2-Classificaon-of-Microorganisms-Polyphasic-Taxonomy.pdf

Classifica(on of Microorganisms: Polyphasic Taxonomy Assoc. Prof. Zeus S. Elumba, Ph.D. MBBGM Division, Institute of Biological Sciences Central Mindanao University Notice: This material is provided to you as a study material/guide in Bio 43 (General Micro...

Classifica(on of Microorganisms: Polyphasic Taxonomy Assoc. Prof. Zeus S. Elumba, Ph.D. MBBGM Division, Institute of Biological Sciences Central Mindanao University Notice: This material is provided to you as a study material/guide in Bio 43 (General Microbiology). You are not permitted to share this material either in electronic or printed forms including uploading it in the public databases. Why identify and classify microorganisms? y o g o l Determine biodiversity. b i Study the functions/roles of microbial species. r o ic MU l M C Species – a basic taxonomic unit. r a S, What is a species? (recallnthe e species I B concept). G e 3 Definition of prokaryotic “a group of strains3 2 species: 0 4 consistency, showing 70%2 that are characterized by a certain degree of phenotypic of DNA–DNA binding and over 97% of 16S ribosomal RNA (rRNA) i ogene-sequence © identity” (Gevers et al., 2005). B Entails Polyphasic Taxonomy.  g y l o i o b o r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B Prokaryotic classification Polyphasic Taxonomy y o g o l A consensus type of taxonomy; no rules. b i r o it considers all available phenotypic and i c U genotypic data and integrates them in a consensus type M M of classification, C framed in a general phylogeny derived from a l16S , rRNA sequence analysis (Vandamme, 1996). e r S e n I B G 023 4 3 2 i o © B  Genotypic g y l o data i o b o r i c M UGeneral Phenotypic data l M C phylogeny r a S , n e I B e G 023 4 3 Polyphasic 2 Taxonomy i o © B Phenotypic data y o g o l b i Cell wall composition (Gram-negative vs. Gram-positive). r o Cellular fatty acids. ic MU Isoprenoid quinones. l M C Polyamines. r a S, e IB Total cellular proteins. e n G 023 4 3 2 i o © B Phenotypic methods g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B (Raina et al., 2019) Genotypic data y o g o l b i DNA base ratios (mol % G+C). (same species < 3% variation; same genus < 10% variation). r o ic MU rRNA gene sequencing (sequence similarity of 16s rRNA sequence = > 97%, same species). l M C r a S, DNA-DNA reassociation (same species > 70% hybridization). n e I B Conserved gene sequence analysis (genes other than 16s rRNA, i.e. rpoA, rpoB, rpoC, etc.). e G 023 4 3 DNA fingerprinting (REP, BOX, ERIC PCR). 2 i o © Multilocus Sequence Typing. (allelic profiling). B In silico phenotyping- predicting phenotypes based on genome sequences. Genotypic methods g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B (Raina et al., 2019) Describing and Reporting a New Species/Taxon y o g o l Key information: b i r o Genome sequences of the type strains of prokaryotes. 16S rRNA gene sequence. ic MU l M C Isolation, habitat, and sample description. r a S, Morphology and growth conditions. Physiology. n e I B Chemotaxonomy. e G 023 Species description (Protologue). 3 2 4 International Publication in the Journal of Systematic and i Evolutionary oMicrobiology. © B Submission of pure cultures in two international culture collections. The Molecular Chronometer y o g o l b a highly conserved protein (e.g. ubiquiTn) or inucleic acid (e.g. an rRNA) whose rate of mutaBon is constant,r o and which can therefore ic MU be used to construct phylogeneBc trees. M l , C (e.i., based on sequence To determine true evoluTonaryarelaTonships similarity of rRNA gene). e r S e n I B G 023 4 3 2 i o © B  g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B LUCA- last universal common ancestor. The university phylogenetic tree of life based on 16s rRNA gene sequence.  g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B © Pearson Education 2019. Brock Biology of Microorganisms. The right chronometer: y o g o l 1. Should be universally distributed across theb i group(s) of interest. r o 2. Must be funcBonally homologous (or i c U of idenTcal funcTon in each organism). M C M l , aalignment 3. Must be amenable to proper e r S with another to idenTfy e n regions of sequence homology I B and sequence variance. G 0which 4. Should have a sequence 2 3 should change at a rate commensurate3with the evoluTonary distance measured. 4 2 i o © B Molecules used as evolutionary chrometers y o g o l Cytochromes (cellular respiration). b i r o Fe-S proteins. ic MU Genes encoding ATPase, RecA, rRNAs. l M C r a S, n e I B e rRNAs: have characteristics G among that 3 are important in studying evolutionary differences 3 0 2 organisms (including prokaryotes). 4 2 i o © B rRNAs are ideal molecular chronometers. y Why? o g o l 1. Universally distributed among organisms.b i r o 2. Exhibit functional constancy. c Ui Key components in the translational M system. M l a S, “antiquity of the protein-synthesizing C machinery” e r 3. n Their sequence is moderately I B well-conserved or constant across phylogenetic lines. e 3 Small molecules3 G 0 which cannot2 tolerate much change and still retain their function. 4 2 o differences Thus:ismall © can be used to determine evolutionary distances B organisms (evolutionary divergence). between  g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B S = Svedberg; a unit of mass  g y l o io 5S rRNA: 1 b ̴ 25 nucleoTde long; too small, limited o informaTon content. i c r U 23S rRNA : 2̴ ,900 nucleoTdes long; M M too large, C more difficult to analyze experimentally. l a S, e r e n 16S rRNA: ̴ 1,500 nucleoTdes long; I B the size is experimentally manageable, more commonly3used. G 0 Its counterpart in3eukaryotes2is the 18S rRNA. 2 originate from the small subunit of the Both 16S rRNA4and 18S rRNA ribosome.i o © B The 16s rRNA gene sequence g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B López-Aladid, R. et al., 2023 PCR amplification of 16s rRNA gene. g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B © Pearson EducaOon 2019. Brock Biology of Microorganisms.  g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B © Pearson EducaOon 2019. Brock Biology of Microorganisms.  g y l o i o o b r ic MU l M C r a S, n e I B e G 023 4 3 2 i o © B © Pearson Education 2019. Brock Biology of Microorganisms. Three Domains: A Comparison y o g o l Characteristics Bacteria Archaea b i Eucarya Peptidoglycan Yes No r o No Lipids Ester linked i clinked M U Ether Ester linked Ribosomes 70S l M 70S C 80S Initiator tRNA Formylmethionine a r S , Methionine Methionine Introns in tRNA No n e I B Yes Yes Ribosomes sensitive to No G 02e 3 Yes Yes diphtheria toxin RNA polymerase 3 One 2 4 (5 subunits) Several (8-12 subunits Three (12-14 subunits i o © each) each) chloramphenicol, B Ribosomes sensitive to Yes No No streptomycin, kanamycin