Taxonomy is one aspect of classification. Organisms are ordered
into groups (taxa) and ranked in a
hierarchy according to established procedures and guidelines.
In this manner, organisms are placed
into taxa of different organizational levels and the inter-relationships
and boundaries between groups are
established.
Nomenclature is another aspect of taxonomy. Names are assigned
to organisms in a systematic
manner.
Identification of an organism is made possible by following the classification
and nomenclature
guidelines and by various scientific approaches. This allows us to
place an organism within its correct
position in the classification scheme.
I. Before scientists had a clear understanding of the nature of microbes
the biological world was
classfied into: plants and animals. Bacteria were
placed with plants. Clearly, this scheme was
inadequate. The electron microscope demonstrated
obvious differences between bacteria and
eukaryotes.
II. Based on nuclear and cellular properties a two kingdom was
also produced: Prokaryotae and
Eukaryote. This was based primarily on presence
of a true nucleus and having organelles sorrounded
or enclosed by a membrane.
III. In 1968 Whittaker proposed his famous 5 Kingdom system
of living organisms. Bacteria were
classified under Kingdom Monera. Members of the
Monera Kingdom were defined as "cells in
which nuclear material is not surrounded by a nuclear
membrane."
IV. The 3 Kingdom of classification proposed by Woese in the
70's utilizes the sequence of the 16S
ribosomal RNA to classify organisms into: Eubacteria,
Archaebacteria and Eukaryota.
The Linnean system of Binomial Nomenclature
Carolus Linneaus developed a scientific system of naming organisms.
The names used by Linneaus in
the Species Plantarum (1753) and the Systema Naturae (1758) are the
basis of the system for plants and animals, respectively. He assigned two
latinized names to each organism:
A genus consists of a group of similar species. Similar genera
are grouped into a family. The species
name or "specific epithet" is unique to the new species. The genus
name is indicated by a capital letter
whereas the species name starts with a lower case letter. By convention
both names are italicized (or
underlined).
Example: Streptococcus pyogenes ---- Once a scientific name has been used in entirety it can subsequently be abbreviated as follows: S. pyogenes
Scientific names should be unique, unchanging and descriptive.
For example, the name may reflect
- the name of the person describing the organism
- the habitat of the organism
- the appearance of the organism
- some names may reflect a disease or infectious
process caused by an organism (e.g., 'pyogenes'
describes the ability to produce pus)
The genetic variability of microbes is further subdivided into subspecies
or types:
a) A strain is equivalent to a clone and represents a population
of genetically identical organisms that
have arisen from a single cell. Some strains of
a bacterial species may be virulent, whereas others are
not.
b) Serovars are antigenically distinct organisms. For example,
over 2,000 serovars of Salmonella
have been identified which are typed according to
their flagellar (H) and somatic (O) antigens.
Classification: The Three Domain System
The Three Domain System, proposed by Woese, is an evolutionary model
of classification based on differences in the sequences of nucleotides
in the cell's ribosomal and transfer RNAs, membrane lipid structure, and
sensitivity to antibiotics. This system proposes that a common ancestor
cell ("Cenancestor") gave rise to three different cell types, each representing
a domain. The three domains are the Archaea (archaebacteria), the
Bacteria
(eubacteria), and the Eukarya (eukaryotes). The
Eukarya are
then divided into 4 kingdoms: Protists, Fungi, Anamalia, and Plantae. A
description of the three domains follows:
1. The Archaea (archaebacteria)
· The Archae are prokaryotic
cells. Unlike the Bacteria and the Eukarya, they have membranes
composed of branched carbon chains attached to glycerol
by ether linkages and have cell walls
which contain no peptidoglycan. While they
are not sensitive to some antibiotics which affect the
Bacteria, they are sensitive to some antibiotics
that affect the Eukarya. The Archae have rRNA and
tRNA regions distinctly different from the Bacteria
and Eukarya. They often live in extreme
environments and include methanogens, extreme
halophiles, and hyperthermophiles.
2. The Bacteria (eubacteria)
· The Bacteria
are prokaryotic cells. Like the Eukarya, they have membranes composed of
straight
carbon chains attached to glycerol by ester linkages.
