Microbiology_23

MICROBIAL
TAXONOMY
 INTRODUCTION
• Taxonomy [Greek taxis, arrangement or order, and
nomos, law, or nemein, to distribute or govern] is
defined as the science of biological classification.
• The term systematics often is used for taxonomy.
However, many taxonomists define it in more
general terms as “the scientific study of organisms
with the ultimate object of characterizing and
arranging them in an orderly manner.”
In a broader sense it consists of three separate but
interrelated parts:
1. Classification: It is the arrangement of organisms
into groups or taxa (sing:taxon) based on mutual
similarity or evolutionary relatedness.
2. Nomenclature: It is the branch of taxonomy
concerned with the assignment of names to
taxonomic groups in agreement with published
rules.
3. Identification is the practical side of taxonomy,
the process of determining that a particular isolate
belongs to a recognized taxon.
 IMPORTANCE OF TAXONOMY
1. Taxonomy allows us to organize huge amounts
of knowledge about organisms because all
members of a particular group share many
characteristics. It is like a giant filing system or
library catalogue that provides easy access to
information. The more accurate the
classification, the more information-rich and
useful it is.
2. Taxonomy allows us to make predictions and
frame hypotheses for further research based
on knowledge of similar organisms. If a relative
has some property, the microorganism in question
also may have the same characteristic.
3.To communicate efficiently: Taxonomy places
microorganisms in meaningful, useful groups
with precise names so that microbiologists can
work with them and communicate efficiently.
4. Taxonomy is essential for accurate
identification of microorganisms.
 MAJOR CONTRIBUTORS OF MODERN
TAXONOMY

ROBERT H. WHITTAKER CARL WOESE CAROLUS LINNAEUS

 TAXONOMIC RANKS
In prokaryotic taxonomy the most commonly used
levels or ranks (in ascending order) are species, genera,
families, orders, classes, and phyla.
 TERMINOLOGIES USED IN TAXONOMY
• Strain: A strain is a population of organisms that is
distinguishable from at least some other populations
within a particular taxonomic category.
• It is considered to have descended from a single
organism or pure culture isolate. Strains within a
species may differ slightly from one another in many
ways.
• Biovars: Biovars are variant procaryotic strains
characterized by biochemical or physiological
differences.
• Morphovars: Morphovars differ morphologically.
• Serovars: Serovars have distinctive antigenic
properties.
• Type Strain: One strain of a species is designated as
the type strain. It is usually one of the first strains
studied and often is more fully characterized than
other strains; however, it does not have to be the
most representative member. The type strain for the
species is called the type species and is the
nomenclatural type or the holder of the species
name.
 CHARACTERISTICS
USED IN TAXONOMY

CLASSICAL MOLECULAR
CHARACTERISTICS CHARACTERISTICS

Physiological
Morphological and Metabolic Ecological Genetic
Characteristics Characteristics Characteristics Analysis
Nucleic Acid
Comparison of Base
Proteins Composition
 CLASSICAL CHARACTERISTICS

• Classical approaches to taxonomy make use of

morphological, physiological, biochemical,
ecological, and genetic characteristics.
• These characteristics have been employed in
microbial taxonomy for many years. They are
quite useful in routine identification and may
provide phylogenetic information as well.
 MORPHOLOGICAL CHARACTERISTICS

