Microbial Systematics
Devlina Sengupta
Department of Microbiology
Kanchrapara College
Introduction
Taxonomy is the science dealing with the description, identification, naming and classification of organism. Bacterial taxonomy
traditionally has focused on practical aspects of identification and description, activities that have
relied heavily on phenotypic comparisons.
It consists of the following 3 interrelated processes:
Classification
Nomenclature
Identification
Classification is the grouping of organisms based on particular characters & is not arranged in an hierarchical order. It is used to
arrange organisms into specific groups called Taxa based on mutual similarity.
Nomenclature is the branch of taxonomy concerned with the assignment of names to taxonomic groups in agreement with
published rules.
Identification is the practical side of taxonomy , the process of determining if a particular isolate belongs to a recognized taxon.
Systematics is the scientific study of organisms with the ultimate objective of characterizing and arranging them in orderly manner.
It links together phylogeny with taxonomy in which organisms are characterized, named, and placed into groups according to
several defined criteria.
At present, the growing use of genetic information, especially DNA sequence data, is increasingly allowing taxonomy to reflect
phylogenetic relationships
as well. This is called Polyphasic approach to classify microbes in which information gleaned from genetic, phenetic &
phylogenetic analysis is incorporated together.
Carolus Linnaeus (1707–1778)
– Swedish botanist credited with founding the science
of taxonomy by Natural classification.
– He introduced the binomial system of nomenclature
– Linnaeus also established a hierarchy of taxonomic
ranks: species, genus, family, order, class, phylum or
division, and kingdom.
– At the highest level, Linnaeus divided all living
things into two kingdoms—plant and animal.
– In his taxonomic hierarchy each organism is
assigned a species name, and species of very
similar organisms are grouped into a
genus and so on.
Three kingdom, five kingdom & six
kingdom concept
• In 1866 Ernst Haeckel proposed the kingdom Protista, to include bacteria, protozoa, algae, fungi. Then in
1890s a third kingdom was introduced
• In 1959, Fungi were placed in their own kingdom
• In 1960s, Robert H. Whittaker founded the five-kingdom system in which prokaryotes were placed in the
kingdom prokaryotae, or Monera and eukaryotes comprised other four kingdoms.
• In 1970s Carl Woese divided the prokaryotes into two as Eubacteria and Archaebacteria. This led to the six
kingdom classification.
A Comparison of the More Notable Classification
Systems of Living Organisms
Haeckel (1894) Whittaker (1959) Woese (1977) Woese (1990)
Three kingdoms Five Six Three
kingdoms kingdoms domains
--------------------------------------------------------------------------------------------------------
-Protista Monera Eubacteria Bacteria
Plantae Archaebacteria
Animalia Protista Protista Archaea
Fungi Fungi
Plantae Plantae Eukarya
• Animalia Animalia
Kingdom Monera & Protista
• Kingdom monera consists of unicellular organisms with a prokaryotic type
of cell organization. They are single celled organisms with no true nuclear
membrane. In the three kingdom system these organisms were included in
the kingdom protists.
• Protists in the five kingdom system are eukaryotes with unicellular
organization, either in the form of solitary cells or colonies of cells lacking
true tissues. They may have ingestive, absorptive or photoautotrophic
nutrition, and they include most of the microorganisms known as algae,
protozoa, and many of the simpler fungi
Carl Woese & FOX
• Proposed classification based on direct
comparison of genetic material and gene Universal ancestor
products using small subunit (SSU) Domain Eukarya
rRNA nucleotide sequences.
• Such system is also called Phyletic or Domain Archaea
Phylogenetic classification. Euryarchaeotes
Crenachaeotes
• It deals with evolutionary development of Nanoarchaeotes
Domain Bacteria
Korarchaeotes
a species.
Proteobacteria
• The prokaryotes were included in a large Chlamydis
Spirochetes
single group for a long time, Carl Woese Cyanobacteria
Gram positive bacteria
in the 1970s showed that prokaryotes are
divided into two distinct lineages or lines
of descent: Archaea and Bacteria.
