phenotypically distinct species (e.g. Bacillus thuringiensis and B. anthracis) • organisms were the ‘universal‘ primers do not fit are not detected • pour method for resolving sub-populations within species

3. Classifing bacteria • Mulitlocus enzyme electrophoresis (MLEE) • classifies bacteria on the basis of the isoforms of a combination of approx. 15 metabolic enzymes • drawback: • low throughput (intensive laboratory work) • not widely used • Multilocus sequence typing (MLST) • based on the partial sequences of 7 housekeeping genes of approx. 450 bp each • high throughput • allows direct comparison between different laboratories • > Database (MLST Public Repository) • drawback: • for some species there is too little sequence variation in the housekeeping genes for a sufficient discrimination • drawback for 16S rRNA and MLST • only limited genome coverage

4. Classifing bacteria • Single-nucleotide polymorphisms (SNPs) • originally developed for use in humans • analysis of single gens • e.g. reconstructing the evolutionary history of S. typhi (82 SNPs) • potential for more general use in bacterial population genetics is still unproven

5. Genomic coverage of genetic typing methods Core genome: encode proteins which are involved in essential functions (replication, transcription and translation) Dispensable genome: encode proteins that facilitate organismal adaptation Neisseria meningitidis: 16S rRNA: 0.07% MLST: 0.2% Salmonella Typhi: SNP: 2%

6. Genetic markers and deviations from population structure Schematic representation of different resolution levels eMLST: extended MLST ST: sequence types ET: electrophoretic types

7. Taxonomic rank modified after: http://en.wikipedia.org/wiki/Taxonomic_rank

8. sequencing technologies Frederick Sanger  developed the Sanger chain-termination method in the late 1970 (Nobel Price)

9. Post-Sanger sequencing technologies

10. Post-Sanger sequencing technologies Roche company reads: 250 bases available since 2005 bad resolution of homopolymer DNA segments (multiple copies of a single base) 400 million high quality bases per 10 hour instrument run Since oct 2008 400 base pairs in length http://www.454.com/about-454/index.asp

11. Post-Sanger sequencing technologies glass slide reads: approx. 25 bases available since 2007 http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/index.htm

12. Post-Sanger sequencing technologies Ilumina inc. reads: approx. 40 bases available since 2006 since June 2009: Full Genome Sequencing Service for $48,000 per genome (first commercial personal genome sequence) http://www.illumina.com

13. Molecular evolutionary mechanisms that shape bacterial species diversity intra-species inter-species population dynamic genetic information of a bacterial species – pan genome Metagenomics (environmental genomics or community genomics) ability to capture genomic diversity within a natural population

14. Pan genome • Pan genome • - core genome • shared by all strains • - dispensable genes • shared by some but not all isolates • - strain-specific genes • unique to each isolate • analysis of 17 Streptococcus pneumoniae genomes core genome of 1,454 genes pan genome of approx. 5000 genes • 142 genomes would need to be sequenced to obtain the complete S.pneumoniae genome (just a assumption) (it is not possible to characterize a species from a single genome sequence) pan genome reflects the selective pressure to generate new adaptive combinations

15. Diversity generating mechanism Evading or avoiding an immune system colonizing a highly variabe enviroment requires diversity! - exchange of DNA - modified clonal growth Simplest mechanism: random change in length (during DNA replication)

16. Diversity generating mechanism Simplest mechanism: random change in length (during DNA replication) Campylobacter jejuni  express different subsets of surface proteins (Heliobacter pylori is also using this mechanism of phase variation)

17. Diversity generating mechanism DNA inversion invertible promoters of Bacteroides fragilis A small section of DNA is inverted by a cleavage-and-ligation reaction that is mediated by a site specific recombinase  control the expression of entire operons

18. Diversity generating mechanism DNA inversion Bacteroides fragilis S specificity M methylation R restriction exchange parts of the coding sequences of expressed genes with sequences from silent cassettes  random expression of different alternative proteins

19. Diversity generating mechanism single base point mutations DNA recombination To see this diversity you need variation between shotgun sequences of the metagenome of a single organism. Can be easly overlooked in clonal cultures. Estimation: >99% of the bacteria in the enviroment cannot be cultured in the laboratory  de novo sequencing is required

20. Applications of the genomic era reverse vaccinology  develop vaccines Rappuoli R. Current Opinion in Microbiology 200, 3:445-450

21. Questions • Thanks for your attention!

22. Definitions Genome: The entire hereditary information of an organism that is encoded by its DNA (or RNA for some viruses) Bacterial typing: A procedure for identifiying types and strains of bacteria. Metagenome: The global genetic repertoire of an environmental niche that is constituted by diverse organisms such as free-living microorganisms in the wild or the commensals of a particular niche in a mammalian host 16S ribosomal RNA: The 16S ribosomal RNA gene is a component of the small bacterial and archaeal ribosomal subunit. The gene includes hypervariable regions that contain species-specific signature sequences which are useful for bacterial and archaeal identification at the species level MLEE: The characterization of bacterial species by the relative electrophoretic mobility of approximately 15 cellular metabolic enzymes MLST: An unambiguous procedure for characterizing isolates of bacterial species using the sequences of internal fragments (usually) seven housekeeping genes. Approximately 450-500 bp internal fragments of each gene are used, as these can be accurately sequenced on both strands using an automated DNA sequencer Pan-genome: The global gene repertoire of a bacterial species that comprises the sum of the core and the dispensable genome SNP: DNA sequence variation that occurs when a single nucleotide in the genome differs between members of a species. Core genome: The pool of genes that is shared by all the trains of the same bacterial species Lateral gene transfer: The mechanism by which an individual of one species transfers genetic material (that is DNA) to an individual of a different species. Metagenomics: The study of the genomic repertoire of all the organisms that live in a particular environment and their activities as a collective. The genomic analysis is applied to entire communities of microorganisms, which bypasses the need to isolate and culture individual microbial species. Reverse vaccinology: A genomic approach to vaccine development that searches the entire genetic repertoire of a pathogen for protective antigens.