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MICROBIAL TAXONOMY
• Phenotypic Analysis
• Genotypic Analysis
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Classification and Taxonomy
• Taxonomy– science of biological classification– consists of three separate but interrelated
parts• classification – arrangement of organisms into
groups (taxa; s.,taxon)
• nomenclature – assignment of names to taxa
• identification – determination of taxon to which an isolate belongs
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Natural Classification
• Arranges organisms into groups whose members share many characteristics
• first such classification in 18th century developed by Linnaeus
– based on anatomical characteristics
• This approach to classification does not necessarily provide information on evolutionary relatedness
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Polyphasic Taxonomy
• Incorporates information from genetic, phenotypic and phylogenetic analysis
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Phenetic Classification
• Groups organisms together based on mutual similarity of phenotypes
• Can reveal evolutionary relationships, but not dependent on phylogenetic analysis
• Best systems compare as many attributes as possible
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Phylogenetic Classification
• Also called phyletic classification systems• Phylogeny
– evolutionary development of a species
• Woese and Fox proposed using rRNA nucleotide sequences to assess evolutionary relatedness of organisms
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Figure 19.7
genus – well defined group of one ormore species that is clearly separatefrom other genera
Taxonomic Ranks and Names
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Taxonomic Ranks and Names
Table 19.3
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Defining Species
• Can’t use definition based on interbreeding because procaryotes are asexual
• Definition of Species
– collection of strains that share many stable properties and differ significantly from other groups of strains
• Also suggested as a definition of species– collection of organisms that share the same
sequences in their core housekeeping genes
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Strains
• Vary from each other in many ways– biovars – differ biochemically and
physiologically
– morphovars – differ morphologically
– serovars – differ in antigenic properties
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Genus
• Well-defined group of one or more strains
• Clearly separate from other genera
• Often disagreement among taxonomists about the assignment of a specific species to a genus
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Techniques for Determining Microbial Taxonomy and Phylogeny
• Classical Characteristics– morphological
– physiological and metabolic
– biochemical
– ecological
– genetic
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Table 14-3
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Table 19.4
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Table 19.5
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Table 14-4
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Molecular Characteristics
• Nucleic acid base composition
• Nucleic acid hybridization
• Nucleic acid sequencing
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Nucleic acid base composition
• G + C content– Mol% G + C =
(G + C/G + C + A + T)100
– usually determined from melting temperature (Tm)
– variation within a genus usually < 10%
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Figure 19.8
as temperature slowlyincreases, hydrogen bondsbreak, and strandsbegin to separate
DNA issinglestranded
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Table 19.6
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Nucleic acid hybridization
• Measure of sequence homology
• Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences
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Fig. 14-20
Hybridizationexperiment:
Same genus,but differentspecies
Organisms tobe compared:
Genomic DNA
Heat todenature
Mix DNA from two organisms—unlabeledDNA is added in excess:
DNApreparation
Genomic DNA
Results andinterpretation:
Same species
Differentgenera 100% < 25%
100 75 50 25 0 Same strain(control)
1 and 2 are likelydifferent generaPercent hybridization
Unhybridized Organism 2 DNAHybridized DNA
Shear and label ( )
Hybridized DNA
Shear DNA
Organism 1 Organism 2
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Genotypic Analysis
• DNA-DNA hybridization
– Provides rough index of similarity between two organisms
– Useful complement to SSU rRNA gene sequencing
– Useful for differentiating very similar organisms
– Hybridization values 70% or higher suggest strains belong to the same species
– Values of at least 25% suggest same genus
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Table 19.7
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Nucleic acid sequencing
• Most powerful and direct method for comparing genomes
• Sequences of 16S and 18S rRNA (SSU rRNAs) are used most often in phylogenetic studies
• Complete chromosomes can now be sequenced and compared
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Comparative Analysis of 16S rRNA sequences
• Oligonucleotide signature sequences found– short conserved sequences specific for a
phylogenetically defined group of organisms
• Either complete or, more often, specific rRNA fragments can be compared
• When comparing rRNA sequences between 2 organisms, their relatedness is represented by an association coefficient of Sab value– the higher the Sab value, the more closely related the
organisms
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Small Ribosomal Subunit rRNA
Figure 19.10
frequently used to create trees showingbroad relationships
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Figure 14.11
Ribosomal RNAs as Evolutionary Chronometers
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oligonucleotidesignaturesequences – specificsequences thatoccur in mostor all membersof a phylo-genetic group
useful forplacingorganisms intokingdom ordomain
Table 19.8
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Genomic Fingerprinting
• Used for microbial classification and determination of phylogenetic relationships
• Used because of multicopies of highly conserved and repetitive DNA sequences present in most gram-negative and some gram-positive bacteria
• Uses restriction enzymes to recognize specific nucleotide sequences– cleavage patterns are compared
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DNA Fingerprinting
• Repetitive sequences amplified by the polymerase chain reaction– amplified fragments run on agarose gel, with
each lane of gel corresponding to one microbial isolate
• pattern of bands analyzed by computer
• widespread application
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Figure 19.11
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Amino Acid Sequencing
• The amino acid sequence of a protein is a reflection of the mRNA sequence and therefore of the gene which encodes that protein
• Amino acid sequencing of cytochromes, histones and heat-shock proteins has provided relevant taxonomic and phylogenetic information
• Cannot be used for all proteins because sequences of proteins with different functions often change at different rates
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Comparison of Proteins
• Compare amino acid sequences
• Compare electrophoretic mobility
• Immunologic techniques can be also used
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Relative Taxonomic Resolution of Various Molecular Techniques
Figure 19.12
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Microbial Phylogeny
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The Evolutionary Process
Evolution: is descent with modification, a change in the genomic DNA sequence of an organism and the inheritance that change by the next generation
Darwin's Theory of Evolution: all life is related and has descended from a common ancestor that lived in the past.
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Assessing Microbial Phylogeny
• Identify molecular chronometers or other characteristics to use in comparisons of organisms
• Illustrate evolutionary relationships in phylogenetic tree
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Molecular Chronometers
• Nucleic acids or proteins used as “clocks” to measure amount of evolutionary change over time
• Use based on several assumptions– sequences gradually change over time– changes are selectively neutral and
relatively random– amount of change increases linearly
with time
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• Cytochromes
• Iron-sulfur proteins
• rRNA• ATPase
• Rec A
Evolutionary Chronometers
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Problems with Molecular Chronometers
• Rate of sequence change can vary over time
• The phenomenon of punctuated equilibria will result in time periods characterized by rapid change
• Different molecules and different parts of molecules can change at different rates
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Creating Phylogenetic Trees from Molecular Data
• Align sequences• Determine number of positions that are
different• Express difference
– e.g., evolutionary distance
• Use measure of difference to create tree– organisms clustered based on relatedness– parsimony – fewest changes from ancestor to
organism in question
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Generating Phylogenetic Trees from Homologous Sequences
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The Major Divisions of Life
• Currently held that there are three domains of life– Bacteria– Archaea– Eucarya
• Scientists do not all agree how these domains should be arranged in the “Tree of Life”
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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Figure 19.14
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Phylogenetic Trees
Figure 19.13
nodes = taxonomic units(e.g., species orgenes)
rooted tree –has node thatserves ascommonancestor
terminalnodes = livingorganisms
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