They have cell walls containing
peptidoglycan, are sensitive to traditional antibacterial
antibiotics, and have rRNA and tRNA
regions distinctly different from the Archaea and
Eukarya. They include: mycoplasmas,
cyanobacteria, Gram-positive bacteria,
and Gram-negative bacteria.
3. The Eukarya (eukaryotes)
· The Eukarya (also spelled
Eucaryota\a) have eukaryotic cells. Like the Bacteria, they have
membranes composed of straight carbon chains attached
to glycerol by ester linkages. If they
possess cell walls, those walls contain no peptidoglycan.
They are not sensitive to traditional
antibacterial antibiotics and have rRNA and tRNA
regions distinctly different from the Bacteria and
the Archaea. They include the following kingdoms:
a. Protista
Kingdom: Protista are simple, predominately unicellular eukaryotic
organisms.
Examples includes slime molds, euglenoids, algae, and protozoans.
b. Fungi
Kingdom: Fungi are unicellular or multicellular organisms with eukaryotic
cell
types. The cells have cell walls but are not organized into tissues. They
do not carry out
photosynthesis and obtain nutrients through absorption. Examples include
sac fungi, club
fungi, yeasts, and molds.
c. Plantae
Kingdom: Plants are multicellular organisms composed of eukaryotic
cells.
The cells are organized into tissues and have cell walls. They obtain nutrients
by
photosynthesis and absorption. Examples include mosses, ferns, conifers,
and flowering
plants.
d. Animalia
Kingdom: Animals are multicellular organisms composed of eukaryotic
cells.
The cells are organized into tissues and lack cell walls. They do not carry
out photosynthesis
and obtain nutrients primarily by ingestion. Examples include sponges,
worms, insects, and
vertebrates.
Two alternative approaches to microbial taxonomy:
I. Phenetic system: groups organisms based on similarity of
shared phenotypic characteristics. For
example, we could place anaerobes in one group and aerobes in another.
This may not always reflect
the correct evolutionary groupings of the organisms.
Bergey's manual is an example of a phenetic system. Microbes
are organized into groups based on
both morphological (staining reactions, cell shape and arrangement,
pigment production, appearance on
media) and physiological (growth requirements, biochemical tests, type
of metabolism). This system can
be useful for identifying an unknown organism as you are doing in our
laboratory.
This classical approach allows one to 'key'out an organism using a series
of mutually exclusive
characteristics. For example: Is the organism gram positive or gram
positive? Is the shape of the
organism a coccus, bacillus or some other morphology? The key eventually
narrows down the organism until an identification is possible.
Numerical Taxonomy
- Calculates the percentage of characteristics that
two organisms or groups have in common
- A large range of traits (morphology, motility,
biochemistry) are considered (at least 50!)
- The result of this classification is a similarity
coefficient (the percentage of the total number of
characters measured that
are common to two organisms. Dendograms are produced to indicate
relatedness....do not worry
about this computers programs can do all this for you!
II. Phylogenetic system: groups organisms based on their shared
evolutionary heritage and descent.
Unlike, a phenetic system, organisms do not have to be phenotypically
similar in order to belong to the
same phylogenetic group. For example, based on genetic and molecular
evidence, Pneumocystis
carinii is now considered to be more closely related to the
fungi
and
is no longer believed to be a
protozoan (although it resembles a protozoan in many respects).
Molecular methods used to type and identify microbes
Two main approaches:
a) comparing DNA or RNA sequences in one or more ways comparing amino
acid sequences of a
protein or proteins
Molecular taxonomy
Uses some key assumptions in order to establish a time-line of evolutionary
relatedness
- genetic mutations are random
- once a mutation occurs, all descendants
of that cell will carry the mutation
- organisms that differ only slightly at the
genetic level have diverged more recently over the course
of evolution than organisms
that differ significantly
a) DNA base composition
- Indicates relatedness of organisms
- Base composition is usually expressed as GC
content
- If the GC content differs by a small percentage
the organisms are not closely related
- The GC content itself does not always mean that
organisms are related. For example,
Mycoplasma and
Bacillus
have similar GC contents but are very different organisms
b) DNA fingerprinting
Comparison of the cleavage pattern (fingerprint) of the DNA from two
organisms (one known, the
other unknown) can determine if they are related.
