Unit 2 Microbial Taxonomy

UNIT 2
MICROBIAL TAXONOMY

Structure
2.1 Introduction Molecular Characterization

Expected Learning Outcomes 2.4 Nomenclature

2.2 Classification 2.5 Bergey’s Manual of

Systematic Bacteriology
History of Classification

Taxonomic Ranks
2.6 Numerical Taxonomy

Classification System 2.7 Summary

2.8 Terminal Questions

2.3 Characterization
Phenotypic or Classical 2.9 Answers
Characterization

2.1 INTRODUCTION
With the estimation of 13 million species existing on Earth, only 1.75 million
species are described which include a description of 1,56,000 microbial
species (Table 2.1). Due to microscopic size, and lack of definite structural
features the classification of microorganisms is a great daunting challenge.
Additionally, with the advent of modern molecular tools, the dataset of
microbial species is going to increase multifold and makes their affiliation more
difficult. But the basic question which may come to your mind is that why do
we need to classify or identify microbes? The answer to this question lies in
the fact that the accurate identification of affiliation of microbe is important
regarding both economic, social and health reasons. Therefore, a repository of
identified microorganisms is required to track causative organisms of a
disease or to find useful microorganisms which have industrial or agricultural
importance. Therefore, we need to have proper identification and classification
system to understand the microbial diversity as well as to have a thorough
reference. This aspect is studied under ‘Taxonomy’ or ‘Systematics.’
Taxonomy (Greek: taxis- arrangement or order, and nomos- law, or to assign,
to distribute or govern) deals with the study of classifying organisms.
Microbial taxonomy may be defined as the study of the diversity of 27
Block 1 History of Microbiology and Classification of Microbes

microorganisms with the aim of organizing and prioritizing in an orderly

manner. The term systematics is often used as a synonym of taxonomy.
There are three separate but integrated components, i.e., classification,
characterization or identification, and nomenclature which constitute
microbial taxonomy. We shall deal these components separately in further
sections.

Table 2.1: Number of described species

Species Number

Bacteria 4000

Protists (algae, protozoa) 80000

Fungi 72000

Others (Plants, animals) 1594000

Expected Learning Outcomes

After studying this unit, you should be able to:

Understand the significance of microbial taxonomy;

Know the components of the microbial taxonomy;

Know how to classify organisms and what are the methods available for
taxonomic affiliation of the microorganisms;

Understand the usefulness of polyphasic taxonomic approaches to

identify and classify organisms, and

Acknowledge the use of numerical taxonomy in microbial

characterization.

2.2 CLASSIFICATION
Classification may be defined as the arrangement of organisms into categories
(taxa) by similarities or relationships.

2.2.1 History of Classification

The oldest mention of classification comes with Aristotle (384-322 BC) who
divided living organisms into plants and animals. After Aristotle, Carl von Linné
or Carolus Linnaeus (1707-1778), a Swedish botanist, grouped organisms
based on their characteristics. This type of method is known as a natural
classification system. He classified all living organisms in animals and plants
as kingdoms which were further grouped into five levels, i.e., class, order,
genus, species, and variety. Owing to his contribution in classifying and
naming organisms Carl is also called as father of taxonomy.

It was Haeckel (1866) who included microorganisms for the first time in
classification system and proposed a separate Kingdom ‘Protista’ to include
unicellular organisms. Further, the classification system gained much attention
28
Unit 2 Microbial Taxonomy
was five kingdom concept (Fig. 2.1) proposed by Robert H. Whittaker
(1969). The five kingdoms namely Monera, Protista, Plantae, Fungi, and
Animalia, were propounded based on (i) cell type, i.e., prokaryotic or
eukaryotic, (ii) level of organization, i.e., solitary and colonial unicellular or
multicellular organization, and (iii) type of nutrition. A brief description of
these kingdoms is given below.

Fig. 2.1: Representation of five kingdoms as per classification system of

Whitakker. Figure adopted from web page.

Monera: (i) Members of monera are most abundant microorganisms which

include prokaryotic organisms such as bacteria, cyanobacteria, and
mycoplasma.

(ii) They are primarily unicellular and have cell walls (except mycoplasma
which lacks a cell wall) and are of varied shapes (spherical, rod-shaped,
comma-shaped, spiral, etc.) and size.

(iii) They include both autotrophic (phototrophic and chemotrophic), and

heterotrophic microorganisms. Some of them are parasites and cause deadly
diseases.

Protista: (i) It includes single cellular eukaryotic organisms represented by

Chrysophytes, Dinoflagellates, Euglenoids, Slime molds, and Protozoans.

(ii) Majority of them are aquatic and have an autotrophic or heterotrophic mode
of nutrition.

(iii) Protists exhibit both sexual and asexual mode of reproduction.

Fungi: (i) They are heterotrophic eukaryotic microorganisms which include

unicellular yeast, multicellular but microscopic molds, macroscopic
mushrooms, etc.

(ii) Most of them exhibit an absorptive mode of nutrition and live on dead
organisms and thus, termed as saprophytes

(iii) They reproduce by vegetative and sexual mode of reproduction. Some of

them form fruiting bodies.

(iv) Many fungal species live in symbiotic relationship with algae and called as
lichens.
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Block 1 History of Microbiology and Classification of Microbes
Plantae: (i) These includes all plants which are multicellular organisms having
cells wall made of cellulose.

(ii) They are autotrophic (photosynthetic) and synthesize their food. However,
some of them such as some epiphytes and insectivorous plants (Venus flytrap,
bladderwort) are partially heterotrophic.

(iii) They show alternation of generation, i.e., the diploid sporophytic and the
haploid gametophytic stage– that alternate with each other.

Animalia: (i) It includes all multicellular eukaryotic organisms which lack a cell
wall.

(ii) They are heterotrophs and depend directly or indirectly on plants. They
show the ingestive type of nutrition.

Despite the systematic classification system, five kingdom concept is not

accepted by many biologists as it lacks distinction between archaea and
bacteria. Therefore, it followed proposal of six kingdom concept which
included archaea.