• Morphology is easy to study and analyze, particularly

in eukaryotic microorganisms and the more complex
prokaryotes.
• In addition, morphological comparisons are
valuable because structural features depend on the
expression of many genes, are usually
genetically stable, and normally (at least in
eukaryotes) these do not vary greatly with
environmental changes. Thus morphological
similarity often is a good indication of
phylogenetic relatedness.
• Many different morphological features are
employed in the classification and
identification of microorganisms .
 PHYSIOLOGICAL AND METABOLIC
CHARACTERISTICS
• Physiological and metabolic
characteristics are very
useful because they are
directly related to the
nature and activity of
microbial enzymes and
transport proteins.
• Since proteins are gene
products, analysis of these
characteristics provides an
indirect comparison of
microbial genomes.
 ECOLOGICAL CHARACTERISTICS
• Many properties are ecological in nature since they
affect the relation of microorganisms to their
environment. Often these are taxonomically
valuable because even very closely related
microorganisms can differ considerably with
respect to ecological characteristics.
• Microbes living in various parts of the human body
markedly differ from one another and from those
growing in freshwater, terrestrial, and marine
environments.
• Some examples of taxonomically important
ecological properties are :
1. The nature of symbiotic relationships.
2. The ability to cause disease in a particular host.
3. Habitat preferences such as requirements for
temperature, pH, oxygen, and osmotic
concentration and growth requirements
 GENETIC ANALYSIS
• As most eukaryotes are able to reproduce sexually,
genetic analysis has been of considerable usefulness in
the classification of these organisms. Although
prokaryotes do not reproduce sexually, the study of
chromosomal gene exchange through transformation
and conjugation is sometimes useful in their
classification.
• TRANSFORMATION: Transformation can occur
between different prokaryotic species but only
rarely between genera. The demonstration of
transformation between two strains provides evidence
of a close relationship since transformation cannot
occur unless the genomes are fairly similar.
• Despite transformation’s usefulness, its results are
sometimes hard to interpret because an absence of
transformation may result from factors other than major
differences in DNA sequence.
• CONJUGATION: Conjugation studies also yield
taxonomically useful data, particularly with the enteric
bacteria. For example, Escherichia can undergo
conjugation with the genera Salmonella and Shigella but
not with Proteus and Enterobacter. These observations
fit with other data showing that the first three of these
genera are more closely related to one another than to
Proteus and Enterobacter.
 MOLECULAR CHARACTERISTICS

• Some of the most powerful approaches to

taxonomy are through the study of proteins and
nucleic acids.
• Because these are either direct gene products or
the genes themselves, comparisons of proteins
and nucleic acids yield considerable information
about true relatedness. These more recent
molecular approaches have become increasingly
important in prokaryotic taxonomy.
 COMPARISON OF PROTEINS
• The amino acid sequences of proteins are direct
reflections of mRNA sequences and therefore
closely related to the structures of the genes coding
for their synthesis. For this reason, comparisons of
proteins from different microorganisms are very
useful taxonomically. There are several ways to
compare proteins.
COMPARISON OF
PROTEINS

INDIRECT
DIRECT APPROACH
APPROACH

PROTEIN ELECTROPHORETIC IMMUNOLOGIC PROPERTIES

SEQUENCING MOBILITY TECHNIQUES OF ENZYMES
PROTEIN SEQUENCING:The most direct approach is
to determine the amino acid sequence of proteins with
the same function. The sequences of proteins with
dissimilar functions often change at different rates;
some sequences change quite rapidly, whereas others
are very stable.
• Nevertheless, if the sequences of proteins with the
same function are similar, the organisms possessing
them are probably closely related.
• The sequences of cytochromes and other electron
transport proteins, histones, heat-shock proteins,
transcription and translation proteins, and a variety of
metabolic enzymes have been used in taxonomic
studies.
Because protein sequencing is slow and expensive,
more indirect methods of comparing proteins
frequently have been employed.

ELECTROPHORETIC MOBILITY: The

electrophoretic mobility of proteins is useful in
studying relationships at the species and subspecies
levels.
IMMUNOLOGIC TECHNIQUES: Antibodies can
discriminate between very similar proteins, and
immunologic techniques are used to compare proteins
from different microorganisms

