IMVI-Microbiologie M1
2009/2010
Taxonomy and Phylogeny
The bacterial groups
Pr A.Klier
Classifying Organisms
Carolus Linnaeus
(1707-1778)
Five Kingdoms
Uni or multicellular
Eukaryotic
Bacteria
Archaea
Eukaryotic
Unicellular
Unicellular
Prokaryotic
Classification phylogénétique
• Premier arbre universel du vivant :
– Subdivision en trois
règnes :
• Plantes.
• Animaux.
• Protistes.
– Organismes microscopiques unicellulaires :
• Question qui se pose
encore aujourd’hui.
Haeckel (1866)
The Three-Domain System
Figure 10.1
Evolution of cell types
Histoire de la vie sur Terre
Formation
de la Terre
Dinosaures
1ers microorganismes
4,5
4,0
3,5
Arthropodes
3,0
2,5
2,0
1,5
1,0
0,5
Milliards d’années
0,0035
Humains
Âge des
microbes
Prokaryotes: 2.5 104 gen/an
H.sapiens: 104 generations
Martin & Embley
Nature 431:152-5.(2004)
The three-domain proposal based on the ribosomal
RNA tree. Woese et al. PNAS. 87:4576-4579. (1990)
The three-domain proposal, with continuous
lateral gene transfer among domains.
Doolittle. Science 284:2124-8. (1999)
The two-empire proposal, separating
eukaryotes from prokaryotes and
eubacteria from archaebacteria.
Mayr, D. PNAS 95:9720-23. (1998).
The ring of life, incorporating lateral gene
transfer but preserving the prokaryote
eukaryote divide.
Rivera & Lake JA. Nature 431: 152-5. (2004)
Summary
First evidence for potential life 3.8 billion yrs ago
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other fossil evidence
molecular fossils
chemolithotrophy vs heterotrophs, who came first?
anoxygenic photosynthesis
oxygenic photosynthesis
Banded iron formations (BIFs)-red beds
Evolution of cell types
Endosymbiosis
Endosymbiotic Theory
Figure 10.2
Figure 10.3
Prokaryotes
Figure 10.6
Palaeomicrobiology
HOW DO WE CLASSIFY
THE MICROBES ???
THE PRINCIPLES OF
TAXONOMY
Importance of taxonomy
• allows scientists to organize huge amounts of
knowledge
• allows scientists to make predictions and frame
hypotheses about organisms
• places organisms into meaningful, useful groups,
with precise names, thus facilitating scientific
communication
• essential for accurate identification of organisms
Systematics
• study of organisms with the ultimate object of
characterizing and arranging them in an
orderly manner
Taxonomic Ranks
• microbiologists
often use
informal names
– e.g., purple
bacteria,
spirochetes,
methaneoxidizing
bacteria
Taxonomic Hierarchy
Figure 10.5
Defining a prokaryotic speciesa difficult task
• can’t use definition based on interbreeding because
procaryotes are asexual
• two definitions suggested:
– collection of strains that share many stable properties and differ
significantly from other groups of strains
– = taxospecies
– collection of strains with similar DNA G + C composition and 
70% sequence similarity in their DNA
– = genomic species
Type strain
• usually one of first strains of a species
studied
• often most fully characterized
• not necessarily most representative member
of species
Binomial system of
nomenclature
• devised by Carl von Linné (Carolus Linnaeus)
• each organism has two names
– genus name – italicized and capitalized (e.g.,
Escherichia)
– species epithet – italicized but not capitalized (e.g.,
coli)
• can be abbreviated after first use (e.g., E. coli)
Classification
• Natural classification
– arranges organisms into groups whose members share many
characteristics
– most desirable system because reflects biological nature of organisms
two methods for construction
– phenetically
• grouped together based on overall similarity, and ignoring their past
– phylogenetically
• grouped based on probable evolutionary relationships. Always
thought impossible to achieve until recently
a) Phenetic Classification
• groups organisms together based on mutual
similarity of present day phenotypes
• can reveal evolutionary relationships, but not
dependent on phylogenetic analysis
• best systems compare as many attributes as
possible, with no weighting
• general purpose classifications
• Known as POLYTHETIC classifications
