1. MICROBIAL TAXONOMY
Phenotypic Analysis
Genotypic Analysis

2. Classification and 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

3. 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

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

7. Taxonomic Ranks and Names
genus – well defined group of one or
more species that is clearly separate
from other genera
Figure 19.7

8. Taxonomic Ranks and Names
Table 19.3

9. 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

10. Strains Vary from each other in many ways
biovars – differ biochemically and physiologically
morphovars – differ morphologically
serovars – differ in antigenic properties

11. 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

12. Techniques for Determining Microbial Taxonomy and Phylogeny
Classical Characteristics
morphological
physiological and metabolic
biochemical
ecological
genetic

13. Table 14-3

14. Table 19.4

15. Table 19.5

16. Table 14-4

17. Molecular Characteristics
Nucleic acid base composition
Nucleic acid hybridization
Nucleic acid sequencing

18. 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%

19. increases, hydrogen bonds break, and strands begin to separate DNA is
as temperature slowly
increases, hydrogen bonds
break, and strands
begin to separate
DNA is
single
stranded
DNA melting curve
Figure 19.8

20. Table 19.6

21. Nucleic acid hybridization
Measure of sequence homology
Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences

22. Fig. 14-20 Organisms to be compared: Hybridization experiment:
Genomic DNA
Genomic DNA
DNA preparation
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 genus, but different species
Same species
Different genera
100%
< 25%
Same strain (control)
1 and 2 are likely different genera
Percent hybridization

23. 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

24. Table 19.7

25. 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

26. 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

27. Small Ribosomal Subunit rRNA
Red dots mark positions where bacteria and archaea differ.
frequently used to create trees showing
broad relationships
Figure 19.10

28. Ribosomal RNAs as
Evolutionary
Chronometers
Figure 14.11

30. 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

32. 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

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

34. Figure 19.11

35. 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

36. Comparison of Proteins
Compare amino acid sequences
Compare electrophoretic mobility
Immunologic techniques can be also used

37. Relative Taxonomic Resolution of Various Molecular Techniques
Figure 19.12

38. 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

41. 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

42. Evolutionary Chronometers
Cytochromes
Iron-sulfur proteins
rRNA
ATPase
Rec A

43. 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

44. 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

45. Generating Phylogenetic Trees from
Homologous Sequences

47. 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”

48. Copyright © The McGraw-Hill Companies, Inc
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Figure 19.14

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