Today these groups are considered to
form two out of three domains of life.
Classification of organisms
Taxonomic Ranks
• Microbes are placed in Hierarchical taxonomic levels with each level or
rank sharing a common set of specific features.
• Highest rank is domain
Bacteria & Archaea Microbes only
Eukarya- microbes & macroorganisms
• Within domain
Phylum, class, order, family, genus, species, epithet, some microbes have
subspecies
• In higher organisms sexually compatible organisms are considered as
species but this definition fails for bacteria(as they do not reproduce
sexually).
• A collection of microbial strains that share many properties and differ
significantly from other groups of strains. Species are identified by
comparison with known “Type strains”: well characterized pure cultures.
Nomenclature
• Casual or Common Name:
• e.g. "typhoid bacillus"
• Scientific or International Name:
• Salmonella typhi
• Salmonella london
• Staphylococcus aureus
• Clostridium tetani
• Mycobacterium bovis
• Borrelia burgdorferi
Classification Systems
1. Artificial or phonetic Classification
•The earliest systems of classification which
remained dominant from 300 B.C. up to about
1830 were artificial systems, which were based on
one or a few easily observable characters
of microbes based on habitat, cell metabolism or
morphology. Phylogenetic or evolutionary related
information is not obtained.
•It was initially used for classification of plants
based on their floral characters(particularly the
number of stamens and carpels), venation,
habit(trees, shrubs, herbs) etc.
•Such types of classification using some arbitrary
or at least easily observable characters, often
irrespective of their affinity, is called artificial.
•Example of artificial classification such as
grouping bacteria according to Gram reaction of
the organism; whether it is acid-fast; its motility;
the arrangement of its flagella; the presence of
spores, capsules, and inclusion bodies; and, of
course, its shape.
2. Natural Classification
• These systems of classifications are
based upon overall resemblances,
mostly in gross morphology, thus,
utilizing as many taxonomic characters
as possible, to group taxa.
• Larger the number of characters shared
by different taxa, the more closely
related they are to each other. This is
the basis of modern classification.
• A natural system never uses habit as
criteria for grouping.
• It studies homology in all characters
including morphology, metabolomics,
nutrition, cytotaxonomy, molecular
systematics, etc.
3. Phylogenetic Classification
• An evolutionary & genetic
arrangement of species in addition
to natural characters.
• Possible by Molecular
Methods
– Genetic Homology:
• Base composition (GC
ratio)
• Nucleic acid
hybridisation.
• Ribosomal RNA (rRNA)
sequence analysis
• Protein profiles and
amino acid sequences
How do new prokaryotic species arise?
•The process of periodic purges and selection within cell population
causes new species to arise.
•Imagine a population of bacteria that originated from a single cell
and that occupies a particular niche in a habitat. In theory, these cells
are genetically identical. If cells in this population share a particular
resource (for example, a key nutrient), the population is considered
an ecotype.
•However, within each ecotype, genes mutate at random over time
as the cells grow. Most of these mutations are neutral and have no
effect. However, if there is a beneficial mutation (one that increases
fitness) in a cell in one of the ecotypes, that cell will produce more
progeny over time, and this will purge the population of the original,
less well-adapted cells.
•Repeated rounds of mutation and selection in this ecotype lead it to
become more and more distinct genetically from the other ecotypes.
Then, given enough time, cells in this lineage will carry a
sufficiently large set of unique traits that they emerge as their own
species.
Genes employed in phylogenetic analysis
• Most widely used and useful for defining relationships are the genes encoding 16S rRNA & its
counterpart in eukaryotes 18S rRNA.
• These are called small subunit ribosomal RNA (SSU RNA) pioneered by Carl Woese.
• They are significant because:
1) universally distributed
2) functionally constant
3) sufficiently conserved (slowly changing)
4)of adequate length (not too large to be sequenced & compared)
• Ribosomal Database Project II (RDP-II) contains a collection of such sequences & variety of analytical
programs.
• LSU-rRNA are also helpful but they are longer sequences which are time consuming for homology
alignment studies.
