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

Nomenclature is another aspect of taxonomy. Names are assigned to organisms in a systematic

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

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

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
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

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
    -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
    - 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:

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

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
-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

· 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
· 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