The most recent classification has been proposed by Carl R. Woese (1998)
who grouped all living life forms in three domains, i.e., Eukarya, Bacteria,
and Archea based on the sequence of rRNA genes, a component of
ribosomal structure (Fig. 2.2). In this classification system, Eukarya includes
three kingdoms namely plants, animals, and fungi. The kingdom Bacteria has
several kingdoms which include phototrophic, chemotrophic, pathogenic
(disease-causing) as well as non-pathogenic bacteria. The Archea includes
prokaryotes that lack peptidogly can in their cell walls. They are usually
extremophiles which can survive the extreme conditions such as high
temperature (thermophiles), high salt concentration (halophiles), and pH
(very low or very high), etc. Methane-producing (methanogenic) anaerobic
bacteria are also the member of Archea.

Fig. 2.2: Three domain system as proposed by Carl R. Woese based on rRNA
gene sequence. Figure adapted from Benjamin Cummings, an imprint
30 of Addison Wesley Longman, Inc.
Unit 2 Microbial Taxonomy
A brief account of different characteristics of these three classes in shown in
Table 2.2.

Table 2.2: Comparison of Bacteria, Archaea, and Eucarya

Property Bacteria Archaea Eucarya

Membraene- Absent Absent Absent

Enclosed Nucleus
with Nucleolus

Complex Internal Absent Absent Absent

Membranous
Organelles

Cell Wall Almost always Variety of types, no No muramic acid

have peptidoglycan muramic acid
containing muramic
acid

Membrane Lipid Have ester-linked, Have ether-linked, Have ester-linked,

straight-chained branched aliphatic straight-chained
fatty acids chains fatty acids

Gas Vesicles Present Present Absent

Transfer RNA Thymine present in No thymine in T or Thymine present

most tRNAs N- TΨC arm of tRNA Methionine carried
formylmethionine Methionine carried by initiator tRNA
carried by initiator by initiator tRNA
tRNA

Polycistronic RNA Present Present Absent

mRNA Introns Absent Absent Present

mRNA Splicing, Absent Absent Present

Capping, and Poly
A Tailing

Ribosomes

Size 70S 70S 80S (ctoplasmic

ribosomes)

Elongation factor 2 Does not react with Reacts Reacts

diphtheria toxin

Senstivity to Sensitive Insensitive Insensitive

chloramphenicol
and kanamycin

Sensitivity to Insensitive Sensitive Sensitive

anisomycin

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Block 1 History of Microbiology and Classification of Microbes

DNA-Dependent RNA Polymerase

Number of One Several Three

enzymes

Structure Simple subunit Complex subunit Complex subunit

pattern (4 subunits) pattern similar to pattern (12-14
eukaryotic subunits)
enzymes (8-12
subunits)

Rifampicin Sensitive Insensitive Insensitive

sensitivity

Polymerase II Absent Present Present

Type Promoters

Metabolism

Similar ATPase No Yes Yes

Methanogenesis Absent Present Absent

Nitrogen fixation Present Present Absent

Chlorophyll-based Present Absent Presenta

photosynthesis

Chemolithotrophy Present Present Absent

a
Present in chloroplasts (of bacterial origin)

2.2.2 Taxonomic Ranks

The microorganisms are arranged in different hierarchical or taxonomic level
based on the similarity of traits. In a taxonomic hierarchy, the lowest level is
considered to be species. The species definition of microbes (bacteria in
particular) is debatable and different from the eukaryotic species (higher
organisms) the latter of which are referred to the members of interbreeding
population producing fertile progeny. The bacterial species is defined as a
collection of strains that share stable properties and differ significantly from
another group of strains. The strain is descendant of a single pure microbial
culture of species. The strains may further be categorized as biovar (variant
based on biochemical or biophysical properties), morphovars (morphological
variant), phagovars (variants with different phage susceptibility) and serovars
(variants differing in antigenic properties). Often, a type strain is used for
identification or characterization of the new microbe. The type strain is one of
the first and fully characterized strains which can be used as a reference point
of the given species. However, the type strain may not necessarily be a
32 representative strain of a species.
Unit 2 Microbial Taxonomy
Genus lies just above the species in hierarchical level which is a collection of
similar species. Similarly, members of similar genera are grouped in a Family,
similar family in Order, the similar order in Class, and so on (Fig. 2.3). With
the flooding sequence data of rRNA gene and protein-coding genes, the
inclusion of superphylum just below the kingdom has been recommended
recently. There are many sequences which do not match with the current
database of known sequences and indicate the possibility of new taxonomic
levels in the future.

Hierarchical Baker’s Yeast M. okinawensis E. coli

Level
Domain Eukarya Archaea Bacteria
Kingdom Fungi NA NA
Phylum Ascomyta Euryarcheota Proteobacteria
Class Hemiascomycetes Methanococci Gammaproteobacteria
Order Saccharomycetales Methanococcales Enterobacteriales
Family Saccharomyceaceae Methanococcaceae Enterobacteriaceae
Genus Saccharomycetes Methanococcus Echerichia
Species cerevisiae okinawensis Coli

Fig. 2.3: The hierarchy of taxonomy: taxonomic representation of the

representative organisms belonging to three domains proposed by
Carl R. Woese.

2.2.3 Classification System

Microorganisms, primarily bacteria, can be grouped or assigned to taxa based
on different classification systems which include phenotypic features,
genotypic character, and/or phylogenetic relationship. Currently, the taxonomic
assignments are done employing polyphasic taxonomy which employs all
three, i.e., phenotypic (phenetic), genotypic, and phylogenetic characteristics.
To better understand how these data are useful in taxonomic criteria, we
discuss these components individually in brief.

Phenetic Classification: This is one of the most common and old methods to
identify and classify organisms. This classification system employs screening
of phenotypically observable characteristics such as the morphological
structure of the cell, colony morphology, biochemical and physiological traits
which can be assayed or observed. This includes analysis of as many as
possible traits (more than 50) for robust classification. Methods used in
phenetic classification are described in section 2.3.1. Though this method can
be used to establish an evolutionary relationship, this is not always the case.
For instance, not all flagellated bacteria are evolutionarily related.