• Several highly conserved proteins are also compared for their evolution including EF-Tu, HSPs (HSP
60), tRNA synthetases etc. Many proteins like Rec A’s homology is used only for bacterial
phylogenetic analysis but not for Archaea and Eukarya as this protein is absent in those domains.
Analytical methods for evolutionary analysis
For obtaining DNA sequences:
• Genomic DNA extracted from an organism is subjected to PCR ( polymerase chain reaction) to obtain
sufficient copies of a gene for efficient sequencing.
• Specific primers are designed to amplify DNA of a specific gene which is a determining step.
• In many cases universal primers are used such as primers 27f, 1492r for 16s rRNA amplification.
• After PCR, the DNA is subjected to Agarose gel electrophoresis.
• The separated DNA strand is excised from the gel, extracted and purified from the agarose, and then
sequenced using the same oligonucleotides as primers for sequencing.
For sequence homology:
• After sequencing, homology is analysed amongst several organisms by several web-based tools such
as BLAST ( Basic Local Alignment search tool) of NCBI. This is done automatically.
• Homologous sequences are downloaded from GenBank
• Alignment is critical as assignment of mismatches and gaps determine how the organism has diverged
from a common ancestral sequence.
• Protein coding genes usually are aligned with the aid of their inferred amino acid sequences.
• 3D arrangement & folding of 16s rRNA is also checked for homology apart from sequence similarity.
Analytical methods for evolutionary analysis
Phylogenetic trees:
• Tree construction from observed nucleotide sequence differences is composed of nodes, branches and
internodes.
• The intermodal points represent ancestors, the tips of the branches are nodes that represent individual
strains of bacterial species that exist now and from which sequence data has been obtained.
• The branches define 1) Analytical methods for evolutionary analysis the order of descent & 2) the
ancestry of the nodes
• Branch lengths define the number of changes that have occurred along the branch.
• Trees can be rooted or unrooted whereby unrooted trees are without any information related to
evolutionary path whereas rooted trees have evolutionary pathway defined from ancestor to each
species.
Tree construction:
• It uses Cladistics for the purpose which is a method based on character-state relationship
• Variations in nucleotide at individual positions for common proteins are characters that define a
monophyletic classification
• A widely used cladistics method are Parsimony ( based on assumption that evolution is most likely to
have proceeded by the path requiring fewest changes), Maximum likelihood method and Bayesian
analysis (based on assumption that certain kinds of nucleotide changes are frequent than others) done
through applications like PAUP ( phylogenetic analysis under parsimony and other methods)
Construction of an unrooted tree using parsimony
method
(using
Where 0= Pleisomorphic or ancestral given
character data ormatrix
1= apomorphic )
derived character
We construct parsimony tree based on shared derived character state or synapomorphic character
state (where two taxa share derived characteristics) Solution
Characters Taxa Determination of outgroup
As the matrix reveals zero no of shared
A B C D derived character states fir taxa A, hence
1 0 1 1 1 taxa A is considered an outgroup
A B C D
2 0 0 1 0
A - 0 0 0
3 0 0 0 1
B - 1 1
4 0 0 1 1
C - 2
5 0 1 0 0
D -
Construction of all possible networks/unrooted trees
Nu = where n= [Link] taxa
Nu= [Link] possible unrooted tree
Here n=4
So, Nu=
=
=
=3
So, there would be possible 3 unrooted trees for 4 different taxa as follows:
A outgroup B
C A B A
B D D D C
C
Tree 1 Tree 2 Tree 3
Determination of total number of evolutionary changes in each possible tree:
5 1 5
1 1
2 3 2
4 4
4 3
3 4
4 2
5
Showing 5 evolutionary changes Showing 6 evolutionary changes Showing 6 evolutionary changes
By the number of evolutionary changes, tree 1 has the least number of mutation and it is the most probable tree by
parsimonious method
Application of SSU rRNA phylogenetic methods
Signature sequences
• These are short oligonucleotide sequences unique t certain groups of organisms, eg for each of the
domains of cellular life or specific group within a domain.
