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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