1

MICROBIAL TAXONOMY

• • Phenotypic Analysis Genotypic Analysis

Classification and Taxonomy

2 •

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

Natural Classification

3 • •

Arranges organisms into groups whose members share many characteristics

•

first such classification in 18 th century developed by Linnaeus

–

based on anatomical characteristics This approach to classification does not necessarily provide information on evolutionary relatedness

4

Polyphasic Taxonomy

•

Incorporates information from genetic , phenotypic and phylogenetic analysis

5

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

6

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

Taxonomic Ranks and Names

7 Figure 19.7

genus – well defined group of one or more species that is clearly separate from other genera

Taxonomic Ranks and Names

8 Table 19.3

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

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biovars – differ biochemically and physiologically

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morphovars – differ morphologically

–

serovars – differ in antigenic properties

Genus

11 • • •

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

12 Techniques for Determining Microbial Taxonomy and Phylogeny •

Classical Characteristics

– – – – –

morphological physiological and metabolic biochemical ecological genetic

Table 14-3

13

Table 19.4

14

Table 19.5

15

Table 14-4

16

17

Molecular Characteristics

• • •

Nucleic acid base composition Nucleic acid hybridization Nucleic acid sequencing

Nucleic acid base composition

18 •

G + C content

–

Mol% G + C = (G + C/G + C + A + T)100

–

usually determined from melting temperature (T

m

)

–

variation within a genus usually < 10%

Figure 19.8

19 as temperature slowly increases, hydrogen bonds break, and strands begin to separate DNA is single stranded

Table 19.6

20

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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

Fig. 14-20 Organisms to be compared: Organism 1 Genomic DNA DNA preparation Shear and label ( ) Organism 2 Genomic DNA Shear DNA Heat to denature Hybridization experiment: Mix DNA from two organisms —unlabeled DNA is added in excess: Hybridized DNA Hybridized DNA Unhybridized Organism 2 DNA Results and interpretation: Same species Same genus, but different species Different genera 100% < 25% 100 75 50 25 0 Percent hybridization Same strain (control) 1 and 2 are likely different genera

22

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

23

Table 19.7

24

Nucleic acid sequencing

25 • 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

Comparative Analysis of 16S rRNA sequences

26 • • •

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 S ab

–

value the higher the S ab value, the more closely related the organisms

Small Ribosomal Subunit rRNA

Figure 19.10

27 frequently used to create trees showing broad relationships

28 Ribosomal RNAs as Evolutionary Chronometers Figure 14.11

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oligonucleotide signature sequences – specific sequences that occur in most or all members of a phylo genetic group useful for placing organisms into kingdom or domain Table 19.8

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31

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 32

33

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

Figure 19.11

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Amino Acid Sequencing

35 • • •

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

Relative Taxonomic Resolution of Various Molecular Techniques Figure 19.12

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Microbial Phylogeny

39

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.

40

Assessing Microbial Phylogeny

•

Identify molecular chronometers or other characteristics to use in comparisons of organisms

•

Illustrate evolutionary relationships in phylogenetic tree

Molecular Chronometers

41 • •

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

42

Evolutionary Chronometers

• • • • • Cytochromes Iron-sulfur proteins

rRNA

ATPase Rec A

Problems with Molecular Chronometers

43 • • •

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

Creating Phylogenetic Trees from Molecular Data

44 • • • •

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

45 Generating Phylogenetic Trees from Homologous Sequences

46

The Major Divisions of Life

47 •

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 .

Figure 19.14

48

Phylogenetic Trees

nodes = taxonomic units (e.g., species or genes) terminal nodes = living organisms rooted tree – has node that serves as common ancestor Figure 19.13

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