Classification of

Microorganisms
Prepared by
Rajesh M.Patel
Assistant Professor,
Department of Pharmaceutical Biotechnology,
Ganpat University.
Email:rajmit_120@yahoomail.com
 Contents of the chapter
Terminology
Taxonmomy: Introduction and History
Classification
Nomenclature
Taxonmic methods for classification of
microorganisms
Intuitive method
Genetic methods
% G+C content
Nucleic acid hybridization
DNA chip technique
 Terminology
Taxonomy
Classification of living organisms into groups
Phylogenetic Classification System:
Groups reflect genetic similarity and evolutionary
relatedness
Phenetic Classification System:
Groups do not necessarily reflect genetic
similarity or evolutionary relatedness. Instead,
groups are based on convenient, observable
characteristics.
 Terminology
Species: A collection of microbial strains that
share many properties and differ significantly
from other groups of strains
Strain: a population of organisms descended
from a single organism or pure culture
isolate.
OR
a culture derived from a single parent that
differs in structure or metabolism from other
cultures of that species
 Terminology
Type strain: One of the first strain among the
species, studied and fully characterised than
other strains.
Biovars procaryotic variant strains
characterized by biochemical or physiological
differences
Morphovars procaryotic variant strains
characterized by morpholigical characteristics
Serovars procaryotic variant strains
characterized by distinctive antigenic
properties
 Taxonomy
Organizing, classifying and
naming living things
Formal system originated by
Carl von Linn (1701-1778)
Identifying and classifying
organisms according to
specific criteria
Each organism placed into a
classification system
 Taxonomy

Taxa: order or arrangement

Nomos: law
Nemein: to distribute or govern
The branch of biology dealing with the
classification of life.
Taxonomy / Systematics can be
understand by general examples
Classification Identification
Organization into groups Distinguishing features
Car Engine size
Truck Mileagae
SUV Number of passengers
Van Type of transmission

Nomenclature
Providing a formal name
Genus & species
Ford Crown Victoria
Chevy Impala
Toyota Camry
Honda Civic
 Taxonomy: History
1700s 2 kingdoms: plant and animal
1800s 3 kingdoms: plant, animal, and
protista
1950-1990s 5 kingdoms: plant, animal,
protista, fungi, monera
Present: 6 kingdoms: eubacteria,
archaebacteria, protista, animal, plant, fungi
Taxonomy consists of 3 parts
Classification
Identification
Nomenclature
Classification

Arrangement into groups based on

mutual similarity or evolutionary
relatedness
Ordering of organisms into groups.
 Classification
Natural classification: arranges organisms
into groups whose members share many
characteristics and reflects as much as
possible the biological nature of organisms.
Linnaeus developed the first natural
classification, based largely on anatomical
characteristics.
E.g. classification of humans as mammals
denotes that they have hair, self-regulating
body temperature,and milk-producing
mammary glands in the female.
Polyphasic classification
Taxonomy

Domain
Kingdom
Phylum
Class
Order
Family
Genus
species
3 Domains
Eubacteria
true bacteria, peptidoglycan
Archaea
odd bacteria that live in extreme
environments, high salt, heat, etc. (usually
called extremophiles)
Eukarya
have a nucleus & organelles (humans,
animals, plants)
3 Domains system
Five-Kingdom System of Biological
Classification
Proposed in 1969 by Robert Whitaker :
1. Kingdom Procaryotae (Monera): Oldest known cells.
Lived over 3.5 billion years ago. Lack a nucleus and
membrane bound organelles.
The other four kingdoms are eucaryotes. Have a
true nucleus and membrane bound organelles.
2. Kingdom Protista: Mostly unicellular, lack tissue
organization. Most have flagella during life.
3. Kingdom Fungi: May be unicellular (yeasts) or
multicellular (molds). Many are saprotrophs.
4. Kingdom Plantae: Multicellular, photosynthetic.
5. Kingdom Animalia: Multicellular, heterotrophs that
ingest food through a mouth or oral cavity.
Five-Kingdom Classification
System (Whittaker)
Scientific
Nomenclature
Developed in the eighteenth century.
Bionomial nomenclature:
Genus and specific epithet(species)
Eg. Escherichia coli, Salmonella typhi,
Bacillus subtilis
Rules for naming are set by international
committees
International Code of Zoological Momenclature
International Code of Botanical Nomenclature
Bacteriological Code and Bergeys Manual
 Scientific Names
Scientific Binomial Source of Genus Source of
Name Specific Epithet
Klebsiella Honors Edwin Klebs The disease
pneumoniae
Pfiesteria piscicida Honors Lois Pfiester Disease in fish