Genotypic Classification: As the name suggests, this system is based on

similarity in the sequence of evolutionarily conserved genes or whole
genomes. Majority of genotypic classification is based on small subunit rRNA
gene sequence. The genetic classification is well suited for studying
evolutionary relatedness among the organisms.
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Block 1 History of Microbiology and Classification of Microbes

Phylogenetic Classification: The phylogenetic classification system

compares organisms based on their evolutionary relationship. The term
‘phylogeny’ is originated from Latin word ‘phylon,’ i.e., tribe or race, and
‘genesis,’ i.e., generation or origin. Most of the evolutionary studies are based
on the fossil record which is not available for the majority of microbes.
Therefore, the phylogenetic classification could be employed only after the
development of microbial identification based on 16S rRNA gene sequence.

2.3 CHARACTERIZATION
Another component of taxonomy is characterization which means a
description of detailed features of given organism. The comprehensive
characterization can enable the accurate identification of an organism.
Several approaches are there for characterizing the microbes. All the
approaches can be broadly divided into phenotypic and molecular
characterization. One or more of these approaches can be employed to
characterize the microbe. These methods differ in their resolution. Greater the
resolution, better identification on different taxonomic rank. The method which
can differentiate microbe at strain level will be considered to have the highest
resolution. We shall have a brief description of various methods used to
characterize microbe at phenotypic or molecular level. Majority of the
descriptions are focused to prokaryotes, and the description specific to
eukaryotic microbe will be mentioned wherever necessary.

2.3.1 Phenotypic or Classical Characterization

This includes methods which analyze morphological, biochemical,
physiological, and ecological features of an organism. These methods form
the basis of phenetic classification. The phenotypic characteristics are useful
and sometimes essential for assigning to the different taxonomic levels such
as phylum, class, order, family, or even species. Some of the important
phenotypical properties which are analyzed for characterization are given
below. The resolution of phenotypic methods is shown in Fig. 2.4.

Morphology and Growth behavior: The primary information about the test
microbe can be obtained from morphological data which is based on cell or
colony morphology and growth behavior. Morphological information is valuable
as it is stable and guided by genetic functions.

Growth conditions: Bacteria are analyzed based on their requirement of the

oxygenic or anoxygenic condition, CO2, and the composition of media, upon
which organisms can grow.

Colony and cell morphology: Information related to colony morphology such as

colour, edge, texture, elevation, opacity, motility on a solid surface, and
production of extra-colonial pigments are used as primary visual data.
However, there is a limited number of characteristics which can be analyzed
with unaided eyes. Further cellular structure, the formation of prosthecae,
branching, endospores, presence and insertion of flagellae, etc., are
determined by light and electron microscopy, which provide valuable data for
34 morphological description.
Unit 2 Microbial Taxonomy
Staining characteristics of cells: Staining methods such as Gram staining,
acid-fast staining, etc. are used to differentiate prokaryotes based on
differential cell wall or membrane composition.

Phenotypic Methods Family Genus Species Sub Strain

species

Serotyping

Raman spectroscopy

SDS-PAGE

MALDI-ToF

Chemotaxonomy (e.g.
FAME profiling)

Phenotyping (growth,
morphololgy, API)

BIOLOG, Omnilog, Vitek

Genotypic Methods

Genome sequencing

16S rRNA, rpoB gene

sequencing

Mol%GC

DNA-DNA Hybridization

MLSA/MLST

Whole cell protein profiling

DNA fingerprinting (BOX-,

ERIC-, REP-PCR

Fig. 2.4: Phenotypic and genotypic methods for the characterization of

prokaryotes and the approximate respective taxonomic levels of
resolution. Light grey region denotes limited resolution of given
method at given taxonomic level. Some of the methods such as
serotyping, SDS-PAGE, and whole cell protein profiling is not
discussed in the text but they are used for typing and identification of
prokaryotes. MLSA and MLST are expanded as Multilocus sequence
alignment and Multilocus sequence typing respectively.
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Block 1 History of Microbiology and Classification of Microbes

Chemical characterization (“chemotaxonomy”): The difference in a

structural component of the cell wall, cell membrane or cytoplasm can provide
useful information for identification and classification. Differences in
peptidoglycan, teichoic acids, mycolic acids, fatty acids, polar lipids,
respiratory lipoquinones, pigments and polyamines (Tindall et al., 2010) are
used in microbial identification. Such cellular features offer systematic
identification often shows the evolutionary relationship. However, it may differ
regarding resolution. For instance, FAME (fatty acid methyl ester) analysis
is one of the most common methods used which is used to identify bacteria
based on type and proportion of fatty acid present in the cell membrane. The
membrane lipid of bacteria varies in species to species, and they differ in
chain length, presence or absence of a double bond, rings, branched rings,
etc. In this method, the fatty acids are extracted from bacteria grown under
standard conditions, chemically treated to generate methyl derivative, and
analyzed by gas chromatography. The fatty acid profile is matched with known
database and identified. FAME profiling is one of the most common methods.
However, its application is limited with the fact that the fatty acid profile
changes with environmental and growth factors (Fig. 2.5).

Fig. 2.5: Workflow of microbial identification by FAME analysis. Figure adapted

from Brock Biology of Microorganism 13th edition, Pearson.

With the development of mass spectrometric methods, fast and accurate

identification is possible by analyzing microbial molecules. A spectrometric
method known as MALDI-ToF (Matrix-assisted laser desorption/ionization-
time of flight) identifies a microbe by analyzing masses of microbial proteins
after comparing with the protein database available (Fig. 2.6). In this method,
microbes are grown in specific/standard conditions, transferred to a sample
target, dried, and analyzed. This method is applied even to those organisms
36 which are difficult to culture.
Unit 2 Microbial Taxonomy

Fig. 2.6: Schematics showing methodogy for bacterial identification using

MALDI-ToF Mass spectrometry. Figure adapted from web source.

Physiological and Metabolic characteristics

Physiological testing is based on the growth properties, the ability to utilize

certain substrates or to show features that provide information about the basic
metabolic activities of prokaryotes. The physiological and metabolic properties
provide useful information which is directly related to the nature and activity of
microbial proteins. There are many commercial products available that claims
reliable identification of microorganisms which includes BiOLOG’s carbon
utilization test. Conventionally, BiOLOG plates contain 96 wells each
containing different carbon sources and a redox dye. One or more wells do not
have any carbon source and act as a control. After inoculation and incubation
of test microbe under standard conditions, the wells are analyzed for the
development of pink colour which indicates the ability to utilize given carbon
source (Fig. 2.7). The profile is then matched with the microbial database, and
the bacterium is identified using numerical taxonomic approach (discussed
in section 2.6). In this system, different plates are used for Gram-positive and
Gram-negative bacteria.

