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 phylogntique
Premier arbre universel du vivant :
Subdivision en trois rgnes :
Plantes. Animaux. Protistes.

Organismes microscopiques unicellulaires :

Question qui se pose encore aujourdhui.
Haeckel (1866)

The Three-Domain System

Figure 10.1

Evolution of cell types

Histoire de la vie sur Terre

Formation de la Terre 1ers microorganismes Dinosaures Arthropodes

4,5

4,0

3,5

3,0

2,5

2,0

1,5

1,0

0,5

Milliards dannes
0,0035

ge des microbes

Humains

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 two-empire proposal, separating eukaryotes from prokaryotes and eubacteria from archaebacteria. Mayr, D. PNAS 95:9720-23. (1998).

The three-domain proposal, with continuous lateral gene transfer among domains. Doolittle. Science 284:2124-8. (1999)

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
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

cant 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 Linn (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??????????
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!!!!!!

Direct correlation of the changes in the sequence of this molecule to a time scale is not possible A cell may contain more than one copy of the 16S rDNA gene, each with a different sequence 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

Bergeys Manual of Systematic Bacteriology

Prokaryotes into 25 phyla
Archaea
2

Bacteria
23

Consensus of experts

Bergeys 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 Bergeys 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)