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 two kingdoms: plant and animal.
Bacteria were placed into plant kingdom. Clearly,
this scheme was inadequate. The electron microscope
demonstrated obvious differences between
bacteria and eukaryotes.
II. In 1968 Whittaker proposed his famous 5 Kingdom system
of living organisms. Bacteria were
classified under Kingdom Prokaryotae (aka Monera).
Prokaryotes were defined as "cells in which
nuclear material is not surrounded by a nuclear
membrane."
The Linnean system of Binomial Nomenclature
The Swedish naturalist, 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.
c) Biovars are organisms which can differ physiologically. For
example, they may possess differing
forms of enzymes
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
Eucarya) 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 we 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
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, humans 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 ans 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)
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
16S ribosomal RNA sequences
- 16S rRNA (about 1500 nucleotides long) is found
in the 30S ribosomal subunit of bacterial
ribosomes.
- 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.
Dr. Carl Woese and colleagues at the University of Illinois examined
the 16S rRNA sequences of
hundreds of organisms and divided the organisms into three domains
based on their signature sequences
and their related properties. The domain is now the highest level of
organization in the biological world.
The Domains are termed:
Eukarya
Eubacteria
Archaea
Microbial phylogeny as revealed by ribosomal RNA sequencing
Cellular life has evolved along three major lineages, two of
which are composed only of prokaryotic
cells (Bacteria and Archaea); the third constitutes the eukaryotic
lineage (Eukarya); called domains
as the highest biological taxon. Plants, animals, fungi, and protists
are all kingdoms within the domain
Eukarya
The universal tree of life:
-Eukarya are not of recent origin, but are as ancient as either of
the prokaryotic lineages;
organelle-less eukaryotes shared common ancestry
with the other two evolutionary lines
-the root of the tree represents a common ancestor; first the
Bacteria and Archaea-Eukarya split,
then the Archaea and Eukarya diverged (Archaea and
Eukarya are more related to each other than
either is to Bacteria)
-Archaea are the most primitive of organisms, Eukarya are the
least primitive; presence of Archaea in
extreme environments reflect those under which life
orginated
-none of the organisms living today are primitive; all are modern organisms
a) Bacteria:
- at least 12 distinct phylogenetic lineages (kingdoms)
- most are composed of species with a mixture of physiologies, morphologies,
and other phenotypic
properties
-Aquifex is the branch closes to the universal ancestor; hyperthermophilic,
chemolithotrophic
hydrogen-oxidizer, consistent with the early environment
of Earth
b) Archaea:
- consists of three major groups (kingdoms)
- physiologies are again consistent with early earth conditions
- Korarchaeota is closest to the root; as a lineage this was only identified
through 16S rRNA obtained
by cloning genes from natural samples; they have
not yet been cultured
c) Eukarya:
- sequences are derived from the 18S rRNA; analysis suggests evolution
in this lineage may have
occurred in major epochs
- early eukaryotes were probably similar to present-day microsporidia
and diplomonads, all obligate
parasites (e.g. the human pathogen Giardia)
- these have nuclei but no mitochondria; and have small genomes for
eukaryotic cells
- rapid evolutionary radiation, as determined by fossil records, occurred
about 1.5 billion years ago;
when it was thought that significant oxygen levels
had accumulated in the atmosphere (development
of ozone shield allowed for a much larger number
of suitable surface habitats)
Characteristics of the primary domains
Cell walls:
· Virtually
all Bacteria have cell walls containing peptidoglycan, which can
be considered a signature
molecule for Bacteria
· Eukarya and Archaea lack peptidoglycan
· If cell walls are present in eukaryotes,
they are usually composed of cellulose or chitin
· Cell walls of Archaea consist of
pseudopeptidoglycan,
polysaccharide, protein, or glycoprotein
Lipids:
· chemical nature of membrane lipids
may be the most useful of all nongenetic criteria for differentiating
Archaea from Bacteria
· Bacteria and eukaryotes have lipids
with a backbone of fatty acids hooked in ester linkage to
a
molecule of glycerol
· Archaea have ether linkages,
and the hydrocarbons are not fatty acids
RNA polymerase:
· Bacteria contain only one type
of RNA polymerase; four polypeptides with a variety of different
sigma factors
· Archaeal RNA polymerases are of
several types, and are more complex structurally than those of
Bacteria
· Eukarya also have several RNA polymerases;
one that makes messenger RNA (highly complex),
other RNA polymerases are for rRNA and tRNA
· The antibiotic choramphenicol (interferes
with the beta subunit of Bacterial RNA polymerase) acts
only on Bacteria; Archaea and Eukarya lack this
form of RNA polymerase.
Features of protein synthesis:
· while ribosomes of Archaea and
Bacteria are the same size (70S), steps in archaeal protein synthesis
more strongly resemble those in eukaryotes
than in Bacteria
· Bacteria incorporate formylmethionine
at the start codon; the initiator tRNA in eukaryotes and
Archaea carry methionine
· exotoxin of Corynebacterium
diptheriae ADP-ribosylates an elongation factor required to
translocate the ribosome along the mRNA making it
inactive
in eukaryotes
· diptheria toxin inhibits protein
synthesis in Archaea but not Bacteria
· most antibiotics that specifically
affect protein synthesis in Bacteria do not affect Archaea or Eukarya