MICROBIAL TAXONOMY
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1
Phenotypic Analysis
Genotypic Analysis
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
2
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
3
Polyphasic Taxonomy
• Incorporates information from
genetic, phenotypic and phylogenetic
analysis
4
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
5
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
6
Taxonomic Ranks and Names
genus – well defined group of one or
more species that is clearly separate
from other genera
Figure 19.7
7
Taxonomic Ranks and Names
Table 19.3
8
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
9
Strains
• Vary from each other in many ways
– biovars – differ biochemically and
physiologically
– morphovars – differ morphologically
– serovars – differ in antigenic
properties
10
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
11
Techniques for Determining Microbial
Taxonomy and Phylogeny
• Classical Characteristics
–
–
–
–
–
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morphological
physiological and metabolic
biochemical
ecological
genetic
Table 14-3
13
Table 19.4
14
Table 19.5
15
Table 14-4
16
Molecular Characteristics
• Nucleic acid base composition
• Nucleic acid hybridization
• Nucleic acid sequencing
17
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%
18
as temperature slowly
increases, hydrogen bonds
break, and strands
begin to separate
Figure 19.8
19
DNA is
single
stranded
Table 19.6
20
Nucleic acid hybridization
• Measure of sequence homology
• Genomes of two organisms are
hybridized to examine proportion of
similarities in their gene sequences
21
Fig. 14-20
Organisms to
be compared:
DNA
preparation
Organism 1
Organism 2
Genomic DNA
Genomic DNA
Shear and label (
)
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
100
75
Same genus,
but different Different
genera
species
50
25
Percent hybridization
22
0
100%
< 25%
Same strain
(control)
1 and 2 are likely
different genera
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
24
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
25
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
27
frequently used to create trees showing
broad relationships
Ribosomal RNAs as
Evolutionary
Chronometers
Figure 14.11
28
29
oligonucleotide
signature
sequences –
specific
sequences that
occur in most
or all members
of a phylogenetic 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
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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
33
Figure 19.11
34
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
35
Comparison of Proteins
• Compare amino acid sequences
• Compare electrophoretic mobility
• Immunologic techniques can be also
used
36
Relative Taxonomic Resolution of
Various Molecular Techniques
Figure 19.12
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Microbial Phylogeny
38
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.
39
Assessing Microbial Phylogeny
• Identify molecular chronometers or
other characteristics to use in
comparisons of organisms
• Illustrate evolutionary relationships
in phylogenetic tree
40
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
41
Evolutionary Chronometers
42
•
Cytochromes
•
Iron-sulfur
proteins
•
rRNA
•
ATPase
•
Rec A
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
43
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
45
46
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”
47
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 19.14
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Phylogenetic Trees
nodes =
taxonomic units
(e.g., species or
genes)
terminal
nodes = living
organisms
Figure 19.13
49
rooted tree –
has node that
serves as
common
ancestor