Classification of Classification of Microorganisms Microorganisms

Prepared byPrepared by

Rajesh M.PatelRajesh M.Patel

Assistant Professor,Assistant Professor,

Department of Pharmaceutical Biotechnology,Department of Pharmaceutical Biotechnology,

Ganpat University.Ganpat University.

Email:rajmit_120@yahoomail.comEmail:rajmit_120@yahoomail.com

Contents of the chapterContents of the chapter TerminologyTerminology Taxonmomy: Introduction and HistoryTaxonmomy: Introduction and History ClassificationClassification NomenclatureNomenclature Taxonmic methods for classification of Taxonmic methods for classification of

microorganismsmicroorganisms Intuitive methodIntuitive method Genetic methodsGenetic methods % G+C content% G+C content Nucleic acid hybridizationNucleic acid hybridization DNA chip techniqueDNA chip technique Numerical classification methodssNumerical classification methodss

TerminologyTerminology TaxonomyTaxonomy

Classification of living organisms into groupsClassification of living organisms into groups Phylogenetic Classification System:Phylogenetic Classification System:

Groups reflect genetic similarity and evolutionary Groups reflect genetic similarity and evolutionary relatednessrelatedness

Phenetic Classification System:Phenetic Classification System: Groups do not necessarily reflect genetic Groups do not necessarily reflect genetic

similarity or evolutionary relatedness. Instead, similarity or evolutionary relatedness. Instead, groups are based on convenient, observable groups are based on convenient, observable characteristics.characteristics.

TerminologyTerminology Species: A collection of microbial strains that Species: A collection of microbial strains that

share many properties and differ significantly share many properties and differ significantly from other groups of strainsfrom other groups of strains

Strain: a population of organisms Strain: a population of organisms descended descended from a from a single organism or pure culture single organism or pure culture isolate.isolate.

OROR a culture derived from a single parent that a culture derived from a single parent that

differs in structure or metabolism from other differs in structure or metabolism from other cultures of that speciescultures of that species

Terminology Type strain: One of the first strain among the Type strain: One of the first strain among the

species, studied and fully characterised than species, studied and fully characterised than other strains.other strains.

Biovars Biovars procaryotic variant strains procaryotic variant strains characterized by biochemical or physiological characterized by biochemical or physiological differencesdifferences

Morphovars Morphovars procaryotic variant strains procaryotic variant strains characterized by morpholigical characteristicscharacterized by morpholigical characteristics

Serovars Serovars procaryotic variant strains procaryotic variant strains characterized by distinctive antigenic characterized by distinctive antigenic propertiesproperties

Taxonomy

Organizing, classifying and Organizing, classifying and naming living thingsnaming living things

Formal system originated by Formal system originated by Carl von LinnCarl von Linné (1701-1778) é (1701-1778)

Identifying and classifying Identifying and classifying organisms according to organisms according to specific criteria specific criteria

Each organism placed into a Each organism placed into a classification system classification system

TaxonomyTaxonomy

Taxa: order or arrangementTaxa: order or arrangement

NNomos: lawomos: law

Nemein: to distribute or governNemein: to distribute or govern

The branch of biology dealing with the The branch of biology dealing with the classification of life.classification of life.

Taxonomy / Systematics can be Taxonomy / Systematics can be understand by general examplesunderstand by general examples

NomenclatureNomenclature Providing a formal nameProviding a formal name Genus & speciesGenus & species

Ford Crown VictoriaFord Crown Victoria Chevy ImpalaChevy Impala Toyota CamryToyota Camry Honda CivicHonda Civic

• Classification • Organization into groups

• Car• Truck• SUV• Van

• Identification • Distinguishing features

• Engine size• Mileagae• Number of passengers• Type of transmission

Taxonomy: History 1700s 2 kingdoms: plant and animal1700s 2 kingdoms: plant and animal 1800s 3 kingdoms: plant, animal, and 1800s 3 kingdoms: plant, animal, and

protistaprotista 1950-1990s 5 kingdoms: plant, animal, 1950-1990s 5 kingdoms: plant, animal,

protista, fungi, moneraprotista, fungi, monera Present: 6 kingdoms: eubacteria, Present: 6 kingdoms: eubacteria,

archaebacteria, protista, animal, plant, fungiarchaebacteria, protista, animal, plant, fungi Taxonomy consists of 3 partsTaxonomy consists of 3 parts

ClassificationClassification IdentificationIdentification NomenclatureNomenclature

ClassificationClassification

Arrangement into groups based on Arrangement into groups based on mutual similarity or evolutionary mutual similarity or evolutionary relatednessrelatedness

Ordering of organisms into groups.Ordering of organisms into groups.