Salmonella Honors Daniel Stupor (typh-) in

typhimurium Salmon mice (muri-)
Streptococcus Chains of cells Forms pus (pyo-)
pyogenes (strepto-)
Penicillium Tuftlike (penicill-) Produces a yellow
chrysogenum (chryso-) pigment
Trypanosoma cruzi Corkscrew-like Honors Oswaldo
(trypano-, borer; Cruz
soma-, body)
Bergeys Manual of
Systematic Bacteriology
First edition published in 1923
Techniques For Determining
Microbial Taxonomy
The Intuitive method
Genetic Homology
DNA/RNA
sequencing
DNA chip
technology
Numerical Taxonomy
Techniques For Determining
Microbial Taxonomy And
Phylogeny
Morphological
characteristics
Morphological features are important .
Morphology is easy to study and analyze, particularly
in eukaryotic microorganisms and the more complex
prokaryotes.
Structural features depend on the expression of many
genes, are genetically stable, and do not vary greatly
with environmental changes.
Many different morphological features are employed in
the classification and identification of microorganisms .
Light microscope : resolution limit of about 0.2 m
reduces its usefulness in viewing smaller
microorganisms and structures.
The transmission and scanning electron microscopes,
with their greater resolution, have immensely aided
the study of all microbial groups
Morphological characteristics
Morphological
characteristics
Differential staining: Gram staining, acid-
fast staining
Biochemical tests: Determines
presence of bacterial enzymes
Morphological characteristics
Biochemical tests
used to identify
selected species of
human pathogens
isolated from marine
mammals.
Assume you isolated
a gm-ve rod that
produces gas from
glucose, is urease
negative and indole
positive. What is the
bacterium?
Morphological characteristics
 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.
Physiological and Metabolic
Characteristics
 Ecological
Characteristics
Ecological properties: affect the relation of
microorganisms to their environment.
Taxonomically valuable because even very closely
related microorganisms can differ considerably with
respect to ecological characteristics.
Microorganisms living in various parts of the human
body markedly differ from one another and from those
growing in freshwater and marine environments.
Some examples of taxonomically important ecological
properties are life cycle patterns; the nature of
symbiotic relationships; the ability to cause disease in a
particular host; and habitat preferences such as
requirements for temperature, pH, oxygen, and osmotic
concentration.
 The Intuitive method
It is rely on the study of the properties of the
organisms by taxonomists for several years
which decides to represent one or more
species or genera.
Demerit:
The characteristics of an organism that is
important to one person may not be so important
to another.
Different taxonomists arrive at very different
groupings.
 Nucleic acid base composition
DNA contains four purine and pyrimidine bases: A,
G, C, T.
(G+C)/(A+T) ratio or G+C content, % of G+C in
DNA, reflects the base.
Mol % (G+C)= [(G+C)/(G+C+A+T)]*100
Estimated by determining the melting
temperature of the DNA
Higher G + C gives a higher melting
temperature
Methods to determine base composition
1.HPLC: Hydrolyse the DNA & Analyse with HPLC
2.Melting temperature (Tm): double stranded DNA,
 Nucleic acid base composition
Absorption of DNA:at max 260
nm. Separation of strand Abs.
When a DNA sample is slowly
heated: absorbance as
hydrogen bonds are broken and
reaches a plateau when all the
DNA has become single
stranded
The midpoint : Tm, a direct
measure of the G +C content.
Density of DNA also increases
linearly with G+C content.
%G +C: by centrifuging DNA in
a CsCl density gradient Figure: A DNA Melting Curve. The
Tm is indicated
Nucleic Acid Base Composition
The G+C content of DNA from animals and
higher plants : average 40%, ranges between
30 to 50%.
Prokaryotic G+C content : 25 to 80%.
If two organisms differ in their G + C content
by >10%, : different base sequences.
The G + C content of strains within a
particular species is constant.
Very different base sequences can be
constructed from the same proportions of AT
and GC base pairs.
 Nucleic Acid Base
Composition

Only if two microorganisms also are alike

phenotypically does their similar G + C
content suggest close relatedness.
 G+C contents of microorganisms
Organism %G+C
Actinomyces 5973
Bacillus 3262

