Microbial Evolution:

The microbial evolution has entered a new era with the use of
molecular phylogenies to determine relatedness. Certainly this type of
phylogenetic analysis remains controversial, but it has opened up
possibility of comparing very diverse microbe with a single yardstick
and attempting to deduce their history. Some scientists have opined
the ‘failure’ of molecular methods to find a single unambiguous
evolutionary progression from a single ancestor to the present panoply
of microorganisms. The increasing appreciation of the ubiquity and
frequency of gene transfer events open the possibility of learning quite
essential prokaryotes is by establishing a central core genes that has
not participated in the general energy of gene transfer. The increasing
number of genome sequences may also contribute a better
understanding of the evolutionary history of microbe.

Microbial taxonomy:
Classification, nomenclature and identification comprise taxonomy of
microorganisms. On the basis of common characters or properties, a
set of organisms is considered into groups. There are no formal rules to
define the taxa. On the other hand, nomenclature is the name given
for defined taxa as also governed by bacteriological code of
nomenclature. The features that are used to differentiate various
microorganism often have little to do with the fundamental basis for
arranging the organisms into taxonomic groups.

General methods of classifying Bacteria

(i) The Intuitive method: Since a large array of microbiologists study
the characteristics of organisms, sometimes, it is difficult to assign
an organism based on all the characters because a character may
be important to a particular microbiologist may not be that
important to another, hence, different taxonomists may arrive at
 very different groupings, Sometimes, this approach i.e. intuitive
method is found useful.
(ii) Numerical Taxonomy: This is based on several characteristics for
each strain and each character is given equal weightage. The %
similarity of each strain may determine by the following formula:
%S= NS/(NS+ND)
Where
NS= Number of characteristics for each strains which are
similar or dissimilar.
ND= Number of characteristics that are dissimilar or
different
On the basis of %S,S=Similarity if it is high to each other,
placed into groups larger and so on.
(iii) Genetic Relatedness: This classification is based on genetic
relatedness (DNA and RNA) between organisms. The % G+C
determines the organism whether it is the same or different
species. If two organisms have quite different mol % G+C, then the
species are different and seems to be not related to each other.

Bacterial Nomenclature: Nomenclature is the branch of taxonomy

concerned with the assignment of naming to the taxonomic groups according
to the international rules. After classifying and identification, it is important
to assign name of the bacterium. The name should be such so that it is
accepted internationally.
If we see the history of bacteria and its systematic studies, it was
Robert Koch isolate of tuberculosis which aws called “Koch’s bacillus”. Here is
still a tendency to honour eminent bacteriologists by assigning bacteria in
Latinised forms of their names. For example Border, Bruce, Erwin, Escherich,
King , Lister, Neisser, Pasteure, Ricketts, salmon and Shiga, etc. each have a
bacterial genus named after them. On the other hand, some of the bacterial
species are after the names of Byod, Stewart and Jenner. It is only Morgan
who had both genus and species names. The another system of
nomenclature was based on disease caused. For example,”Koch’s bacillus
was named later as “The tubercle bacillus”.
It was Carl Linnaeus who gave hierachial classification system which
was already attempted to assign both plants and animals. According to this,
orders of bacteria divided into families and in each family, there are number
of genera, each of which comprises one or more species.
In this process each bacteria was described according to bacterial
nomenclature (genus and species). The genus name is always capitalized,
often to just the first letter is in capital letter and species name is written with
a lower initial name. The care must be taken while abbreviating the common
genera in share the same initial letter then the first few letters are written eg.
Staphylococcus aureus abbreviated as Staph aureus, Streptococcus
pyrogenes abbreviated as Strepto pyrogenes. Generally bacterial names
should be printed in italic letters but if it is not possible then bacterial names
are usually underlined. When a group of bacteria is written , like
staphylococci, the name is neither italicized nor underlined and it is not
written with a initial capital letter.The unnamed species if it is one written as
sp. or if many, the spp. should be used. It (sp. or spp.) should also not be
underlined or italicized.
Currently, the bacterial nomenclature is regulating by a committee on
systematic nomenclature. List of approved names are published in
International Journal of Systematic Bacteriology. It is kept that most of the
bacterial names should be descriptive eg. Mycobacterium tuberculosis cause
tuberculosis. But, sometimes they reflect the microscopic appearance as in
the case of Staphylococcus aureus, which is golden berry that forms cluster
like, grapes.