ClassificationClassification Natural classification: arranges organisms into arranges organisms into

groups whose members share many groups whose members share many characteristics and reflects as much as characteristics and reflects as much as possible the biological nature of organisms. possible the biological nature of organisms. Linnaeus developed the first natural Linnaeus developed the first natural classification, based largely on anatomical classification, based largely on anatomical characteristics.characteristics.

E.g. classification of humans as mammals E.g. classification of humans as mammals denotes that they have hair, self-regulating denotes that they have hair, self-regulating body temperature,and milk-producing body temperature,and milk-producing mammary glands in the female.mammary glands in the female.

Polyphasic classification

TaxonomyTaxonomy

DomainDomain KKingdomingdom PPhylumhylum CClasslass OOrderrder FFamilyamily GGenusenus sspeciespecies

3 Domains3 Domains

EubacteriaEubacteria true bacteria, peptidoglycantrue bacteria, peptidoglycan

ArchaeaArchaea odd bacteria that live in extreme odd bacteria that live in extreme

environments, high salt, heat, etc. (usually environments, high salt, heat, etc. (usually called extremophiles)called extremophiles)

EukaryaEukarya have a nucleus & organelles (humans, have a nucleus & organelles (humans,

animals, plants)animals, plants)

3 Domains system3 Domains system

Five-Kingdom System of Biological ClassificationProposed inProposed in 19691969 by Robert Whitaker : by Robert Whitaker :

1.1. Kingdom Procaryotae (Monera):Kingdom Procaryotae (Monera): Oldest known cells. Oldest known cells. Lived over 3.5 billion years ago. Lack a nucleus and Lived over 3.5 billion years ago. Lack a nucleus and membrane bound organelles.membrane bound organelles.

The other four kingdoms are The other four kingdoms are eucaryoteseucaryotes. Have a . Have a true nucleus and membrane bound organelles.true nucleus and membrane bound organelles.2. Kingdom Protista:2. Kingdom Protista: Mostly unicellular, lack tissue Mostly unicellular, lack tissue

organization. Most have flagella during life.organization. Most have flagella during life.3. Kingdom Fungi3. Kingdom Fungi: May be unicellular (yeasts) or : May be unicellular (yeasts) or

multicellular (molds). Many are saprotrophs. multicellular (molds). Many are saprotrophs. 4. Kingdom Plantae:4. Kingdom Plantae: Multicellular, photosynthetic. Multicellular, photosynthetic.5. Kingdom Animalia:5. Kingdom Animalia: Multicellular, heterotrophs that Multicellular, heterotrophs that

ingest food through a mouth or oral cavity.ingest food through a mouth or oral cavity.

Five-Kingdom Classification Five-Kingdom Classification System (System (Whittaker)Whittaker)

Scientific NomenclatureScientific Nomenclature Developed in the eighteenth century.Developed in the eighteenth century. Bionomial nomenclature:Bionomial nomenclature: Genus and specific epithet(species)Genus and specific epithet(species) Eg. Eg. Escherichia coli, Salmonella typhi, Escherichia coli, Salmonella typhi,

Bacillus subtilisBacillus subtilis Rules for naming are set by international Rules for naming are set by international

committee’scommittee’s International Code of Zoological MomenclatureInternational Code of Zoological Momenclature International Code of Botanical NomenclatureInternational Code of Botanical Nomenclature Bacteriological Code and Bergey’s ManualBacteriological Code and Bergey’s Manual

Scientific Binomial Source of Genus Name

Source of Specific Epithet

Klebsiella pneumoniae

Honors Edwin Klebs The disease

Pfiesteria piscicida Honors Lois Pfiester Disease in fish

Salmonella typhimurium

Honors Daniel Salmon

Stupor (typh-) in mice (muri-)

Streptococcus pyogenes

Chains of cells (strepto-)

Forms pus (pyo-)

Penicillium chrysogenum

Tuftlike (penicill-) Produces a yellow (chryso-) pigment

Trypanosoma cruzi Corkscrew-like (trypano-, borer; soma-, body)

Honors Oswaldo Cruz

Scientific NamesScientific Names

Bergey’s Manual of Systematic Bacteriology

First edition published in 1923

Techniques For Determining Microbial Taxonomy

The Intuitive methodThe Intuitive method Genetic HomologyGenetic Homology

DNA/RNA DNA/RNA sequencingsequencing

DNA chip DNA chip technologytechnology

Numerical TaxonomyNumerical Taxonomy

Techniques For Determining Microbial Taxonomy And Phylogeny

Morphological features are important . Morphology is easy to study and analyze, particularly

in eukaryotic microorganisms and the more complex prokaryotes.