Each organism has a unique restriction digest
profile
c) Hybridization of DNA probes
- The most widely used molecular method used to
determine relatedness or organisms
- ssDNA is separated from ds DNA on a filter
- The DNA of one organism is radiolabelled and mixed
at low concentrations with the
nonradioactive denatured
DNA of the other organism
- The more related the organisms, the higher the
degree of complementary base pairing which can
be detected by a higher
reading of radioactivity.
d) Nucleic acid hybridization
- Two organisms: grow one in [3H] thymine,
the other one without it.
- Harvest and isolate DNA
- Denature DNA from one organism (heating) and bind
it to a filter membrane
- Add denatured DNA from the other organism
- Wash and add S1 nuclease to remove any single
stranded DNA
- Expose to X-ray film.
- If closely related they would anneal (bind) if
conditions are right (60-70 C). You can get binding
using lower temperatures
(35-55 C) but this is just background!!
Other methods for identifying bacteria
Serological tests
- Identify microbes by reactivity with specific
antibodies
- Serotyping developed by Rebecca Lancefield. Designed
A through O system to identify variants
(serovars or serotypes)
of Streptococci
- Enzyme-linked immunosorbent assay (ELISA)
- Immunofluorescent antibody testing (IFAT)
- Western blot
Evolutionary chronometers
Choosing the right chronometer:
· the molecule
should be universally distributed across the group chosen for study
· it must be
functionally homologous in each organism (phylogenetic comparisons
must start with
molecules of identical function)
· the sequence
should change at a rate commensurate with the evolutionary distance to
be
measured; the broader the
phylogenetic distance to be measured, the slower must be the rate at
which the sequence changes
Ribosomal RNAs as evolutionary chronometers:
- It is likely that the protein-synthesizing process
is very old, and so rRNA molecules are very good
for discerning evolutionary
relationships among living organisms
- This rRNA is also found the ribosomes of chloroplasts
and mitochondria and is therefore present
in animals and plants.
- As the ribosome plays a critical role in protein
synthesis most mutations in rRNA are harmful and
tend to occur very infrequently.
- Therefore,16S rRNA is a very useful molecule for
comparing relatedness of organisms over the
course of evolution.
- rRNAs are ancient molecules, functionally constant,
universally
distributed, and moderately
well conserved across
broad phylogenetic distances
-the number of different possible sequences is large,
so similarity in two sequences always indicates
some phylogenetic relationship;
the degree of similarity in rRNA sequences between two
organisms indicates relative
evolutionary relatedness
Ribosomal RNA molecules:
-in prokaryotes they are 5S, 16S, and 23S
-16S and 23S each contain several regions of highly
conserved sequence that allows for proper
sequence alignment, but
contain sufficient sequence variability in other regions to serve as
phylogenetic chronometers
-5S has also been used but it is too small which
limits its information usefulness
-16S is more experimentally manageable than 23S
RNA and has been used extensively for
developing databases
-in eukaryotes, the 18S rRNA counterpart
is the 16S rRNA
Signature sequences: short oligonucleotide sequences unique to
a certain group or groups of
organisms.
-signatures defining each of the three primary domains
have been identified
-other signatures defining the major taxa within
each domain have also been detected
-signatures are generally found in defined regions
of the 16S rRNA molecule, but are only readily
apparent when the computer
scans sequence alignments
-they allow for placing unknown organisms in the
correct major phylogenetic group, and can be
useful for constructing
genus and species-specific nucleic acid probes which are used exclusively
for identification purposes
in microbial ecology and diagnostics
- As the 16SrRNA is so highly conserved organisms
are classified as separate species if their
sequences show less than
98%
homology and are classified as different genera if their sequences
show less than 93% identity.
Specific base sequences in the rRNA known as signature
sequences were commonly
found in particular groups of organisms