CHAPTER 10
CLASSIFICATION OF MICROORGANISMS
The
science of classification is called taxonomy. The objective is to find how
groups of organisms are alike and how they are different. Scientists believe
that most living organisms have not yet been discovered or classified. When
possible new species are discovered, we need a standard to use to determine
whether the organism is sufficiently different from known species to establish
a new species or possibly a new genus for it, or whether it should be placed
into an existing group.
STUDY OF
PHYLOGENETIC RELATIONSHIPS
About
1.7 million living organisms have been identified. All of them share certain
characteristics:
Composed of cells
Plasma membrane
ATP for energy
DNA carries genetic information
That
is not to say that all 1.7 million are alike, because of course there are major
differences. We classify organisms into groups called taxa. Systematics, or
phylogeny, is the study of the evolutionary history of organisms. (Who or what
were the ancestors of the group under study?)
From
the beginning of scientific study, organisms have been sorted and classified.
1. Ancient Greeks on to 1700’s---2
kingdoms, plant and animal, not many groups besides this
2.
1735---Linnaeus also used 2 kingdom system, but established other groups (taxa)
and classified most known organisms into all his groups. Microorganisms didn’t
get a clear place in this system, but their importance was not recognized at
this time.
3.
In 1866, Haeckel proposed adding the kingdom Protista for all microorganisms.
Some accepted this idea but others continued to place bacteria in the plant
kingdom.
4. Beginning in late 1930’s---the next
step was the development of the electron microscope, which allowed detailed
study of the tiny structures within cells for the first time. It was then
realized that there were 2 basic types of cells---the eukaryotic cells with a
distinct, membrane-bound nucleus; and the prokaryotic cells (bacteria all go in
this group) which did not have this separate nucleus. Although it did not happen immediately, it
was realized that these cells were so different that a new kingdom was needed.
5.
1969---Whittaker’s Five-Kingdom system was proposed and became widely
accepted.
a. Kingdom Monera or
Prokaryotae---all prokaryocytes, which means all bacteria
b. Kingdom Protista---unicellular
eukaryocytes---protozoa, unicellular algae, slime molds
c. Kingdom Fungi---unicellular yeasts,
multicellular (but still microscopic)
molds, macroscopic mushrooms. Plantlike but no chlorophyll, so no
photosynthesis.
d. Kingdom Plantae---plants. All
multicellular and all carry on photosynthesis.
e. Kingdom Animalia---animals
As
studies of cells continued, scientists began to concentrate on studies of
ribosomes, since that is the one organelle that all cells have. First, they
discovered that prokaryotic and eukaryotic ribosomes are not identical
(remember 70 S and 80 S ribosomes). More specifically, they then began to study
the sequence of bases in the ribosomal RNA. The more different these are, the
further back in time the organisms shared a common ancestor. This led to the
discovery that there are three distinct types of cells. Differences between the
everyday eubacteria and the strange archaeobacteria had already caused some
question about whether these two groups of bacteria should even be in the same
kingdom. It had been recognized that structure of membrane lipids and several
other characteristics varied considerably, but the rRNA studies were even
stronger evidence. Now most scientists believe that there are 3 different types
of cells. All eukaryotic cells, prokaryotic cells of the more common
eubacteria, and prokaryotic cells of the strange, unusual bacteria called the
archaeobacteria.
Dr. Carl Woese first proposed that what
should be done was establishing a new classification taxon
, the domain, that would be above kingdom.
Those favoring this idea believe that all 3 groups were derived from an
unknown common ancestor way back in time, each taking its own steps in its own
direction. This idea was not immediately accepted, but over nearly 30 years
most scientists have come to believe this is the best way. The 3 domains are :
More
about this domain---these are the strange, unusual bacteria which have also
been called archaeobacteria. Although their appearance through a microscope or
as visible colonies may be very similar or identical to other bacteria, they
have some differences which put them in a group apart:
****1.
Differences in RNA
2. Differences in structure of membrane
lipids
3. Cell wall structure (NO peptidoglycan
but similar substances)
4. Unusual metabolic processes-----the
ability to metabolize unusual substrates and the production of unusual end
products
5. Ability to thrive in extreme
physical conditions
The
archaea are placed in three groups:
1. Methanogens---strict anaerobes that
produce methane (CH4) from carbon dioxide and hydrogen
2. Extreme halophiles---require very high
concentrations of salt
3. Hyperthermophiles----grow well in hot,
acid environments
SCIENTIFIC
NOMENCLATURE
Common
names for organisms are not always the same in every country, or even in
different parts of the same country.