Clostridium 2154
Escherichia 4852
Micrococcus 6475
Neisseria 4754
Proteus 38-41
Pseudomonas 58-70
Salmonella 50-53
Staphylococcus 30-38
Aspergillus niger 52
Nucleic Acid
Hybridization
By mixing ssDNA from two different species
and determining the percentage of the DNA
that can form dsDNA hybrids
The greater the percent hybridization, the
closer the species
 Nucleic Acid
Hybridization
Heat mixture of dsDNA.
cool and hold at a
temperature about 25C.
The base sequences will
reassociate to form stable
dsDNA.
Whereas noncomplementary
strands will remain single .
Incubation of the mixture at 30
to 50C will allow hybrids of
more diverse ssDNAs to form,
at 10 to 15C permits hybrid
formation only with almost
identical strands.
 Nucleic Acid
Hybridization
Nitrocellulose filters with bound nonradioactive
Nitrocellulose filters with bound nonradioactive
DNA strands are incubated with single-
stranded DNA fragments made radioactive
with 32P, 3H, or 14C.
Radioactive hybridize with the membrane-
bound ss-DNA.
Wash to remove any nonhybridized ssDNA
Measure the radioactivity.
The quantity of radioactivity bound to the filter
reflects the amount of hybridization and thus
the similarity of the DNA sequences.
 Nucleic Acid
The Hybridization
degree of similarity is expressed as the %
DNA radioactivity retained on the filter
compared with the % of homologous DNA
radioactivity bound under the same conditions
Two strains whose DNAs show 70%
relatedness and less than a 5% difference in
Tm are considered members of the same
species.
Very different DNA molecules will not form a
stable, detectable hybrid.
More distantly related organisms are
compared by carrying out DNA-RNA
hybridization.
 DNA Chip technology
Quick method to detect a pathogenic
microorganisms in a host by identifying a
gene that is unique to that pathogen.
Protocol:
DNA chip is made up of DNA Probes.
A DNA sample should be collected from
unknown organism is labelled with a
fluorescent dye and added to the chip.
Hybbridization between probe DNA and
sample DNA is detected by fluorescence.
DNA chip Technology
 Nucleic Acid
Sequencing
Genome structures directly compared only by
Genome structures directly compared only by
sequencing DNA and RNA.
The 5S and 16S rRNAs isolated from the 50S
and 30S subunits, respectively.
Purified, radioactive 16S rRNA is treated with
the enzyme T1 ribonuclease.
The fragments are separated and sequenced.
16S rRNA fragments from different
procaryotes are aligned and compared using a
computer, and association coefficients (Sab
values) are calculated.
 Nucleic Acid
Sequencing
Complete rRNAs sequencing
First, RNA is isolated and purified.
Then, revers transcriptase is used to make
cDNA.
Next, the PCR amplifies the cDNA.
Finally, the cDNA is sequenced and the
rRNA sequence deduced from the results.
 Numerical Taxonomy
Peter H. A. Sneath and Robert Sokal have defined
Numerical taxonomy: the grouping by numerical
methods on the basis of their character states.
Information about organisms is converted into
numerical form and compared by means of a
computer.
Each characteristics given equal weight.
At least 50 and preferably several hundred
characteristics compared.
Determine the presence or absence of selected
characters in the group of organisms.
Calculate simple matching coefficient (SSM),
Jaccard coefficient.
Numerical Taxonomy
 Numerical Taxonomy
The simple matching coefficients, or other
association coefficients are then arranged
to form a similarity matrix.
The results of numerical taxonomic
analysis are often summarized with a
treelike diagram called a dendrogram.
Organisms with great similarity are
grouped together and separated from
dissimilar organisms such groups of
organisms are called phenons.
Phenons formed at about 80% similarity
often are equivalent to species.
 Numerical Taxonomy

Clustering and Dendrograms in Numerical Taxonomy. (a) A small similarity matrix

that compares six strains of bacteria. The degree of similarity ranges from none (0.0)
to complete similarity (1.0). (b) The bacteria have been rearranged and joined to
form clusters of similar strains. For example, strains 1 and 2 are the most similar. The
cluster of 1 plus 2 is fairly similar to strain 3, but not at all to strain 4. (c) A dendrogram
showing the results of the analysis in part b. Strains 1 and 2 are members of a 90-
phenon, and strains 13 form an 80-phenon. While strains 13 may be members of a
single species, it is quite unlikely that strains 46 belong to the same species as 13.