2 Microbial Classification and Taxonomy Taxonomy? Refer Pg 389; 17.2Science of biological classificationConsists of three separate but interrelated partsclassification – arrangement of organisms into groups (taxa, sing.taxon)nomenclature – assignment of names to taxaidentification – determination of taxon to which an isolate belongs
3 Natural Classification Natural Classification arranges organisms into groups whose members share many characteristicsFirst such classification in 18th century developed by Linnaeus based on anatomical characteristicsthis approach to classification does not necessarily provide information on evolutionary relatedness in microbesE.g classification of humans as mammals –milk producing, hair, self regulating temp. etc.
4 Polyphasic TaxonomyPolyphasic Taxonomy is used to determine the genus and species of a newly discovered procaryoteincorporates information from phenetic (phenotypic) and phylogenetic analysis
5 Phenetic Classification groups organisms together based on mutual similarity of phenotypescan reveal evolutionary relationships, but not dependent on phylogenetic analysisE.g because motility and flagella are always associated in particular organisms, it is reasonable to suppose that flagella is involved in some types of motility
6 Phylogenetic Classification Phylogenetic, also called phyletic classification systemsPhylogeny is based on evolutionary development of a speciesusually based on direct comparison of genetic material and gene productsthis approach is widely acceptedlarge databases exist for rRNA sequences
7 Taxonomic Ranks and Names microbiologists often use informal names that don’t necessarily have taxonomic significancee.g., purple bacteria, spirochetes, methane-oxidizing bacteriaTable 17.2 shows the levels of taxonomic names
8 Defining Procaryotic Species The basic taxonomic group in microbial taxonomy is the species.Cannot use definition based on interbreeding because procaryotes are asexual.A prokaryotic species is collection of strains that share many stable properties and differ significantly from other groups of strains.Also suggested as a definition of species as a collection of organisms that share the same sequences in their core housekeeping genes (genes required to code for products needed by cells)-bases on sequence data.
10 Strains Descended from a single, pure microbial culture Strains vary from each other in many waysBiovars – differ biochemically and physiologicallyMorphovars – differ morphologicallySerovars – differ in antigenic properties
11 Type Strain Usually one of first strains of a species studied Often most fully characterizedNot necessarily most representative member of species
12 Genus Genus- well-defined group of one or more strains Clearly separate from other generaOften disagreement among taxonomists about the assignment of a specific species to a genus
13 Binomial System of Nomenclature Binomial system was devised by Carolus LinnaeusEach organism has two namesgenus name – italicized and capitalized (e.g., Escherichia)species epithet – italicized but not capitalized (e.g., coli)can be abbreviated after first use (e.g., E. coli)a new procaryotic species cannot be recognized until it has been published in the International Journal of Systematic and Evolutionary Microbiology
14 Techniques for Determining Microbial Taxonomy and Phylogeny Classical characteristicsmorphologicalphysiologicalmetabolicecologicalgeneticMolecular characteristicsnucleic acid base compositionnucleic acid hybridizationnucleic acid sequencinggenomic fingerprintingamino acid sequencing
15 Ecological Characteristics Life-cycle patternsSymbiotic relationshipsAbility to cause diseaseHabitat preferencesGrowth requirements
16 Genetic Analysis Genetic Analysis Study of chromosomal gene exchange by transformation and conjugationplasmids can be used for the analysis of phenotypic traits
17 Nucleic Acid Base Composition Determine the G + C contentWhere G=Guanine, C=Cytosine, A=adenine and T=Thymine ( nucleotide are the DNA base)The G+ C content is often estimated by determining the melting temperature (Tm) of the DNAHigher G + C gives a higher melting temperature
18 Nucleic Acid Hybridization measure of sequence homology( molecular relatedness)common procedure for hybridisation:bind nonradioactive DNA to nitrocellulose filterincubate filter with radioactive single-stranded DNAThe quantity of radioactivity bound to the filter reflects the amount of hybridisation between the 2 DNA and thus similarity of the 2 sequences.
19 …Nucleic Acid Hybridization measure amount of radioactive DNA attached to filter.The degree of similarity is expressed as the % of experimental DNA radioactivity retained on the filter as compared to other sps. of the same genus under the same conditions.Usually less than 5 % difference in melting point ( T m ) is considered as members of same sps.