Each organism needs to be assigned a name that is recognized by all
biologists. Scientific nomenclature began with Linnaeus, who chose Latin, which
was the language of scholars in his day. The name of each organism has 2 parts
(binomial nomenclature). These names are
the same world-wide.
Rules for naming protozoa and parasitic
worms are in the International Code of
Zoological
Nomenclature.
Rules for naming fungi and algae are in the International Code of Botanical
Nomenclature.
Rules for naming newly classified bacteria are
established by the
International Committee on Systematic
Bacteriology and published in the International
Journal
of Systematic Bacteriology. They are then included in the standard of
reference for
bacteria, Bergey’s Manual of Systematic Bacteriology.
New laboratory techniques, involving
analysis of DNA and RNA, have caused some
bacteria to be reclassified. Sometimes
organisms which have been in separate genera are
combined
into one genus; other times organisms previously in the same genus are split
out into separate genera. Even if the genus name is changed, the species name
usually remains the same.
This
is the complete list of taxa (classification groups) which are used to classify
living organisms:
Domain (this is the relatively new one)
Kingdom
Phylum
Division (this one may be used in place of
phylum in botany)
Class
Order
Family
Genus
Species
Organisms
are grouped according to common properties (characteristics) and an estimate as
to how closely related they are to a common ancestor. Some classifications are at least partly
based on fossils, but this type of material is available mostly for the larger
eukaryocytes. Microbes leave very few fossils, so other evidence often must be
used for them.
Bergey’s Manual of Systematic Bacteriology, 2nd
edition, is the standard for classification of prokarocytes. Two domains,
Bacteria and Archaea, are included. Each domain is divided into phyla, based on
similarities in rRNA. Organisms continue to be divided into taxa that are more
and more specific until, eventually reaching the species level. In eukaryocytes, a species is defined as a
group of closely related organisms that breed among themselves. A bacterial species is defined as a
population of cells with similar characteristics. (Bacteria mostly reproduce by
asexual binary fission, so the other definition can’t fit.) Members of the species are VERY similar to
each other, but all may not be totally identical. The designation of strain is
used for these slightly different versions within the same species of bacteria.
Strains are identified by numbers, letters, or names that follow the species
name.
domain: Eukarya---contains 4 kingdoms
kingdom: protista---unicellular eukaryocytes
kingdom: fungi---unicellular yeasts,
multicellular molds, macroscopic fungi--these all absorb organic matter through
their plasma membranes
kingdom: plantae---plants--macroscopic algae,
mosses, ferns, conifers, flowering plants--all are multicellular, all carry on
photosynthesis
kingdom: animalia---animals--sponges, worms,
insects, vertebrates--all ingest nutrients
Virus
classification is a mess. Viruses are not composed of cells, so there are
arguments about whether they are even really alive.
Alive: They have either DNA or RNA, which
must mean they are living
Not alive:
No cellular structure
No metabolism and no
reproduction until they invade a living cell
Do not have DNA and RNA
both—living things have both
Viruses
are described as obligate intracellular parasites. A viral species is a population of viruses
with similar characteristics that occupies a particular ecological niche. (The
ecological niche of a virus is its host cell). There are two hypotheses on the
origin of viruses:
1. They arose from strands of DNA similar
to plasmids
2. They developed from degenerative cells
that lost their ability to function independently
METHODS OF CLASSIFYING AND IDENTIFYING MICROORGANISMS
Microorganisms
are identified and classified for more than one reason. We attempt to identify
and classify all living things, but determining the identity of pathogens is
especially important, because it often makes effective treatment possible.
Characteristics used to classify and identify microbes include:
1.
MORPHOLOGY---This refers to
the form of the organism---in bacteria this is the basic shape, the size, any
characteristic arrangement, the presence of flagella, presence of a capsule,
presence of endospores, etc. This is a beginning in classifying and identifying
microbes, but it is of limited use. Many microbes look exactly the same. Even
eubacteria and archaea may look identical through the microscope.