More recently, BiOLOG has introduced highly automated system with updated
microbial database called Omnilog which enables identification of microbes
including bacteria, yeast, and filamentous fungi based on ability to metabolize
all major classes of biochemicals, in addition to determining other important
physiological properties such as pH, salt, and lactic acid tolerance, reducing
power, and chemical sensitivity. This system allows profiling of more than 50
different microbial sample at a time and provide data in a minute. Similar to
BiOLOG, there are other fast microbial identification system such as Vitek
(manufactured by BIOMÉRIEUX) and API (Analytical Profile Index) system
which exploits physiological properties to identify microbe. 37
 Block 1 History of Microbiology and Classification of Microbes

Inoculation to GN for
(Gram-negative) or GP
(for Gram-positive
Bacteria BIOLOG Plates
containing different
carbon source Pink colour devlopes if the carbon
source is utilized in given well

Obtaining data in BIOLOG reader

Analysis and identification at regular interval (12, 24, 36, 48 h

Fig. 2.7: Schematics showing identification of bacteria using BIOLOG carbon

utilization test. BIOLOG is commercially supplied.

Ecological characteristics

The ability of a microorganism to colonize certain ecological conditions, to

establish symbiotic relationship with specific host, or having requirement of
specific pH, temperature, or oxygen can have taxonomic value.

Why should 2.3.2 Molecular Characterization

molecular methods
be used for Molecular characterization is usually referred to the description or analysis of
identification? an organism based on nucleic acid (DNA or RNA) or protein information. Due
to lack of fossil record, phylogenetic analysis was not possible until the
The phenotypic
methods for nucleic acid sequence analysis developed. The recent advances
methods can be used
have enabled us to sequence bacterial DNA of even non- culturable
for identification but
organisms, i.e. without culturing them in the laboratory which has divulged the
most of these
techniques are time great diversity of microorganisms. There are several methods which are used
consuming and are for molecular characterization or typing or strains. The term ‘typing’ is used
affected by changes for designating a strain to the certain specific pattern of DNA profile. However,
in several the resolution of each method varies and range from domain to species or
environmental even strain level identification. These methods include: (i) determination of
factors. The nucleic acid base composition, (ii) nucleic acid hybridization, (iii) nucleic acid
molecular techniques sequencing and DNA fingerprinting. The resolution of various genotypic
such as sequence methods shown in Fig. 2.4.
based, gel based,
and protein based Nucleic acid base composition
systems are fast and
have high specificity One of the many ways of taxonomic determination involves comparison of
and less chance of bases in genome by determining GC (Guanine and Cytosine) content. The
error. mol%GC can be calculated using following formula:
ୋାେ
%mol G+C= × 100
୅ା୘ାୋାେ

This can be done directly from the sequence data of genome. For bacteria
whose genome sequence is not available, the base composition can be
estimated from DNA melting temperature (Tm) curve. This is based on the
fact that the genome having high G+C content will melt at higher temperature
as three hydrogen bonds are involved in making pair of G with C whereas only
two bonds are there between A (Adenine) and T (Thymine). Melting
temperature (Tm) is the temperature at which 50% of DNA is denatured. For
instance, Tm of Mycoplasma hominis, having 29% G+C, is 65 ˚C whereas it is
38 85 ˚C for Micrococcus luteus which has 79% G+C.
Unit 2 Microbial Taxonomy
For DNA melting analysis, the DNA is slowly heated at increasing
temperature. As the temperature increases, the bonds between the base pair
start breaking until the complete DNA denatures (strands are separated from
one another). While doing this, the absorption of DNA is measured
spectrophotometrically at 260 nm (A260). The absorbance increases with the
separation of strands and reaches to plateau when the denaturation completes
yielding single-stranded DNA (ssDNA). The resultant melting curve is used for
estimation of Tm. The mid-point of rising curves gives Tm (Fig. 2.8).

Fig. 2.8: A melting curve of DNA. A Tm is indicated at mid-point.

The range of GC % in prokaryotes (25 and 80%) is very wide as compared to

the higher eukaryotic organisms (30 to 50). This %G+C content within a
species remain constant and varies very little within the genus. It is estimated
that the two organisms having more than 10% difference in G+C content are
not closely related. However, it may not be applied that if the organisms have
similar G+C content will be closely related to the sequence may vary even if
the composition is similar.

Table 2.2: Range of G + C content in various microbial groups

Microbial Groups %G+C

Eubacteria (Bacteria) 25 to 80
Archebacteria 27 to 62
Fungi 22 to 62
Protozoa 21 to 65
Algae 37 to 68

Nucleic acid hybridization

One of the oldest and direct molecular methods for comparing a pair of
microbes is DNA-DNA hybridization (DDH). In this method, the DNAs are
heated to get single-stranded DNA (ssDNA). Then the ssDNAs of two
microbes are allowed to cool and hold at temperature 25 ˚C below the Tm. The
extent of annealing two strands depends on the similarity between the
39
Block 1 History of Microbiology and Classification of Microbes

sequences of two organisms being compared. By convention, the organisms

showing 70% similarity based on the extent of hybridization are considered a
member of same species. A schematic of DDH is shown in Fig. 2.9.

Fig. 2.9: Methodology for DNA-DNA hybridization to identify an organism. (A)

Genomic DNA of test isolate is labeled (shown here as radioactive
phosphate in the DNA of Organism 1). (B) Excess unlabeled DNA is
added to prevent labeled DNA from reannealing with itself. Following
hybridization, hybridized DNA is separated from unhybridized DNA.
Radioactivity in the hybridized DNA is measured. (C) Radioactivity in
the control (Organism 1 DNA hybridizing to itself) is taken as the 100%
hybridization value. The Figure and legend are adapted form Brock
Biology of Microorganisms, 13th edition with slight modification.

Though the DDH is still used for confirming the taxonomic affiliation of a
microbe, it is cumbersome to use and sometimes crude when compared to
genomic data. The best alternative method of DDH is the estimation of
average nucleotide identity (ANI). In ANI, pair-wise alignments of genome
sequences of the two organisms are done. In this method information of whole
genome sequence is not required. 20% coverage of genome sequence is
sufficient for ANI. In general, an ANI value of 95 to 96% indicates identification
at species level.