b) Phylogenetic Classification
• also called phyletic classification systems
• phylogeny
– evolutionary development of a species
• usually based on direct comparison of genetic
material and/or gene products
• Once thought impossible to achieve for
prokaryotes – no fossils
• Now achieved – a revolution in microbiology
Major Characteristics Used in
Taxonomy
• Needed for classifying and identifying microbes
• Morphology not as useful with bacteria as it is with higher
plants and animals, since little variation
• Two major types:
– Classical/conventional characteristics
– Molecular characteristics
Classical Characteristics
• Morphological – cell shape etc =limited value
• physiological and metabolic = biochemical
attributes
• ecological = genetic analysis = their ability to
carry out DNA recombination with other strains
Ecological characteristics
• life-cycle patterns
• symbiotic relationships
• ability to cause disease
• habitat preferences
• growth requirements
Also use comparisons of selected
chemical components of cells
= CHEMOTAXONOMY
•Cell wall chemistry, including peptidoglycan
chemistry and teichoic acids, and mycolic acids
•Lipids like ubiquinones and menaquinones
•Chemical fingerprinting of cells
Molecular Characteristics
more useful and reliable
• comparison of proteins
• nucleic acid base composition
• nucleic acid hybridization
• nucleic acid sequencing
Nucleic acid base composition
• G + C content
– Mol% G + C =
(G + C/G + C + A + T)100
– usually determined from melting temperature (Tm)
– An exclusionary character i.e. the same G+C value does
not mean the two organisms are necessarily closely
related, but if they differ by more than 5% then they
belong to different species
– An essential piece of information in any description of a
new genus
Range of G+C contents
as temperature slowly
increases, hydrogen bonds
break, and strands
begin to separate
DNA is
single
stranded
Nucleic acid hybridization
• measure of sequence homology
• common procedure- all are difficult
– bind nonradioactive DNA to nitrocellulose filter
– incubate filter with radioactive single-stranded DNA
– measure amount of radioactive DNA attached to filter
Level of DNA:DNA Hybridization used as
benchmark to define a genomic species of a
bacterium
ie strains with >70% DNA:DNA hybridization
belong to same genomic species
but is not used to separate genera
Assessing Microbial Phylogeny
• identify molecular chronometers or other
characteristics to use in comparisons of
organisms
• illustrate evolutionary relationships in
phylogenetic tree
-The “Woesian” Revolution
-1977-
Carl Woese - a chemist working in
relative isolation compares 16S rRNA
sequences and discovers that:
1) “Archaebacteria” represent a new
kingdom
2) A universal and quantitative
phylogeny is possible
Molecular Chronometers
• nucleic acids or proteins used as “clocks” to
measure amount of evolutionary change over
time
Which is the best molecule to use as a
“molecular clock” for phylogenetic
characterization ????
Most people think at the moment
that 16S rRNA is
Conservation and variation in small
subunit rRNA
This diagram shows conserved
and variable regions of the small
subunit rRNA (16S in prokaryotes
or 18S in eukaryotes). Each dot
and triangle represents a position
that holds a nucleotide in 95% of
all organisms sequenced, though
the actual nucleotide present (A,
U, C, or G) varies among species.
Figure by Jamie Cannone,
courtesy of Robin Gutell; data
from the Comparative RNA Web
Site: www.rna.icmb.utexas.edu
Conservation and variation in small
subunit rRNA
The starred region from part A as
it appears in a bacterium
(Escherichia coli), an archaean
(Methanococcus vannielii), and a
eukaryote (Saccharomyces
cerevisiae). This region includes
important signature sequences for
the Bacteria and Archaea. Figure
by Jamie Cannone, courtesy of
Robin Gutell; data from the
Comparative RNA Web Site:
www.rna.icmb.utexas.edu
rRNA
frequently used to create trees showing
broad relationships
WHY??????????