Characters used in Bacterial Identification: The goal of

identification is merely to provide the name of an isolate, most identification
systems depend on first determining a number of morphological,
biochemical, cultural, antigenic or other phenotypic characteristics of the
isolate before the name can be assigned. Noel R. Creig in 2001 stated that,
an ideal universal systems for identifying a pure culture would be one which
provides the name without having to determine these characteristics.
Automatic Cellular Fatty Acid (CFA) analysis and 16S sequencing are two
such systems that have proven to be extremely useful in identification of an
unknown organism. The CFA system depends on saponifying the fatty acids
with NaOH, converting them into their volatile methyl esters and then
separating and quantifying each fatty acid by gas-liquid chromatography.
The 16S sequencing is one of choice at present, is a very rapid method
where 16S sequences of ribosomal RNA can be obtained and analyzed and a
large number of the isolates can be handled in a short time. In this technique,
all or most of the nucleotide sequence of 16S r RNA of the unknown isolate is
determined. In addition to the identification of an unknown isolate, this
method has been found to be very useful for the identification of many
different organisms in a mixed culture.

Traditional tests used in identifying an isolate: In order to identify an

unknown isolate, it is important to determine the phenotypic characteristics
first by studying the staining behaviour (Gram staining, endospore staining,
capsule staining, acid fast staining, flagella staining)), physiological and
metabolic characteristics by performing a series of biochemical tests such as
carbohydrate fermentation, nitrate reduction, casein hydrolysis, starch
hydrolysis, lipid hydrolysis, gelatin liquefaction, catalase test, β-galactosidase
test, oxidase test, urease test, decarboxylase test and the growth
characteristics (eg. Pigmentation, mucoid colonies, swarming or size of cell or
organisms).

Molecular Characteristics used in identifying an isolate: Molecular

approaches such as comparisons of proteins and nucleic acids have become
increasingly important in taxonomy of bacteria. Since amino acids sequences
of proteins are direct reflections of mRNA sequences and are therefore
closely related to the structure of genes coding for their synthesis. Other
methods used to compare microbial genomes is by the determination of G+C
content of DNA. Third molecular technique more oftenly used to compare
DNA or RNA sequences for determining genetic relatedness among
microorganisms is by nucleic acid hybridization technique. Application of DNA
hybridization technique to determine DNA relatedness among bacteria
resulted in the development of a natural, phylogenetic definition of a species.
At present molecular basis, using mol% G+C range, DNA-DNA or rDNA
relatedness and similarity of 16S vs. 18S rDNA sequences, is being used as a
criterion to compare bacterial species with higher organisms.
Identification of a particular species using DNA probes: DNA probes
are used to verify the identification of a bacterial isolate. A probe is a single
stranded DNA sequence that can be used to identify an organism by forming
a hybrid with a unique complimentary sequence on the DNA or rRNA of that
organism. Using probes as a “shot gun” approach to identification of an
isolate, however, is costly and time consuming. DNA probes can even be
used to identify individual cells in mixed culture under a microscope. A
specific DNA probe is conjugated to a fluorescent dye and applied to cells on
a slide. Fluorescing cells could be visualized under a fluorescence
microscope, if hybridization occurs between the probe DNA or rRNA of an
appropriate cell.
Identification of a particular strain of a species: Several methods are
used for differentiating bacterial strains from one another and are
categorized into two types i.e., traditional methods (eg. Serological tests,
Phage typing, Plasmid fingerprinting and Antibiogram) and DNA fingerprinting
which is most specific and recent method for the identification of a particular
strain of a species. DNA fingerprinting can be performed in the following four
ways: DNA fingerprinting using a probe and agarose gel electrophoresis;
Ribityping; DNA fingerprinting using pulsed field gel electrophoresis (PFGE)
and Randomly amplified polymorphic DNA (RAPD) strain typing.