Nucleic acid sequencing-based characterization

A certain stretch of the genome or specific genes are the potential candidate
for identification. These sequences can also be used for analysis of
phylogeny. The desirable properties of a gene or sequence required for being
used as identification marker are as follows:

i) It should be universal, i.e., present in all the organisms.

ii) The sequence should be long enough for identification with high
resolution.

iii) The sequence should be conserved among the organisms so that there
will be measurable relationship even between distantly related
organisms.
40
Unit 2 Microbial Taxonomy
iv) It should have some variable region which is enough for distinguishing
the organisms even if they are closely related, and

v) which are not transferred to another organism through horizontal gene

transfer.

SSU rRNA sequence analysis

Considering properties mentioned above, the rRNA genes appeared to be the

most appropriate candidate for microbial identification. Since rRNA gene is
one of the most conserved genes, present in all organisms, and long enough,
it is routinely used for microbial identification. The rRNA is constituent of
ribosome which has two units namely small subunit (SSU) and large subunit
(LSU). Three types of rRNA namely 5S (120bp), 16S (1542 bp), and 23S
(3200 bp) are present in prokaryotic ribosomes out of which 16S rRNA, a
component of SSU, is the most frequently used for identification due to its
most conserved nature and moderate size for analysis. The 16S rRNA gene
(also called 16S rDNA) consists of conserved sequences flanking several
variable (referred as V1 to V9) region (Fig. 2.10). These variable regions
enable comparison between closely related microbes while the stable
(conserved) regions allow the comparison of distantly related
microorganisms. For identification, usually full length 16S rRNA gene is
amplified by polymerase chain reaction (PCR) using a pair of primers
designed based on conserved region, sequenced in DNA sequencer, and
analyzed using the appropriate computational tool. However, sometimes the
partial sequence of 16S rRNA gene can also be analyzed for tentative
identification. For identification of eukaryotic microorganisms, 18S rRNA
(component of SSU) gene is used for identification. For identification of fungi
at the species level, ITS (inter-transcribed spacer sequence), which is
located between rRNA genes, are used. The arrangement of fungal rRNA
genes is shown in Fig. 2.11. Once the sequence data is obtained, the
sequence is searched for the best match on various databases such as NCBI
(National Center for Biotechnology Information) or Ribosomal Database
project (RDP, https://rdp.cme.msu.edu/) using BLAST (Basic Local Alignment
Sequence Tool).

Fig. 2.10: Schematics showing arrangement of rRNA genes of E. coli. The lower
panel shows various conserved (light grey) and variable regions (dark
grey).
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Block 1 History of Microbiology and Classification of Microbes

Fig. 2.11: A schematic showing rRNA gene arrangement in fungi. ITS1 and ITS2
are inter-transcribed spacer sequence. D1 and D2 refers to domain 1
and 2 whereas IGS (Inter-genic spacer sequence).

The resolution of 16S rRNA sequence-based analysis is at the genus level.

However, it can help identify at species level as well. By convention, but not
necessarily, the organism showing more than 97% similarity in rRNA gene
sequence can be considered a member of same species. The major
advantage of 16S rRNA gene sequence analysis is rapid and accurate
identification. Further, it can also allow identification of those bacteria which
cannot be cultivated in laboratories. This property has enabled us to explore
the diversity of microorganism of given community in a culture-independent
manner. Any uncultivated microorganisms which are identified solely on its
nucleic acid sequence are called a phylotype.

Sequence analysis of other genes than 16S rRNA genes

16S rRNA gene sequence has undoubtedly revolutionized the bacterial

taxonomy. However, there are certain other house-keeping genes (whose
expression is necessarily required for cell growth) whose sequences are also
conserved and can be used for taxonomic affiliation of prokaryotes to
supplement the data of 16S rRNA sequence data. The name of some of these
genes is enlisted in Table 3. A gene rpoB encoding β-subunit of RNA
polymerase is widely used for bacterial identification. It exists as single copy
gene and shows much more discriminatory power than 16S rRNA gene. For
identifying fungi (eukaryotic microbes), genes encoding RNA polymerase
(RPB1 and RPB2), translation elongation factor 1-ɑ (tef1) and β-tubulin
(benA, tubC) are preferred for identification. The benA encodes beta 1, and
beta 2 and tubC encodes beta 3 protein of β-tubulin. Among the alternative
genes, β-tubulin are most commonly used. However, due to lack of an
appropriate database, these genes are not solely used for identification.

Table 2.3: Other genes than 16S rRNA genes which can be used for
bacterial identification.

Genes Function
rpoA, rpoB, rpoC and rpoD Encodes for RNA polymerase a key
enzyme for transcription process
gyrA, and gyrB Encodes gyrase, a topoisomerase
dnaJ It is a molecular chaperone
ppk1 Codes for polyphosphate Kinase 1

Multi-locus sequence alignment (MLSA)

For robust identification of a bacterium at species or even strain level, it is

better to rely on more than one gene. MLSA employs an approach of
42 sequencing and analyzing 5 to 7 house-keeping genes which are relatively
Unit 2 Microbial Taxonomy
more conserved and are usually not horizontally transferred. The selection of
genes may differ based on group of bacteria to be analyzed. MLSA is derived
from the similar technique multi-locus sequence typing (MLST) which was
used to type the strain based on analysis of partial sequence of many house-
keeping genes. The MLSA is also used for phylogenetic analysis with more
confidence.

Oligonucleotide signature sequence

These are short conserved sequence that is identified after comparing

thousands of rRNA genes of organisms and are specific to particular
phylogenetic group. This is useful for grouping or identifying microorganisms
at domain level.

Indels

Indels (insertion/deletion) are specific length of sequences which are inserted

or deleted in many genes and are specific to certain phylum. Indels are
particularly useful in phylogenetic analysis when they are flanked by
conserved region. The indels in housekeeping genes are less prone to
horizontal transfer and can be used for phylogenetic analysis.