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Found in all Prokaryotes
Large information content (1500bp)
The genes encoding it show both conserved and highly variable
regions
This allows phylogenies covering a broad range of relationships from
species to Domain level to be elucidated
A large data base of sequences is available (>25,000)
Gene (ie 16S rDNA) easily sequenced
Probably not as susceptible to lateral gene transfer as some other
genes
But not perfect!!!!!!
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Direct correlation of the changes in the sequence of this molecule
to a time scale is not possible
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A cell may contain more than one copy of the 16S rDNA gene,
each with a different sequence
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Difficult to interpret the taxonomic significance of high
similarities (>98%) of 16S rDNA sequences between two
organisms in terms of whether they are the same or different
species, since only a single gene and not whole genome
compared
rRNA, DNA, and Proteins as
Indicators of Phylogeny
• all are used
• do not always produce the same
phylogenetic trees
Genomic Fingerprinting
An indirect way of comparing the DNA sequences of
different bacteria
All use PCR to copy genomic DNA which is then
fingerprinted in some way
Various methods
•Ribotyping
•RAPD-PCR
•16S-23S spacer region fingerprinting and other amplified DNA restriction
endonuclease digest analysis (ADRDA) methods
•MLST
•VNTR
Direct Nucleic acid sequencing
• usually comparison of rRNA genes ie 16S
rRNA genes
• DNA much easier to sequence than RNA
• increasingly, comparison of entire bacterial
genomes now being used
16S rRNA as evolutionary
chronometer
Creating phylogenetic trees
from molecular data
• align sequences (software like Clustal W) and data
bases like GenBank, EMBL or RDP
• determine number of positions that are different
• express difference
– e.g., evolutionary distance
• use measure of difference to create tree (PHYLIP)
– organisms clustered based on relatedness
– several methods for constructing trees, and most people use
>1
– no agreement on best method for doing this
– maximum liklihood is probably best but very slow
Polyphasic Taxonomy
• use of all possible data to determine phylogeny
– i.e., genotypic and phenotypic information
• data used depends on desired level of resolution
– e.g., serological data – resolve strains
– e.g., protein electrophoretic patterns – resolve species
– e.g., DNA hybridization and % G + C – resolve at
genus and species level
The Major Divisions of Life
• based primarily on rRNA analysis
• currently held that there are three domains of
life
– Bacteria
– Archaea
– Eucarya
Some interesting findings
• minimal genome size
– based on analysis of Mycoplasma genitalium
genome
• smallest procaryotic genome sequenced
• ~108-121 genes not required for growth in laboratory
• ~265-350 genes required for growth in laboratory
More findings…
• many identified genes have unknown
function
– e.g., Mycoplasma genitalium
• 22% have unknown function
– e.g., Haemophilus influenzae
• > 40% have unknown function
– e.g., Methanococcus jannaschii
• a member of Archaea
• 66% have unknown function
– e.g., E. coli
• ~2500 of 4288 genes have unknown function
Approaches to Identification
• Blunderbuss approach-use as many tests as possible. Slow
and expensive
• Dichotomous keys, but one wrong answer gives wrong
identification
• Simultaneous approach using computer based systems eg
Biolog, but only as good as the data base
• Serological methods using O or other antigens
• Molecular techniques like genomic fingerprinting and
USING DNA or RNA targeted probes
Historical Perspective of Bacterial
Phylogenetic Analyses:
Pre-1977
1923 - Bacterial Taxonomy:
Bergey's Manual of Determinative
Bacteriology
“Species” description
Bergey’s Manual of Systematic
Bacteriology
• Prokaryotes into 25 phyla
– Archaea
•2
– Bacteria
• 23
• Consensus of experts
Bergey’s Manual of Systematic
Bacteriology
• The bible for bacterial taxonomists
• Edited by an international committee of
bacterial taxonomists
• Detailed work containing descriptions of all
prokaryotic species currently identified
The First Edition of Bergey’s
Manual of Systematic
Bacteriology
• primarily phenetic
• cell wall characteristics played important role as
characters to group and separate bacteria
• Gram-negative versus Gram-positive bacteria versus
bacteria with no cell walls (Mycoplasmas)