20 Nucleic Acid Sequencing most powerful and direct method for comparing genomessequences of 16S (procaryotes) and 18S (eucaryotes) ribosomal RNA (rRNA) are used most often in phylogenetic studiescomplete chromosomes can now be sequenced and compared
21 …Nucleic Acid Sequencing Comparative Analysis of 16S rRNA Sequences:Oligonucleotide signature sequences are short conserved sequences specific for a phylogenetically defined group of organismseither complete or, more often, specific rRNA fragments can be comparedwhen comparing rRNA sequences between 2 organisms, their relatedness is represented by an association coefficient or Sab valuethe higher the Sab value, the more closely related the organisms
22 Use of DNA Sequences to Determine Species Identity DNA sequences can also be used to determine species strains in addition to genusIt requires analysis of genes that evolve more quickly than rRNA encoding genesMultilocus sequence typing (MLST), the sequencing and comparison of 5 to 7 housekeeping genes instead of single gene is done.This is to prevent misleading results from analysis of one gene.
23 Genomic Fingerprinting Genomic Finger Printing also used for microbial classification and determination of phylogenetic relationshipsGenomic Finger Printing does not involve nucleotide sequencingCan be used because of multicopies of highly conserved and repetitive DNA sequences present in most gram-negative and some gram-positive bacteriaMulticopies can be obtained by Polymerase chain reaction using restriction enzymes
24 …Genomic Fingerprinting uses restriction enzymes (endonucleases) that recognize specific nucleotide sequencesRestriction Enzyme cuts DNA at specific sitesRestriction fragments are compared by Gel Electrophoresis.
25 …Genomic Fingerprinting Repetitive sequences amplified by the polymerase chain reactionamplified fragments run on agarose gel, with each lane of gel corresponding to one microbial isolatepattern of bands analyzed by Gel Document systemcomparison of bands is called restriction fragment length polymorphism (RFLP)It allows for identification to species, subspecies and often allows strain level identificationPCR has a widespread application
27 Amino Acid Sequencingthe amino acid sequence of a protein is a reflection of the mRNA sequence and therefore of the gene which encodes that proteinamino acid sequencing of proteins such as cytochromes, histones and heat-shock proteins has provided relevant taxonomic and phylogenetic informationcannot be used for all proteinscompare protein mass spectra
28 Assessing Microbial Phylogeny evolutionary relationships represented using phylogenetic treesA phylogentic tree is a graph which connects nodes and branches
29 Phylogenetic Trees a. Unrooted tree – b. Rooted treehas node that serves ascommonancestorFigure 17.11
30 The Major Divisions of Life Currently held that there are three domains of lifeDomain BacteriaDomain ArchaeaDomain Eucaryascientists do not all agree how these domains should be arranged in the “Tree of Life”
32 Impact of Horizontal Transfer extensive horizontal gene transfer has occurred within and between domainspattern of microbial evolution is not as linear and treelike as once thought
33 Universal Phylogenetic Tree with Lateral Gene Transfer Figure 17.13
34 Domain EucaryaThe domain Eucarya is divided into four kingdoms by most biologists:Kingdom Protista, including the protozoa and algaeKingdom Fungi, the fungi (molds, yeast, and fleshy fungi)Kingdom Animalia, the multicellular animalsKingdom Plantae, the multicellular plants
35 Domain Archaea Phylogeny of domain Archaea Based primarily on rRNA sequence data, domain Archaea is divided into two phylaPhylum CrenarchaeotaPhylum Euryarchaeota
37 Bergey’s Manual of Systematic Bacteriology In 1923, David Bergey, bacteriologist, Univ of Pennsylvania and 4 other colleagues published a classification of bacterial sps.It is an accepted system of procaryotic taxonomyDetailed work containing descriptions of all procaryotic species currently identified
38 The First Edition of Bergey’s Manual of Systematic Bacteriology The first edition, published in 1984 and is currently in its ninth editionIt contained descriptions of all known procaryotic species then identified, mostly based on phenotypic characters i.e phenetic
39 The Second Edition of Bergey’s Manual of Systematic Bacteriology largely phylogenetic rather than phenetic ; 5 volumes.procaryotes are divided between two domains and 25 phyla
40 BibliographyLecture PowerPoints Prescott’s Principles of Microbiology-Mc Graw Hill Co.https://files.kennesaw.edu/faculty/jhendrix/bio3340/home.html