Classification of Microorganisms

Why do we want to classify microorganisms?
Microorganisms are classified into taxa (taxonomic categories) to facilitate research, scholarship and communication. Biologists try to look for a natural classification system which are based on ancestral relationships. The hierarchy of the taxa reveals the evolutionary or phylogenetic relationships between microorganisms.

Five kingdom system
This system was proposed in 1969 by Robert Whittaker. This is the system most of us are familiar with. The kingdoms include i) Monera ( Procaryotae), the bacteria, ii) Protista, the unicellular Eukaryotes, iii) Fungi, iv) Plantae and v) Animalia.

Three domain system
This system was proposed in 1978 by Carl Woese. Molecular biology and biochemistry provide the basis of this newly proposed system. Based on distinct differences in the ribosomal RNA sequences two distinct taxons were discovered among the kingdom Monera. The three domains are Eubacteria, Archaea, and Eucarya. The kingdoms Animalia, Plantae, Fungi, and Protista are kingdoms in the domain Eucarya.

Taxonomic hierarchy
Domain, kingdom, phylum (division), class, order, family, genus and species. Species definition in Eucarya is relatively simple compared to bacteria since it relies on the sexual reproduction and isolation of individuals within a species. A bacterial species is defined are a population of cells with similar characteristics — either phenotypic or genotypic.

How are organisms named?
Binomial nomenclature where each organism has two names — a genus name and a specific epithet or species name. Both are always either underlined or italicized and the genus is always capitalized and the species is lowercase. Example Escherichia coli

Rules for naming bacteria are outlined by the International committee on Systematic Bacteriology. Descriptions of new species of bacteria are first published in International Journal of Systematic Bacteriology before they are incorporated into Bergey’s Manual of Systematic Bacteriology, the bible of systematic bacteriology.

Methods to classify and identify microorganisms

Bergey’s manual of determinative bacteriology is the bible for the identification of bacteria. You will find many tables of characteristics of identified and classified bacteria that appear in Bergey’s Manual of Systematic Bacteriology. In addition to the properties listed in Bergey’s, the source and habitat of microorganisms is also important to consider when identifying microorganisms.

Morphological characteristic
Useful for higher organisms but not so for microorganisms. Hundreds of different species of microorganisms are either rod or cocci shaped. Other morphological characteristics such as endospores and flagella are useful.

Staining
Gram and acid-fast staining are very useful in identifying microorganisms. Doctors can start prescribing antibiotics given the gram stain of a potential pathogen.

Biochemical tests
Enzymatic activities are widely used to differentiate and identify species of bacteria. Many different rapid, miniaturized testing systems have been commercialized. Examples include the API strips, Enterotubes, and BIOLOG. Each of these have a variety of tests that are done simultaneously on a single isolate. BIOLOG can do 95 different tests, API can do 20 tests and Enterotubes can do 14-17 different tests. Some of these have been somewhat automated and the results analyzed by computers.

Serology
Antibodies against specific microorganisms can be used for the identification of many microorganisms. Slide agglutination are used to rapidly identify potential pathogens using antiserum made against the pathogen. Positive identification is indicated by agglutination or clumping of the bacteria and negative is indicated by no clumping of the bacteria. Enzyme-linked immunosorbent assay (ELISA) is a rapid approach. Antibodies are coated on microtiter plates, bacteria are added, a second antibody with an enzyme linked to it is added, and a substrate is added. If the bacteria is recognized by the antibodies the enzyme will work on the substrate and you get a positive well. Another serological test is called Western blotting, where proteins of the patient are separated by electrophoresis and blotted onto a membrane. Antibodies with enzymes linked to them are used to "probe" the membrane with the patient’s proteins. If the protein of interest is there, the antibodies will bind and lightup with the substrate for the enzyme linked to the antibody.

Phage typing
Bacteriophage will lyse specific strains of bacteria. To determine the identity of a bacteria, it is plated out on a general medium. Before the bacteria grows up, drops of phage containing solutions are placed in various spots on the plate. If the cell is susceptible to the phage, the phage will lyse the cells resulting in clearing zones called plaques. By looking at the phage sensitivity patterns, you can identify the bacteria.

Amino acid sequencing
The amino acid sequence can be determined from any protein. If you compare the same protein from different bacteria, the more distantly related the bacteria are the more differences you will find in the amino acid sequences of the protein. The converse of this is that the more closely related the bacteria are the more similar the proteins will be.

Fatty acid profiles
The fatty acids of the membranes of bacteria are extracted and separated using a gas chromatograph. The fatty acids can be identified using internal known standards. The fatty acid profiles of a species is pretty constant so you can develop a data base to compare unknown bacteria with known strains of bacteria for identification. Commercial systems have been developed — Sherlock of MIDI for example.

DNA base composition
DNA base composition is usually expressed as percentage guanine plus cytosine. Related organisms that are genetically related - have many identical or similar genes - should have similar G+C %. Unrelated organisms predictably would have dissimilar G+C%, but they could also have similar G+C% by chance. Other types of analysis should be performed to support relationships.

DNA fingerprinting
DNA sequencing is becoming more of a reality but still is very costly and time consuming therefore it is not used routinely for determining relationships between organisms. But there is an approach that compares base sequences of organisms. Restriction enzymes - restriction endonucleases - cut DNA at specific sequences. For example the restriction enzyme EcoRI cuts DNA at G - AATTC (between the G and first A). DNA from two or more organisms can be digested and the resulting restriction fragments separated by electrophoresis. The patterns after electrophoresis can be compared to determine relatedness. Related organisms should have more similar patterns than unrelated organisms. This is routinely used in epidemiological studies of bacteria and viruses.

Ribosomal RNA sequencing
Sequencing the 16S ribosomal RNA gene. Used to determine the phylogenetic relationships among bacteria and determine diversity among communities of bacteria. Several advantages: All cells contain rRNA; Closely related species have fewer differences than distantly related species; rRNA genes are highly conserved compared to other gene sequences; rRNA sequencing doesn’t require culturing the organism. The gene can be obtained using the polymerase chain reaction.

Polymerase chain reaction
Specific sequences of DNA can be amplified using site specific DNA primers and a DNA polymerase called Taq polymerase. See figure 9.14 for details.

Nucleic acid hybridization
Double stranded DNA can be denatured either chemically or thermally. Thermally denatured DNA will reform native double stranded DNA if cooled slowly since the two strands are complements of each other. If you have DNA from two different organisms mixed and denatured the degree at which they will form a double stranded helix (hybridize) depends on their relatedness.

Southern blotting relies on the same principle. See figure 9.12 DNA is probed with a piece of DNA that is labelled either with an enzyme or radioactive nucleotide.

REVIEW Table 10.5

Dichotomous keys are used for identification of microorganisms based on data from analysis described above, especially biochemical type.

Cladograms
Tree like structure used to show evolutionary relationships.