PROPERTIES OF ENZYMES: The physical,

kinetic, and regulatory properties of enzymes have
been employed in taxonomic studies. Because enzyme
behaviour reflects amino acid sequence, this approach
is useful in studying some microbial groups, and
group-specific patterns of regulation have been found.
 NUCLEIC ACID BASE COMPOSITION
• Microbial genomes can be directly compared, and
taxonomic similarity can be estimated in many ways.
• The first, and possibly the simplest, technique to be
employed is the determination of DNA base
composition.
• GC CONTENT: DNA contains four purine and
pyrimidine bases: adenine (A), guanine (G), cytosine
(C), and thymine (T). In double-stranded DNA, A pairs
with T, and G pairs with C. Thus the (GC)/(AT) ratio
or GC content, the percent of GC in DNA, reflects the
base sequence and varies with sequence changes as
follows:
Mol % of (G+C)= (G+C) X 100 /(A+T+G+C)
• The base composition of DNA can be determined in
several ways. Although the GC content can be
ascertained after hydrolysis of DNA and analysis of its
bases with high-performance liquid chromatography
(HPLC), physical methods are easier and more often
used.
• The GC content often is determined from the melting
temperature (Tm) of DNA. In double-stranded
DNA three hydrogen bonds join GC base pairs, and
two bonds connect AT base pairs.
• As a result DNA with a greater GC content will have
more hydrogen bonds, and its strands will separate
only at higher temperatures—that is, it will have a
higher melting point.
• DNA melting can be easily
followed
spectrophotometrically
because the absorbance of 260
nm UV light by DNA
increases during strand
separation.
• When a DNA sample is
slowly heated, the absorbance
increases as hydrogen bonds
are broken and reaches a
plateau when all the DNA has
become single stranded.
The GC content of DNA from animals and higher plants
averages around 40% and ranges between 30 and 50%. In
contrast, the DNA of both eukaryotic and prokaryotic
microorganisms varies greatly in GC content; prokaryotic
GC content is the most variable, ranging from around 25
to almost 80%. Despite such a wide range of variation, the
GC content of strains within a particular species is
constant. If two organisms differ in their GC content by
more than about 10%, their genomes have quite different
base sequences.
But, it is not always proper to assume that organisms with
very similar GC contents also have similar DNA base
sequences. Only if two microorganisms also are alike
phenotypically then their similar GC content suggest close
relatedness.
GC content data are taxonomically valuable in at least
two ways.
First, they can confirm a taxonomic scheme developed
using other data. If organisms in the same taxon are too
dissimilar in GC content, the taxon probably should be
divided.
Second, GC content appears to be useful in
characterizing procaryotic genera since the variation
within a genus is usually less than 10% even though
the content may vary greatly between genera.
 NUCLEIC ACID HYBRIDIZATION
• The similarity between genomes
can be compared more directly
by use of nucleic acid
hybridization studies. If a
mixture of single stranded DNA
formed by heating dsDNA is
cooled and held at a temperature
about 25°C below the Tm,
strands with complementary base
sequences will reassociate to
form stable dsDNA, whereas
non-complementary strands will
remain single.
In hybridization techniques, nitrocellulose filters with
bound nonradioactive DNA strands are incubated at the
appropriate temperature with single-stranded DNA
fragments made radioactive with 32P, 3H, or 14C.
After radioactive fragments are allowed to hybridize
with the membrane-bound ssDNA, the membrane is
washed to remove any nonhybridized ssDNA and its
radioactivity is measured. The quantity of radioactivity
bound to the filter reflects the amount of hybridization
and thus the similarity of the DNA sequences. The degree
of similarity or homology is expressed as the percent of
experimental DNA radioactivity retained on the filter
compared with the percent of homologous DNA
radioactivity bound under the same conditions.
• Two strains whose DNAs show at least 70%
relatedness under optimal hybridization conditions and
less than a 5% difference in Tm often are considered
members of the same species.
• Therefore DNA-DNA hybridization is used to study
only closely related microorganisms.
• More distantly related organisms are compared by
carrying out DNA-RNA hybridization experiments
using radioactive ribosomal or transfer RNA. The
technique is similar to that employed for DNA-DNA
hybridization: membrane-bound DNA is incubated
with radioactive rRNA, washed, and counted.
 NUCLEIC ACID SEQUENCING
• Despite the usefulness of GC content determination
and nucleic acid hybridization studies, genome
structures can be directly compared only by
sequencing DNA and RNA, for which there are
different techniques available.
• Most attention has been given to sequences of the 5S
and 16S rRNAs isolated from the 50S and 30S
subunits, respectively, of prokaryotic ribosomes. The
rRNAs are almost ideal for studies of microbial
evolution and relatedness since they are essential
to a critical organelle found in all
microorganisms.
• Their functional role is the same in all ribosomes.
Furthermore, their structure changes very slowly
with time, presumably because of their constant
and critical role.
• Because rRNA contains variable and stable
sequences, both closely related and very distantly
related microorganisms can be compared.

16 S rRNA gene with variable and conserved regions

• There are several ways to sequence rRNA.
Ribosomal RNAs can be characterized in terms of
partial sequences by the oligonucleotide cataloging
method as follows.
• Purified, radioactive 16S rRNA is treated with the
enzyme T1 ribonuclease, which cleaves it into
fragments. The fragments are separated. The
sequences of corresponding 16S rRNA fragments
from different prokaryotes are then aligned and
compared using a computer, and association
coefficients (Sab values) are calculated.
• Complete rRNAs now are sequenced. The RNA is
isolated and purified. Then, reverse transcriptase is
used to make complementary DNA (cDNA) using
primers that are complementary to conserved
rRNA sequences. Next, the polymerase chain
reaction amplifies the cDNA. Finally, the cDNA
is sequenced and the rRNA sequence deduced from
the results.