Stepping stones towards a new electronic prokaryotic taxonomy. The ultimate goal in taxonomy. Pragmatic towards diagnostics

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1 Stepping stones towards a new electronic prokaryotic taxonomy - MLSA - Dirk Gevers Different needs for taxonomy Describe bio-diversity Understand evolution of life Epidemiology Diagnostics Biosafety... Laboratory of Microbiology Pragmatic towards diagnostics A classification that is of little use to microbiologists no matter how fine a scheme or who devised it, will soon be ignored or significantly modified The ultimate goal in taxonomy An objective delineation of species based upon the natural relationships of organisms, i.e. based upon evolutionary evidence E. Stackebrandt Classification of strains into recognizable functional units never static as new info is constantly being generated current practice DDH Polyphasic description of species observation 2 observation 3 observation 1 16S rrna gene sequencing initial grouping using a polyphasic approach 1

2 mic taxono d n quicksa Landscaping the microbial diversity How many bacteria Which bacteria Seazonal influence Location dependency om Taxon P. Dawyndt y Within a timespan of weeks, not years Future is easier to invent than to predict Whole genome sequencing is the future Future needs Fast (large biodiversity to explore) Although whole genome sequences are the blueprint of the organism and clearly contains detailed taxonomic information on the strain, it is not realistic to think that we will have whole genome sequences for all new strains in the near future. Portable ( online taxonomy ) For now, 16S rrna gene sequencing is the standard in environmental studies and a more pragmatic alternative could be provided by a multi locus sequencing approach, providing: Resolution (1) An increase in phylogenetic signal/accuracy Robust (study populations) Functional (2) Multiple genes provide a buffer against the distorting phylogenetic signals at a single locus, such as effects of recombination, gene conversion, and horizontal gene transfer What is MLSA? Sequencing of multiple protein-coding genes (loci) and subsequent phylogenetic analysis of the(se) (concatenated) sequence alignment(s) to characterize the genetic relationships among the strains analyzed. MLST is for genotypic characterization of prokaryotes at the infraspecific level using only the allelic mismatches of usually 7 housekeeping genes (typing, epidemiology). In order to be applicable for more diverse groups of strains, the use of simple clustering procedures based on the number of allelic mismatches is invalidated. 2

3 current future practice genotype vs. phenotype DDH 16S multilocus rrna gene sequencing...classification must be a reflection of the natural relationships of bacteria, i. e. their degree of DNA similarity... Wayne et al., 1987 In the end we want biological meaningful groups, i.e. groups that reflect phenotype and the evolutionary process that have been shaping their genotype initial grouping using a polyphasic approach A 3-step process (1) 16S rrna sequencing to assign an unknown strain to a group (genus or family). (2) This defines which genes and primers are to be used for MLSA to assign the strain to a species (3) Polyphasic approach for a valid description of species How does a few genes correlate with the whole-genome-based relatedness? Konstantinidis, unpublished Konstantinidis et al., AEM (in press) 3

4 Which genes to sequence? Bacterial core genes (universal, house keeping) Which genes to select? Not duplicated Sufficient length Congruent with the overall genome phylogeny With universally conserved regions for primers Unlinked in the genome (e.g. to avoid hitch-hiking of (Zeigler, 2003) DNA exchange (Santos and Ochman, 2004) selection and recombination events) Which genes to sequence? Group specific approach Not duplicated Sufficient length Congruent with the overall genome phylogeny With universally conserved regions for primers Unlinked in the genome (e.g. to avoid hitch-hiking of Combining gene sequences? How to combine data from a different taxonomic range? 0 true biological distance (Zeigler, 2003) (Santos and Ochman, 2004) DNA exchange clonal level species level genus level... selection and recombination events) gene 1 gene 2 16S rrna intra- vs interspecies diversity interspp. intrasp. 0 4

5 intra- vs interspecies diversity interspp. intrasp. 0 MLSA is attractive... Objective method for classification Allows a more natural species definition Fast (large biodiversity to explore) Portable ( online taxonomy ) Increased resolution = solution to the burden of routine species identification MLSA has its uncertainties and flaws... Link between species and level of similarity? (lack of theory-based concept) A single definition possible for groups in which different biological processes have caused speciation (Neisseria vs. Mycobacterium) Link with the phenotype will always be important MLSA has its uncertainties and flaws... MLSA has its uncertainties and flaws... Link between species and level of similarity? (lack of theory-based concept) A single definition possible for groups in which different biological processes have caused speciation (Neisseria vs. Mycobacterium) Link with the phenotype will always be important It ignores the variable part, potentially the genetic basis of ecological differentiation On a larger scale it might not be possible to delineate groups as a result of a continuous spectrum of genotypic variation How to apply to uncultured material? 5

6 an MLSA platform! Acces to all sequences and analysis tools Suggest genes and enforce the use of the same genes in order to obtain an unprecedented scale enlargement (necessary to evaluate robustness, study concept,...) Incorporate organisms biology (ecological data, source, phenotype,...) connectivity Landscaping the bacterial diversity understanding relationships experimental data understanding patterns classification methods understanding principles Reflection - 1 Reflection - 2 Should we calibrate each new method to yield the clusters previously determined by phenotypic criteria? - How to tackle the lack of theory-based concept? Should the complete genome be used as the reference standard? DDH is a measure for genomic relatedness, but is it a measure for evolutionary relationship... and thus... is there a core that represents organismal phylogeny... is there a true phylogeny possible Kunin GR 2005 Not all genes in the genome tell the same phylogenetic history How to determine true organismal phylogeny? DNA exchange 6

7 - Laboratory of Microbiology - Reflection - 3 What with our nomenclature - named species are not meaningful from an evolutionary standpoint Em. Prof. Dr. ir. Jean Swings Prof. Dr. Peter Vandamme Prof. Dr. Paul De Vos Prof. Dr. Anne Willems Dr. Peter Dawyndt Dr. Marc Vancanneyt Dr. Sabri Naser 7