• They are useful for their exclusivity which helps in determining newly isolated or misclassified
organism into their correct phylogenetic group
• They help in designing specific nucleic acid probes called phylogenetic probes (labelled DNA/ rRNA
strands to complementarily hybridize nucleic acid from a mixture)
• FISH (fluorescent in-situ hybridization) is a technique to apply probes directly to microbial culture or
environment without extracting DNA or rRNA. Fluoresceing cells are directly observed under
microscope.
Microbial community analysis by PCR & Ribotyping
• PCR amplified known rRNA genes can be sequenced and aligned with unknown organisms to infer
differences in sequences and thus construct a phylogenetic tree
• That is how if a new microbial community is established, the amplified DNA sequence might reveal
key differences in structure, biochemical functions etc.
• Ribotyping is a technique which utilizes differences in banding patterns of DNA fragments generated
as the DNA of a particular organism is digested by different REs. Every species has its own unique
banding pattern or ribotype which discriminates between species and strains.
• These digested fragments of variable length are separated by gel electrophoresis, separated DNA
fragments transferred from gel onto nylon membrane via blotting technique and membrane
hybridized with rRNA gene probe.
• The fluorescing pattern then generated is digitized and computer is used to make comparisons of
this pattern with patterns from reference organisms available from database (NCBI, EMBL).
Phyletic classification
Protists are a paraphyletic group because animals, fungi, and plants are the
crown groups evolved from different lineages of the protists. They aren't
included in the same group as protists
INTRASPECIES CLASSIFICATION
• Biotypes
– Biochemical properties.
• Serotypes
– Antigenic features.
• Phage Types
– Bacteriophage susceptibility.
• Colicin Types
– Production of bacteriocins.
Nomenclature
• Naming of microorganisms in accordance with the
agreement with published rules.
• The fourth International congress of bacteriology held
• Rules published in the International Code of
Nomenclature of Bacteria.
• The International Journal of Systematic Bacteriology
Rules for the Nomenclature of
Microorganisms
• There is only one correct name for an organism.
• Names that cause error or confusion should be rejected.
• All names in Latin or are latinized.
– The first word (genus) is always capitalized.
– The second word (species or specific epithet) is not capitalized.
– Both genus and species name, together referred to as species, are either
underlined or italicized when appearing in software.
– The correct name of a species or higher taxonomic designations is
determined by valid publication, legitimacy of the name with regard to
the rules of nomenclature, and priority of publication.
Identification
• Biologists often use a taxonomic key to
identify organisms according to their
characteristics.
• Dichotomous key
– most commonly used in identification.
– has paired statements describing
characteristics of organisms.
Methods used for Identification of
Bacteria
• Cellular morphology
• Staining characteristics
• Motility
• Growth characteristics
• Biochemical characteristics
• Serological tests
• Analysis of metabolic end products or structural
components of organisms by different methods (e.g.
GLC)
• Genetic analysis using nucleic acid probes and other
molecular techniques (e.g. PCR)
TEST
Organism Gram Shape Catalase Indole
B. subtilis + Rod + -
C. freundii - Rod + -
E. faecium + Coccus - -
P. vulgaris - Rod + +
S. aureus + Coccus + -
• Dichotomous Key
Gram reaction
+ -
morphology indole
rods cocci + -
B. subtilis P. C. freundii
vulgaris
catalase
+ -
S. aureus E. faecium
Enterotube
• Bergey's Manual
– Methods for distinguishing and identifying
bacteria are assembled into Bergey's
Manual of Determinative Bacteriology
– Bergey's Manual of Systematic
Bacteriology
• Provides description of physical & chemical
characteristics and system of identification of
medically important members of selected
sections of bacteria
Species concept in Microbiology
•The issue of what actually constitutes a prokaryotic species remains
controversial. As species are the fundamental units of biological diversity, how
the concept of species is defined in microbiology determines how we
distinguish and classify the units of diversity that make up the microbial world.
