Microbial Taxonomy

Microbial Classification and Taxonomy

• Taxonomy
• Science of biological classification
• Consists of three separate but interrelated parts
• classification – arrangement of organisms into groups (taxa, sing.taxon)
• nomenclature – assignment of names to taxa
• identification – determination of taxon to which an isolate belongs

Natural Classification

Natural Classification arranges organisms into groups whose members share many characteristics

• First such classification in 18^th century developed by Linnaeus based on anatomical characteristics
• this approach to classification does not necessarily provide information on evolutionary relatedness in microbes
• E.g classification of humans as mammals –milk producing, hair, self regulating temp. etc.

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Polyphasic Taxonomy

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• Polyphasic Taxonomy is used to determine the genus and species of a newly discovered procaryote
• incorporates information from phenetic (phenotypic) and phylogenetic analysis

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Phenetic Classification

• groups organisms together based on mutual similarity of phenotypes
• can reveal evolutionary relationships, but not dependent on phylogenetic analysis
• E.g because motility and flagella are always associated in particular organisms, it is reasonable to suppose that flagella is involved in some types of motility

Phylogenetic Classification

• Phylogenetic, also called phyletic classification systems
• Phylogeny is based on evolutionary development of a species
• usually based on direct comparison of genetic material and gene products
• this approach is widely accepted
• large databases exist for rRNA sequences

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Taxonomic Ranks and Names

• microbiologists often use informal names that don’t necessarily have taxonomic significance
• e.g., purple bacteria, spirochetes, methane-oxidizing bacteria

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• Table 17.2 shows the levels of taxonomic names

Defining Procaryotic Species

The basic taxonomic group in microbial taxonomy is the species.

• Cannot use definition based on interbreeding because procaryotes are asexual.
• A prokaryotic species is collection of strains that share many stable properties and differ significantly from other groups of strains.
• Also suggested as a definition of species as a collection of organisms that share the same sequences in their core housekeeping genes (genes required to code for products needed by cells)-bases on sequence data.

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Figure Hiearchical arrangement in Taxonomy

Strains

• Descended from a single, pure microbial culture
• Strains vary from each other in many ways
• Biovars – differ biochemically and physiologically
• Morphovars – differ morphologically
• Serovars – differ in antigenic properties

Type Strain

• Usually one of first strains of a species studied
• Often most fully characterized
• Not necessarily most representative member of species

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Genus

• Genus- well-defined group of one or more strains
• Clearly separate from other genera
• Often disagreement among taxonomists about the assignment of a specific species to a genus

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Binomial System of Nomenclature

Binomial system was devised by Carolus Linnaeus

• Each organism has two names
• genus name – italicized and capitalized (e.g., Escherichia)
• species epithet – italicized but not capitalized (e.g., coli)
• can be abbreviated after first use (e.g., E. coli)
• a new procaryotic species cannot be recognized until it has been published in the International Journal of Systematic and Evolutionary Microbiology

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Techniques for Determining Microbial Taxonomy and Phylogeny

Classical characteristics

• morphological
• physiological
• metabolic
• ecological
• genetic

Molecular characteristics

• nucleic acid base composition
• nucleic acid hybridization
• nucleic acid sequencing
• genomic fingerprinting
• amino acid sequencing

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Ecological Characteristics

• Life-cycle patterns
• Symbiotic relationships
• Ability to cause disease
• Habitat preferences
• Growth requirements

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Genetic Analysis

Genetic Analysis

• Study of chromosomal gene exchange by transformation and conjugation
• plasmids can be used for the analysis of phenotypic traits

Nucleic Acid Base Composition

• Determine the G + C content

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Where G=Guanine, C=Cytosine, A=adenine and T=Thymine ( nucleotide are the DNA base)

• The G+ C content is often estimated by determining the melting temperature (T_m) of the DNA
• Higher G + C gives a higher melting temperature

Nucleic Acid Hybridization

Nucleic Acid Hybridization

• measure of sequence homology

( molecular relatedness)

• common procedure for hybridisation:
• bind nonradioactive DNA to nitrocellulose filter
• incubate filter with radioactive single-stranded DNA
• The quantity of radioactivity bound to the filter reflects the amount of hybridisation between the 2 DNA and thus similarity of the 2 sequences.

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…Nucleic Acid Hybridization

• measure amount of radioactive DNA attached to filter.
• The degree of similarity is expressed as the % of experimental DNA radioactivity retained on the filter as compared to other sps. of the same genus under the same conditions.
• Usually less than 5 % difference in melting point ( T _m ) is considered as members of same sps.

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Nucleic Acid Sequencing

Nucleic Acid Sequencing

• most powerful and direct method for comparing genomes
• sequences of 16S (procaryotes) and 18S (eucaryotes) ribosomal RNA (rRNA) are used most often in phylogenetic studies
• complete chromosomes can now be sequenced and compared

…Nucleic Acid Sequencing

Comparative Analysis of 16S rRNA Sequences:

• Oligonucleotide signature sequences are short conserved sequences specific for a phylogenetically defined group of organisms
• either complete or, more often, specific rRNA fragments can be compared
• when comparing rRNA sequences between 2 organisms, their relatedness is represented by an association coefficient or S_ab value
• the higher the S_ab value, the more closely related the organisms

Use of DNA Sequences to Determine Species Identity

DNA sequences can also be used to determine species strains in addition to genus

• It requires analysis of genes that evolve more quickly than rRNA encoding genes
• Multilocus sequence typing (MLST), the sequencing and comparison of 5 to 7 housekeeping genes instead of single gene is done.
• This is to prevent misleading results from analysis of one gene.

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Genomic Fingerprinting

• Genomic Finger Printing also used for microbial classification and determination of phylogenetic relationships
• Genomic Finger Printing does not involve nucleotide sequencing
• Can be used because of multicopies of highly conserved and repetitive DNA sequences present in most gram-negative and some gram-positive bacteria
• Multicopies can be obtained by Polymerase chain reaction using restriction enzymes

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…Genomic Fingerprinting

• uses restriction enzymes (endonucleases) that recognize specific nucleotide sequences
• Restriction Enzyme cuts DNA at specific sites
• Restriction fragments are compared by Gel Electrophoresis.

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…Genomic Fingerprinting

• Repetitive sequences amplified by the polymerase chain reaction
• amplified fragments run on agarose gel, with each lane of gel corresponding to one microbial isolate
• pattern of bands analyzed by Gel Document system
• comparison of bands is called restriction fragment length polymorphism (RFLP)
• It allows for identification to species, subspecies and often allows strain level identification
• PCR has a widespread application

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Figure Genomic Finger Printing

Amino Acid Sequencing

• the amino acid sequence of a protein is a reflection of the mRNA sequence and therefore of the gene which encodes that protein
• amino acid sequencing of proteins such as cytochromes, histones and heat-shock proteins has provided relevant taxonomic and phylogenetic information
• cannot be used for all proteins
• compare protein mass spectra

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Assessing Microbial Phylogeny

• evolutionary relationships represented using phylogenetic trees
• A phylogentic tree is a graph which connects nodes and branches

Phylogenetic Trees

a. Unrooted tree –

b. Rooted treehas node that

serves as

common

ancestor

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Metagenomics and its applications