Based
on morphology alone, it is sometimes even quite difficult to determine which
group of microbes an organism belongs to. An example of this is an
opportunistic pathogen found mainly in AIDS patients, Pneumocystis jiroveci,
previously known as Pneumocystis
carinii. At first, this organism was believed to be a protozoan, but more
recently it has been found to be more closely related to fungi. This is
important because it may allow more effective treatment.
2.
DIFFERENTIAL STAINING---this
typically begins with a gram stain. The acid-fast stain is another example.
3.
BIOCHEMICAL TESTS---since
so many bacteria look so much alike, it is frequently necessary to resort to
biochemical tests for identification. What these tests are really looking for
is the ability of an organism to produce certain enzymes. They do this by
testing for the ability of a species to make use of certain nutrients, and also
what waste products are produced in the process.
One
group of microbes which often must be identified by biochemical tests is a
group of related gram-negative rods whose natural habitat is the digestive tract of humans and other
animals. Some of these are considered normal flora (organisms that live in or
on the body and usually cause no harm) and some are pathogens. Genera included
are Escherichia, Enterobacter, Shigella,
Citrobacter, and Salmonella. Shigella and Salmonella are pathogens, which can be distinguished from normal
flora organisms by relatively simple biochemical tests. Selective and
differential media may also aid in identification.
We
will perform some of these biochemical tests and use some special media in our
lab, using the relatively old-fashioned method of preparing separate test tubes
and Petri dishes of various media. In a
commercial or hospital lab, special test kits which contain a number of
different media in a tube-like container would be used. One system does 15 tests. The bacteria are
incubated into all the compartments at once.
Since
characteristics vary with different strains, the more tests that are done the
better, and the more certain the identification.
Serological
testing can even distinguish between strains of the same species. Strains with different antigens are called
serotypes, serovars, or biovars. Streptococci were originally sorted out by
this method. Today, two serotypes of Streptococcus
pyogenes have been isolated as the causative agent from cases of
necrotizing fasciitis (flesh-eating bacteria).
Another
widely used test is the enzyme-linked immunosorbent assay (ELISA). Known
antibodies are mixed with unknown bacteria. Observing which antibodies the
bacteria react with can identify them. This test is also used to detect the
presence of AIDS antibodies.
7. DNA FINGERPRINTING—this is a
more complicated test. It is time-consuming and expensive. However, it
actually determines the sequence of bases in an organism's DNA and can give
much more conclusive proof of relatedness. Restriction enzymes cut a molecule
of DNA everywhere a specific base sequence occurs. One specific restriction
enzyme would cut DNA everywhere these sequences occur:
DNA
samples from two different organisms are treated separately with the same
restriction enzyme. The resulting fragments are separated by electrophoresis,
and the number and sizes of the fragments are compared. The more similar the
patterns, the more closely related the organisms.
8.
RIBOSOMAL RNA SEQUENCING--this
is another technique used to determine the relationships among organisms. Ribosomal RNA is believed to change less than
other nucleic acids over time, because if an organism has a drastic change in
its rRNA it would be unable to assemble proteins and would die. A study of the
similarity of rRNA of two different organisms is believed to show how far back
in the past the two organisms branched off a common ancestor.
Nucleic
acid hybridization assumes that if 2 organisms are closely related, their
nucleic acid sequences will be quite similar. Less closely related organisms
will have less similarity. This is the basis for several means of testing:
1. DNA single strands from 2 different
organisms are mixed, and the more the strands that pair up the more similar the
DNA, so the more closely related the organisms are.
2. Since RNA is transcribed from a single
strand of DNA, it will pair up with the proper section of single-strand DNA if
the 2 are mixed. RNA from one organism
can be mixed with separated strands of DNA from another and relatedness can be
determined by observing the amount of pairing that occurs.
3. The technique can be used to identify unknown
organisms by a technique called Southern blotting.
4. DNA probes can be prepared using sections
of the DNA of an organism. The DNA probes are single strands which are stained
with a fluorescent dye or made radioactive. These probes are then mixed with
single-strand DNA which has been prepared from a sample suspected of containing
the organism. If the two match, they will hybridize and this can be detected by
looking for the radioactivity or the dye.