•Prokaryotic species is defined operationally as a group of strains sharing a
high degree of similarity in several independent traits. Traits currently
considered most important for grouping strains together as a species include:
a)70% or greater genomic DNA–DNA hybridization and
b)97% or greater identity (3% difference) in 16S rRNA gene sequence
•16S rRNA gene sequence differences of more than 5% from all other
organisms is considered good evidence that an organism constitutes its own
genus
•For better species-level resolution gyrB, the gene encoding DNA gyrase
subunit B, and luxABFE, a series of genes encoding luminescence enzymes
are used in addition to the 16srRNA gene.
Newer approaches for exploring unculturable bacteria
Discovery
Phenotypic Genotypic
FAME Whole cell protein
analysis via mass Plasmid analysis or
analysis spectroscopy ribotyping
Molecular Taxonomy & Phylogeny
The classification of organism on the basis of molecular substances present
inside them.
Molecular phylogenetics deals with the study of evolutionary relationships
among biological entities by using a combination of molecular data (such as
DNA & protein sequences, presence or absence of transposable elements)
and statistical techniques.
Molecular markers: can be characterized as type I & type II markers.
Type I markers are associated with genes of known function.
TypeII markers are associated with genes of unknown function.
Allozyme markers are type I markers as the protein they encode are
associated with some function.
Microsatellites and other neutral markers are type II markers unless they are
associated with gene of some known function.
*Mitochondrial DNA markers are non-nuclear DNA having inherited
maternally with a haploid genome. They do not undergo recombination &
thus are homologous markers.
Single Nucleotide Polymorphism: SNPs arise due to single nucleotide
substitutions(transitions/transversions) or single nucleotide
insertions/deletions.
SNPs are the most abundant polymorphisms in the genome of any organism.
These single nucleotide variation can be detected using PCR, microchip arrays
or fluorescence technology.
Molecular Chronometers
It is a phylogenetic marker whose rate of mutation is constant & can be used
to construct phylogenetic tree.
Must be present in all the organisms to be compared
-Same function in all organisms to be compared
o Part of ribosome
o Protein synthesis
-Has sufficient amount of sequence conservation to allow alignment yet
enough variability to provide information at many levels- species to Domain-
Rate of change should reflect the evolutionary distance
Ribosomal RNAs act as evolutionary chronometers.
Present in all organisms
-rRNA has variable and conserved regions
-Use variable regions to compare closely related organisms i.e. same species,
genus or family
-Use conserved regions to compare distantly related organisms i.e. from
different kingdoms or domains
Chemotaxonomy
FAME analysis or fatty acid methyl esters analysis measures the types and
proportions of fatty acids present in cytoplasmic membrane lipids and the outer
membrane lipids of gram negative bacteria.
The fatty acid composition of Bacteria varies from species to species in chain length and
in the presence or absence of double bonds, rings, branched chains, or hydroxy groups.
Procedure: For the analyses, fatty acids extracted from cell hydrolysates of a culture
grown under standardized conditions are chemically derivatized to form their
corresponding methyl esters. These now volatile derivatives are then identified by gas
chromatography. A chromatogram showing the types and amounts of fatty acids from the
unknown bacterium is then compared with a database containing the fatty acid profiles of
thousands of reference bacteria grown under the same conditions. The best matches to
that of the unknown are then selected .
Cytochrome analysis: Cytochromes are membrane bound hemoproteins that
contain heme groups and carry out electron transport processes. Cytochrome
a,b, c, d.
Gram +ve cytochrome: Cytochrome bcaa3
Facultative anaerobic Gram +ve : Cytochrome C
Clostridium: No cytochrome
Gram –ve cytochrome: cytochromes bdoa1
Phototroph: Cytochrome bc
Polyphasic Bacterial Taxonomy
• More data will become available, more bacteria will be
identified, there will be more information, and software
development will need to address the combination
and linking of the different databases.
• A polyphasic approach to bacterial classification
includes:
– Methods to phylogenetically allocate bacteria
– Methods to compare and group large numbers of strains
into clusters of similar bacteria
– DNA-DNA hybridization to determine the relationships
between
represnetativies withing and between each of those clusters
– And descriptive methods which will provide further genotypic
and phenotypic information.