DNA fingerprinting

The bacterial strains can be identified or typed by sequencing-independent

approaches that analyze DNA profiling which can either be generated by
restriction digestion or by amplification of repetitive DNA present in the
genome. Some of the DNA or genomic fingerprinting techniques are described
below.

rRNA gene-based fingerprinting

There are two methods by which profiling of genome based on rRNA gene is
done. One method called RFLP (Restriction fragment length polymorphism)
employs digestion of PCR-amplified rRNA gene with restriction enzymes. The
digested product is then analyzed by gel electrophoresis. The differential
pattern of DNA fragments is generated due to variable sequence of rRNA
which can lead to identification or typing of a strain using type strain as a
reference (Fig. 2.12a). This method is also called ARDRA (Amplified rDNA
restriction analysis).

Another method is PCR-independent as it is based on hybridization approach.

In this method, a genome is digested with one or more restriction enzymes,
electrophoretically separated on agarose gel, transferred on nylon membrane,
and hybridized with rRNA gene-specific molecular probe (a labeled
oligonucleotide). After hybridization, the developed image of DNA profile is
analyzed. This method is called ribotyping (Fig. 2.12b).

Repetitive sequence (Rep)-PCR fingerprinting

Bacterial genomes possess multiple copies of repetitive DNAs dispersed

across the genome. They are usually located in the intergenic region. Based
on the sequences, three families of repetitive DNAs namely BOX, ERIC
(Enterobacter Repetitive Intergenic Consensus), and REP (Repetitive Extra-
43
Block 1 History of Microbiology and Classification of Microbes

genic Palindrome) have been recognized and used for typing and
identification. The length of BOX, ERIC, and Rep is 154, 124-127, and 35-40
bp respectively. The corresponding protocol is referred to BOX-PCR, ERIC-
PCR, and REP-PCR respectively. The length and sequence of these repetitive
DNAs are same across the majority of Gram-positive and negative bacteria.
However, they differ in their location and number of repeats which form the
basis of variation among the bacteria. In rep-PCR approach, primers specific
to the repetitive sequences are used to amplify DNA fragments between
repetitive elements. The resulting amplified products are separated by gel or
capillary electrophoresis. The given profile is then analyzed using a computer
program. The resolution of this method of identification is at the species level.

Fig. 2.12: Fingerprinting based on rRNA gene. Methodology for (A) RFLP of 16S
rRNA or ARDRA, (B) Ribotyping.

2.4 NOMENCLATURE
Nomenclature is the assigning names to the taxonomic units which have
been characterized and classified. Like in higher organisms, the
microorganisms are also named following binomial nomenclature system
proposed by Linnaeus. Accordingly, every organism is assigned two names in
which the first indicates genus and the second, species. The names are
written in italics or are underlined as specified by nomenclature rules. The
initial letter of genus name is capitalized, and that of the species name is in
small letter. For example, in Escherichia coli, Escherichia is a genus and coli is
species. Sometimes the species name is followed by subspecies name. For
example, Salmonella enterica serovar Typhimurium. Here Typhimurium is a
serovar, an epithet at the subspecies level. There may be a different
consideration for the naming of the species. It can be based on the location
where it was isolated from, the disease it causes, the host it colonizes, etc. For
example, Streptococcus pneumoniae (Streptococcus: chain of round shaped
bacterium that causes pneumonia); Azospirillum brasilense (Azo-nitrogen,
44 spirillum- spiral in shape, brasiliense, i.e., in Brazil), etc.
Unit 2 Microbial Taxonomy
The naming of an organism follows specific and strict guidelines. These
guidelines or rules are published by a specific organization which reviews the
nomenclature proposed for new organisms. Rules for assigning names for
protozoa and parasitic worms are published in International Code of
Zoological Nomenclature, whereas it is International Code of Botanical
Nomenclature for assigning names to algae and fungi. Similarly,
International Committee on Systematics of Prokaryotes publishes rules for
naming prokaryotes in Bacteriological Code. Bacteriological Code publishes
the name of newly discovered or characterized (both genotypically and
phenotypically) bacteria after its review and publication in International Journal
of Systematic and Evolutionary Microbiology (IJSEM). Finally, the bacterium is
listed and described in Bergey’s Manual of Systematic Bacteriology.
Microorganisms that have been sufficiently characterized to assign
provisionally at genus and species level but have not been cultivated in pure
culture are preceded by the term Candidatus which means candidate. For
instance, a photosynthetic bacterium belonging to phylum ‘Acidobacterium’
which can only be grown as coculture with another isolate, has been named
as Candidatus Chloracidobacterium thermophilum (here genus and species
are not italicized). Recently, “The All-Species Living Tree" Project (LTP)
(https://www.arb-silva.de/projects/living-tree/) has been started with the
partnership of five international agencies together with the journal Systematic
and Applied Microbiology (SAM). This project provides a valuable resource,
particularly for microbial taxonomists. The project aimed to reconstruct
separate and curated 16S and 23S rRNA datasets and trees spanning all
sequenced type strains of the hitherto classified species of Archaea and
Bacteria.

With the development of new and sensitive molecular and metabolic tools, the
organisms can be renamed and assigned to new taxonomic ranks. Sometimes
a genus can be divided into two new genera, or two genera can be merged to
one based on the differences of characteristics. For example, genera
Diplococcus and Streptococcus were merged to one whereas a genus
Enterococcus was separated from the Streptococcus. Therefore,
“Streptococcus faecalis” and “Streptococcus faecium” were renamed as
Enterococcus faecalis and Enterococcus faecium respectively. This example
also indicates that the name of species is more stable than the name of
genera as the species name did not change even after the formation of new
genera.

2.5 BERGEY’S MANUAL OF SYSTEMATIC

BACTERIOLOGY
Bergey’s manual of Determinative Bacteriology was first published as a
guide to identify different species of bacteria by Professor Bergey and his
collaborators in 1923. It described the classification system for bacteria and
provided information which could be used for bacterial identification. The last
edition (9th) of this book was published in 1994. In 1984, the first edition of
Bergey’s manual of systematic bacteria with its four volumes published. It
had descriptions of all the bacterial and archaeal species identified till then.
The traits or charactersmentioned in this book for the classification of bacteria 45
Block 1 History of Microbiology and Classification of Microbes

was phenetic. Further in 2001, the second edition of Bergey’s manual of

systematic bacteria with its five volumes published. Each volume covered a
specific group of organisms. The classification system adopted for a
description of the taxons were based on genotypic and phylogenetic
approaches. The brief outline of the different volumes of this book is given
below.