Microbial Phylogeny and Current Classification of

Bacteria/ BERGEY’S MANUAL OF SYSTEMATIC
BACTERIOLOGY
The studies conducted by Carl Woese and his collaborators on
ribosomal RNA sequencing has revealed a previously unsuspected
phylogeny of living organisms, a phylogeny quite different from
previous ones based primarily on phenotypic relationships. All living
 organisms are grouped into three major groups: the Bacteria, the
Archaea and the Eucarya. The Bacteria and Archaea first diverged,
then the eukaryotes developed. These three primary groups are called
domains of life, the domain being the highest of biological taxons.
Thus, Protists, Fungi, Plants and Animals are all kingdoms within the
domain Eucarya. A summary of the features distinguishing the three
domains i.e, bacteria, Archaea and Eucarya are listed below:

Major differentiating features among three domains

Characteristics Bacteria Archaea Eucarya

Procaryotic cell Yes Yes No
structure
Membrane Absent Absent Present
enclosed
nucleus with
nucleolus
Gas vesicles Present Present Absent
Plasmids Yes Yes Rare
Histone No Yes Yes
proteins
Presence of Yes No No
muramic acid
Membrane Ester-linked Ester-linked Ester-linked
lipids
Ribosomes 70S 70S 80S
Initiator tRNA N- Methionine Methionine
formylmethionine
Operons Yes Yes No
Presence of No Yes Yes
introns in some
tRNA genes
DNA-Dependent
RNA
polymerase One Several Three
Number of 4 sub units 8-12 sub units 12-14 sub units
enzymes Sensitive Insensitive Insensitive
Structure
Rifampicin
sensitivity
Polymerase II Absent Present Present
type promoters
Metabolism
Similar ATPase No Yes Yes

Methanogenesi Absent Present Absent

s
Present Present Absent
Nitrogen
fixation Present Absent Present

Chlorophyll
based Present Present Absent
photosynth
esis

Chemolithothro
phy

In 1923, David Bergey, professor of bacteriology at the university of

Pennsylvania and four colleagues published a classification of bacteria
that could be used for identification of bacterial species, the Bergey’s
Manual of Determinative Bacteriology. The first edition of Bergey’s
Manual of Systematic Bacteriology, a more detailed work that contains
descriptions of all prokaryotic species currently identified. The first
volume of the second edition was published in 2001. This section
briefly describes the current edition of Bergey’s Manual of Systematic
Bacteriology.
The Bergey’s Manual of Systematic Bacteriology has four
volumes, that contain the internationally recognized names and
descriptions of bacterial species. The details of the informations are
given below:
Vol I: (Sections 1-11) 1984: Gram negative bacteria
Vol II: (Sections 12-17) 1986: Gram positive bacteria; Phototrophic and other
specialized bacteria
Vol III: (Section 18-25) 1989: Archaeobacteria
Vol IV: (Sections 26-33) 1991: Actinomycetes and other filamentous bacteria
Division I. Gracilicutes
Division II. Fimicutes
Division III. Tenericutes
Division IV. Mendosicutes
Section 1. The Spirochetes
Order I Spirochaetales
Family I Spirochaetaceae eg. Spirochaeta
Family II Leptospiraceae eg. Leptospira
Section 2. Aerobic/ Microaerophilic, motile, helical/vibroid gram-negative
bacteria
Section 3. Non-motile, gram-negative, curved bacteria
Family I. Spirosomaceae, eg. Spirosoma
Section 4. Gram-negative Aerobic rods and cocci
Family I. Pseudomonadaccae
Family II. Azotobacteriaceae
Family III. Rhizobiaceae
Family IV. Methylococcaeae
Family V. Halobacteriaceae
Family VI. Acetobacteriaceae
Family VII. Legionellaceae
Family VIII. Neisseriaceae
Section 5. Facultatively Anaerobic Gram-negative Rods
Family I. Enterobacteriaceae
Family II. Vibrionaceae
Family III. Pasteuellaceae
Section 6. Anaerobic Gram-negative, curved and helical rods
Family I. Bacteriodaceae
Section 7. Sulphur reducing bacteria
Section 8. Anaerobic Gram-negative Cocci
Section 9. The Rickettsias and Chlamydias
Section 10. The Mycoplasmas
Section 11. Endosymbionts
Section 12. Gram-positive cocci
Section 13. Endospore forming Gram-positive rods and cocci
Section 14. Regular, non sporing, Gram-positive rods
Section 15. Irregular, non-sporing, Gram-positive
Section 16. The mycobacteria
Section 17. Nocardioforms
Section 18. An oxygenic, phototrophic bacteria
I. Purple bacteria
II. Green sulphur bacteria
III. General Incertae Sedis

