Transcript Taxonomy, Phylogeny & Molecular Chronometers

Prokaryote Taxonomy & Diversity
Classification, Nomenclature & Identification
Phenetic & Numerical Classification
Molecular Phylogenetic Approach
Note: No Kingdom
Basics to Know
• A grouping based on similar characters is called a taxon.
• Hierarchy of Prokaryote Taxa often omits Kingdom. Phylum often
referred to as a Section.
• Bergey’s Manual of Systematic Bacteriology
– 1st ed. (1989); mostly phenetic classification; 4 volumes
– 2nd ed. (2001-2003); mostly phylogenetic classification; 5 volumes
• Species defined differently for prokaryotes (no sexual reproduction) =
a collection of genetically unique populations with many stable
characters distinctly different from other groups.
• Within a species there may be strains, populations characterized by
minor variations in biochemical/ physiological properties (biovars),
antigenic distinctions (serovars), shape (morphovars), or viral
susceptibility (phage-typing).
The focus here is on best
techniques for
distinguishing strains of
the same species.
Phenetic Characters:
Very useful in identification!
1) Ecological Characters
2) Genetic Characters
3) Morphological
Characters
4) Physiological and
Metabolic Characters
Phenetic Identification
Use of dichotomous keys for bacteria
Phenetic Identification
Use of multi-test kits and their databases.
The combination of positive results from an unknown is
entered into a database of results form known bacteria. A
computer model predicts the most probable match and
level of certainty. Intended use is for clinical isolates.
api® substrate utilization tests
Molecular Characters
• Fatty acid profiles (FAME analysis)
• Proteins
– Electrophoretic Mobility
– Immuno-Reactivity
– A.A. Sequence Data
• Nucleic Acids
– Nucleotide composition (G+C content ≈ Tm)
– Degree of Hybridization (>70% ≈ species)
– Nucleotide Sequence Data
FAME – MIS
(microbial identification system)
Tm ≈ G+C
dsDNA
ssDNA
dsDNA
ssDNA
DNA-DNA Hybridization
•
May be performed in solution but most quantitative on solid substrate.
•
Compare denatured genomic DNA fragments of one species to others:
1) Bind one species DNA (non-radioactive) to nitrocellulose paper.
2) Hybridize with other species radioactive labeled DNA fragments.
3) Amount of radioactivity is relative to the amount of similarlity; i.e.
degree of hybridization (100% for same species; e.g. N. meningitidis)
 min. # characters? 50
 Use all characters? SM or J?
 80% phenon ≈ species
 Similarity matrix? All pairings
 Graph as a dendrogram (tree).
Organism A
Numerical
Classification
Cluster Analysis
E.g. six different bacteria
* All possible pairs are evaluated to generate SSM values.
* Generation of a similarity matrix (numerical).
* Clusters together organisms of similar SSM values (colored).
Dendrogram
Construction &
Interpretation
• Graphical representation of the cluster analysis.
• Arrange pairs in order of decreasing similarity values.
• Length of horizontal line reflects % similarity.
• Nodes (branches points) represent a point at, and
below, which the characteristics are indistinguishable.
Phylogenetic Classification
Molecular Chronometers
• Phylogeny refers to grouping based on
evolutionary relatedness; regardless of phenetic
characters.
• Phylogeny is inferred from changes in protein or
DNA sequence over evolutionary time.
• Attributes of an Ideal “Molecular Chronometer”:
–
–
–
–
Universally distributed.
Functionally homologous.
Ease of analysis.
Rate of sequence change commensurate with
evolutionary distance measured.
Why the 16SrRNA gene?
• All cellular life has ribosomes with rRNAs
• All rRNAs have the same function (transcription) in all cellular life.
• The 16SrRNA gene provides enough information (# sequence positions)
and not too big that its cloning and sequencing is inefficient (time & $).
• There are only 7 regions that are considered highly variable most of the
molecule’s sequence is very conserved so to maintain critical functions.
Small subunit rRNA of the
three domains of life.
Bacteria
(Mitochondria &
Chloroplasts)
16SrRNA
Archaea
16SrRNA
Eucarya
18SrRNA
Phylogenetic Distance Matrix Tree
• Sequence molecular chronometer of choice for all
organisms studied.
• Align sequences based on highly conserved
sequence regions.
• Pair-wise determination of differences.
• Adjustments may be made for multiple mutations as
particular sites.
• Construction of tree requires massive computer
calculations to determine the best fit of the data
(similar to dendrogram).
Phylogenetic
Distance Matrix Tree
(continued)
• Nodes represent divisions in
taxonomic units.
• Relative evolutionary distance is
the sum of line distance.
• Scale is in units of “fixed-point
mutations per sequence position”.
•Trees can be rooted or unrooted
(rooted trees require more
complex calculations as there are
a greater combination of possible
outcomes)
Phylogenetic Tree of Life:
The RDP II
Rooted Tree based on 16SrRNA and 18SrRNA sequence data.
Signature Sequences of 16SrDNA
Used for design of
taxon specific
oligonucleotides.
Used as probes in
identification of
unknowns
Used as primers in
PCR amplification of
unknowns for further
phylogenetic study.
The later has led to
discovery of viable
but non-culturable
prokaryotes!
Whole Genomes Cloning
Bioinformatics
Fig 15.6
Prescott
Computers are
required to deal with
massive data
manipulations:
• Aligning & Editing
sequences
•Annotation
- ORP
- rRNA & tRNA
- Transposons
- Ori C
• Phylogenetic
Analysis between
organisms.
e.g. Haemophilus influenzae (1995)