Volume 1: It deals with all Archaea and deeply branching and phototrophic
bacteria. It includes a description of two phyla of Archaea (Crenarachaeota
and Euryarchaeota), nine of deeply branching and two of phototrophic
bacteria.

Volume 2: It is divided into parts A and B which describe a single phylum

proteobacteria. It represents the largest group and contains most of the
Gram-negative bacteria which include pathogens (Escherichia, Salmonella,
Vibrio, Helicobacter, Yersinia, Legionellales, and many other notable genera),
many free-living and nitrogen-fixing bacteria.

Volume 3: This volume describes a single phylum Firmicutes which includes

majorly Gram-positive bacteria having low mol% G + C content. They produce
endospores, are resistant to desiccation, and survive extreme conditions.

Volume 4: This volume covers the description of 12 different phyla namely

Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria,
Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes,
Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes.

Volume 5: It covers a single phylum Actinobacteria which includes Gram-

positive bacteria having high mol% G + C content. Many actinobacteria are
agriculturally and medically important as actinomycetes are known to
decompose organic material and produce antibiotics.

2.6 NUMERICAL TAXONOMY

Numerical taxonomy is a classification system which employs numerical
algorithms to quantify and compare various traits of microorganisms. Sneath
and Sokal define numerical taxonomy as “the grouping by numerical methods
of taxonomic units into taxa by their character states.” Each trait is termed as
OTU (operational taxonomic unit) which is used as an input in numerics.
Thus, the information about the properties (OTUs) of organisms is converted
into a form suitable for numerical analysis and then compared using a
computer. The result of similarity of characters between the microbes is used
for grouping. Each character or OTU is given equal weightage. For instance, if
the oxidase activity is an OTU, presence, and absence of this activity will be
assigned as 1 and 0 respectively. Here oxidase activity is an OTU or character
to be compared. Similarly, as many characters as possible (more than 50 or
even upto thousands) are considered for comparison and robust classification.
It is best to include many different kinds of data based on morphological,
biochemical, and physiological characters.
46
Unit 2 Microbial Taxonomy
Following data input obtained from different tests, an association coefficient
is calculated for each pair of organisms. The association coefficient is a
function that measures the agreement between characters possessed by two
organisms. There are several methods for calculating association coefficient
out of which two most common are simple matching coefficient (SSM), and
Jaccard coefficient (SJ). The SSM is the proportion of characters that match
regardless of whether the attribute is present or absent whereas SJ is
calculated by ignoring those characters which are absent in both organisms.
Both coefficients are represented between 0.0 and 1.0 where 0.0 indicates no
match and 1.0 represents 100% match. The SSM and SJ are calculated as
given below:

SSM= a + d / a + b + c + d

SJ = a / a + b + c

Where a= number of characters (OTU) present (shown as 1) in both

organisms; b and c= number of characters differing between the two
organisms (0,1 or 1,0, i.e., absent in one organism and present in other or vice
versa); d= number of characters absent in both (shown as 0)

The values of association coefficient measured for each pair of organisms are
then used to construct similarity matrix which forms the basis of clustering the
organisms (Fig. 2.13). The clusters are shown in a tree-like representation
called as dendrogram using suitable computer software. The organisms
having great similarity are grouped and separated from dissimilar organisms in
the dendrogram. Such groups are called ‘phenon.’ Phenons formed at 70or
more percent are treated as members of same species.

Fig. 2.13: Clustering analysis of 6 bacterial isolates based on the similarity

matrix obtained by calculating simple matching coefficient. (a) The
similarity matrix is comparing a pair of bacteria. For instance, strain 1
shows the coefficient of 0.92 with strain 2 which indicates 92%
similarity. The coefficient value 1 means 100% similarity whereas 0
shows no similarity at all. (b) Based on the % similarity, the bacterial
strains are rearranged and grouped with the different colour given
based on the range of % similarity. (c) A dendrogram constructed
based on the grouping made in ‘b,’ For instance, strain 1 and 2 shows
the highest similarity with 92% (phenon-92) thus shown as the closest
from one another. The Figure is adopted from Prescott’s
Microbiology, 7th Edition, McGraw Hill Publication.
47
Block 1 History of Microbiology and Classification of Microbes

SAQ 1
Tick [√] mark the correct statement:

a) Species concept is applied same to all organism including bacteria

[True/False]

b) A bacterium can be identified based on its ability to used different carbon

sources. [ True/False]

c) Bacteria are placed under kingdom Protista as per Whittaker’s five

kingdom concept. [ True/False]

d) 16S rRNA sequence analysis can be used for identification of protozoan

parasites. [ True/False]

SAQ 2
Fill in the blanks with appropriate words:

a) Binomial nomenclature was introduced by……………………..

b) The organism causing malaria belongs to domain …………………

c) A descendant of single pure microbial culture is called …………

d) The FAME profiling used for identification of microbe analyzes

differences in the type of ………………present.

e) A tree-like presentation which shows the differences among microbial

isolates is called……………….

f) 16S and 18S rRNA used for microbial identification is component of

…………subunit of the ribosome.

g) …………..gene sequence analysis can be used as an alternative rRNA

gene cluster for identification for fungi.

h) Families of repetitive DNA namely………, …………., and …………..are

present in bacteria which can be used for DNA fingerprinting analysis.

2.7 SUMMARY
• Microbial taxonomy or systematics is defined as the study of the diversity
of microorganisms with the aim of organizing and prioritizing in an
orderly manner.

• It constitutes three separate but integrated components namely

classification, characterization/identification, and nomenclature.

• The first classification system which included microorganisms was

proposed by Robert H. Whittaker who introduced five kingdom concept
and placed microorganisms under kingdom Monera (bacteria,
cyanobacteria, and mycoplasma) Protista (algae and protozoa), and
Fungi.
48
Unit 2 Microbial Taxonomy
• The most recent classification proposed by Carl Woese introduced three
domain concepts, i.e., Bacteria, Archaea, and Eucarya based on the
differences in rRNA gene sequence.