Section 19. Oxygenic Photosynthetic Bacteria

Family. Prochloraceae
Section 20. Aerobic Chemolithotrophic Bacteria and associated organisms
A. Nitrifying Bacteria
B. Colourless Sulphur bacteria
C. Obligately Chemolithotrophic Hydrogen Bacteria
D. Iron and mangnese oxidizing bacteria
E. Magnetotactic bacteria
Section 21. Budding and Appendaged bacteria
Section 22. Sheathed Bacteria
Section 23. Non-photosynthetic, Non-fruiting gliding bacteria
Section 24. Fruiting gliding bacteria
Section 25. Archaeobacteria
Section 26. Nocardioform Actinomycetes
Section 27. Actinomycetes with multicellular sporangia
Section 28. Actinoplanetes
Section 29. Streptomycetes and related genera
Section 30. Maduromycetes
Section 31. Thermomonospora and Related Genera
Section 32. Thermoactinomycetes
Section 33. Other Genera

Ribosomal RNA and its sequencing

Ribosomal RNA proved to be a universal tool for the phylogenetic

analysis and interrelationship among the organisms. It is ancient,

universally distributed and most conserve region in the genome of the

microorganisms. Although in prokaryotic ribosomes, threr are three

ribosomal RNAs i.e. 5S, 16S and 23S but only 16S sequence is used

because nucleotides are neither less nor more in length and easy to

sequence. About 1500 nucleotides are present in 16S r RNA which are

determined by using polymerase chain reaction (PCR) and gene

sequencer. The 5S r RNA contains only approx. 120 nucleotides that

are too small to conclude any fruitful relationship, whereas 23S

contains approx. 2900 nucleotides which are quite high in number;

therefore it is not suitable for experiments. In eukaryotes, 18S r RNA is

used for phylogenetic studies. The database of r RNA sequences of

both prokaryotic and eukaryotic microorganisms can be accessed on

internet.

Ribosomal RNase is easily extracted from cells. About 300-500

mg cells are taken in a centrifuge tube, mixed with appropriate

quantity of DNase to break down and isolate RNA for sequencing. The

heated cells are mixed with phenol for the removal of proteins and

carbohydrates etc. to release the RNA which are precipitated by using

alcohol and salt. The RNA so obtained is mixed and taken equally with

DNA primer complementary to the region in 16S r RNA. The enzyme

reverse transcriptase is added along with P-labelled deoxynucleotide

32

triphosphate. In each tube, a small amount of different ddNTP is added.

Reverse transcriptase determines the 16S r RNA template and

terminates the DNA copy. The fragments are then separated by gel

electrophoresis. The sequence of 16S r RNA is then determined by

using standardf c DNA sequence.

Now a days ribosomal RNA sequencing using the polymerase

chain reaction to yield many copies of 16S ribosomal gene is amplified

and the methodology given by Sanger (1977) is shown below:

Methodology for the determination of sequences