• The organisms are arranged in various taxonomic ranks which include

domain, superphylum, phylum, class, order, genus, and species (top to
bottom).

• The microorganisms can be classified as phenetic, genotypic, and

phylogenetic. However, the best approach applied for classification is
polyphasic taxonomy which employs all the classification approaches
mentioned above.

• The microorganisms can be characterized employing observable traits

(phenotypic) and/or genotypic traits which lead to the identification of
microorganisms.

• The phenotypic methods are based on the morphology of cell or colony,

physiological features such as substrate utilization, metabolic
characteristics, and growth environment. On the other hand, the
genotypic characterization is based on genome sequence, nucleotide
base composition (mol%G + C), DNA-DNA hybridization, DNA
fingerprinting (ARDRA, Ribotyping, Rep-PCR, etc.), and rRNA or other
protein-coding gene sequence analysis.

• For measuring relatedness of microorganisms, the data of different traits

are converted into a matrix which gives a value calculated using
numerical taxonomic approach.

• The nomenclature of microorganisms follows binomial nomenclature

system which indicates the name of genus and species. The guidelines
for naming microorganisms are published in International codes for
nomenclature after review of the proposed name.

• The description of various cultivated bacteria is provided in Bergey’s

manual of systematic bacteriology.

2.8 TERIMINAL QUESTIONS

1. What is taxonomy and what are its components?

2. How can the cell membrane component used for bacterial identification?

3. What is the principle of BIOLOG microbial identification system?

4. Diagrammatically represent rRNA gene cluster of fungi which can be

used for fungal identification.

5. Why is rRNA gene sequence analysis the most preferred approach for
microbial identification?

6. What is the principle of bacterial DNA fingerprinting?

49
Block 1 History of Microbiology and Classification of Microbes

7. What do you mean by numerical taxonomy and how are simple

matching and Jaccard coefficient calculated?

8. What is molecular charaterization? Describe few major methods which

can be used under this approach.

9. Describe five kingdom concept of Whittaker.

10. Describe phenotypic methods which can be used for identification of

microbes.

11. Describe sequencing-based approaches for microbial identification.

12. Describe DNA-DNA hybridization method and its best alternative for
bacterial identification.

13. What is the significance of indels in microbial identification?

2.9 ANSWERS
Self-Assessment Questions

1. a) False

b) True

c) False

d) False

2. a) Carl Linnaeus

b) Eucarya

c) Strain

d) Fatty acid

e) Dendrogram

f) Small

g) β-tubulin

h) BOX, ERIC, and REP

Terminal Questions
1. Microbial taxonomy may be defined as the study of the diversity of
microorganisms with the aim of organizing and prioritizing in an orderly
manner. It is also known as systematic. Three components of taxonomy
are classification, characterization or identification, and nomenclature.

2. FAME (fatty acid methyl ester) analysis is used to identify bacteria based
on type and proportion of fatty acid present in the cell membrane. The
50 membrane lipids of bacteria vary from species to species, and they differ
Unit 2 Microbial Taxonomy
in chain length, presence or absence of a double bond, rings, branched
rings, etc. In this method, the fatty acids are extracted from bacteria
grown under standard conditions, chemically treated to generate methyl
derivative, and analyzed by gas chromatography. The fatty acid profile is
matched with known database and identified

3. BIOLOG has introduced highly automated system with updated

microbial database called Omnilog which enables identification of
microbes including bacteria, yeast, and filamentous fungi based on
ability to metabolize all major classes of biochemicals, in addition to
determining other important physiological properties such as pH, salt,
and lactic acid tolerance, reducing power, and chemical sensitivity. This
system allows profiling of more than 50 different microbial sample at a
time and provide data in a minute.

4. Please refer to Fig 2.11

5. rRNA gene is one of the most conserved genes, present in all

organisms, and long enough, it is routinely used for microbial
identification.

6. The bacterial strains can be identified or typed by sequencing-

independent approaches that analyze DNA profiling which can either be
generated by restriction digestion or by amplification of repetitive DNA
present in the genome. The digested product is then analyzed by gel
electrophoresis. The differential pattern of DNA fragments is generated
due to variable sequence which can lead to identification or typing of a
strain using type strain as a reference.

7. Numerical taxonomy is a classification system which employs numerical

algorithms to quantify and compare various traits of microorganisms. For
more details, please refer to the section 2.6.

8. Molecular characterization is usually referred to the description or

analysis of an organism based on nucleic acid (DNA or RNA) or protein
information. Methods include: (i) determination of nucleic acid base
composition, (ii) nucleic acid hybridization, (iii) nucleic acid sequencing
and DNA fingerprinting.

9. Robert H. Whittaker propose five kingdom concept. The five kingdoms

namely Monera, Protista, Plantae, Fungi, and Animalia, were
propounded based on (i) cell type, i.e., prokaryotic or eukaryotic, (ii) level
of organization, i.e., solitary and colonial unicellular or multicellular
organization, and (iii) type of nutrition.

10. Serotyping, RAMAN spectroscopy, SDS PAGE, MALDI-Tof,

chemotaxonomy are some of the phenotypic methods which can be
used for identification of microbes

11. A certain stretch of the genome or specific genes are the potential
candidate for identification. These sequences can also be used for
analysis of phylogeny. These include genome sequence analysis,
sequence analysis of 16S rRNA and some other such as rpoB encoding
51
Block 1 History of Microbiology and Classification of Microbes

β-subunit of RNA polymerase, genes encoding RNA polymerase (RPB1

and RPB2), translation elongation factor 1-ɑ (tef1) and β-tubulin (benA,
tubC) and determination of GC content.

12. One of the oldest and direct molecular methods for comparing a pair of
microbes is DNA-DNA hybridization (DDH). In this method, the DNAs
are heated to get single-stranded DNA (ssDNA). Then the ssDNAs of
two microbes are allowed to cool and hold at temperature 25 ˚C below
the Tm. The extent of annealing two strands depends on the similarity
between the sequences of two organisms being compared. By
convention, the organisms showing 70% similarity based on the extent
of hybridization are considered a member of same species.

13. Indels (insertion/deletion) are specific length of sequences which are

inserted or deleted in many genes and are specific to certain phylum.
Indels are particularly useful in phylogenetic analysis when they are
flanked by conserved region.

52