Structural features depend on the expression of many genes, are genetically stable, and do not vary greatly with environmental changes.

Many different morphological features are employed in the classification and identification of microorganisms .

Light microscope : resolution limit of about 0.2 m reduces its usefulness in viewing smaller microorganisms and structures.

The transmission and scanning electron microscopes, with their greater resolution, have immensely aided the study of all microbial groups

Morphological characteristics

Morphological characteristics

Morphological characteristics

Differential staining:Differential staining: Gram staining, acid- Gram staining, acid-fast stainingfast staining

Biochemical tests:Biochemical tests: Determines Determines presence of bacterial enzymespresence of bacterial enzymes

Morphological characteristics Biochemical tests Biochemical tests

used to identify used to identify selected species of selected species of human pathogens human pathogens isolated from marine isolated from marine mammals.mammals.

Assume you isolated Assume you isolated a gm-ve rod that a gm-ve rod that produces gas from produces gas from glucose, is urease glucose, is urease negative and indole negative and indole positive. What is the positive. What is the bacterium?bacterium?

Morphological characteristics

Physiological and metabolic characteristics are very useful because they are directly related to the nature and activity of microbial enzymes and transport proteins.

Since proteins are gene products, analysis of these characteristics provides an indirect comparison of microbial genomes.

Physiological and Metabolic Characteristics

Physiological and Metabolic Characteristics

Ecological properties: affect the relation of microorganisms to their environment.

Taxonomically valuable because even very closely related microorganisms can differ considerably with respect to ecological characteristics.

Microorganisms living in various parts of the human body markedly differ from one another and from those growing in freshwater and marine environments.

Some examples of taxonomically important ecological properties are life cycle patterns; the nature of symbiotic relationships; the ability to cause disease in a particular host; and habitat preferences such as requirements for temperature, pH, oxygen, and osmotic concentration.

Ecological Characteristics

The Intuitive methodThe Intuitive method

It is rely on the study of the properties of the It is rely on the study of the properties of the organisms by taxonomists for several years organisms by taxonomists for several years which decides to represent one or more which decides to represent one or more species or genera.species or genera.

Demerit: Demerit: The characteristics of an organism that is The characteristics of an organism that is

important to one person may not be so important important to one person may not be so important to another.to another.

Different taxonomists arrive at very different Different taxonomists arrive at very different groupings.groupings.

Nucleic acid base composition

DNA contains four purine and pyrimidine bases: A, G, C, T.

(G+C)/(A+T) ratio or G+C content, % of G+C in DNA, reflects the base.

Mol % (G+C)= [(G+C)/(G+C+A+T)]*100 Estimated by determining the melting Estimated by determining the melting

temperature of the DNAtemperature of the DNA Higher G + C gives a higher melting temperatureHigher G + C gives a higher melting temperature

Methods to determine base composition

1.HPLC: Hydrolyse the DNA & Analyse with HPLC

2.Melting temperature (Tm): double stranded DNA, A=T, G = C, High G +C = high melting point.

Nucleic acid base composition Absorption of DNA:at max 260

nm. Separation of strand Abs. When a DNA sample is slowly

heated: absorbance as hydrogen bonds are broken and reaches a plateau when all the DNA has become single stranded

The midpoint : Tm, a direct measure of the G +C content.

Density of DNA also increases linearly with G+C content.

%G +C: by centrifuging DNA in a CsCl density gradient

Figure: A DNA Melting Curve. The Tm is indicated

Nucleic Acid Base CompositionNucleic Acid Base Composition

The G+C content of DNA from animals and higher The G+C content of DNA from animals and higher plants : average 40%, ranges between 30 to 50%. plants : average 40%, ranges between 30 to 50%.

Prokaryotic G+C content : 25 to 80%. Prokaryotic G+C content : 25 to 80%. If two organisms differ in their G + C content by If two organisms differ in their G + C content by

>10%, : different base sequences.>10%, : different base sequences. The G + C content of strains within a particular The G + C content of strains within a particular

species is constant. species is constant. Very different base sequences can be constructed Very different base sequences can be constructed

from the same proportions of AT and GC base from the same proportions of AT and GC base pairs. pairs.

..

Nucleic Acid Base Nucleic Acid Base CompositionComposition

Only if two microorganisms also are alike Only if two microorganisms also are alike phenotypically does their similar G + C phenotypically does their similar G + C content suggest close relatedness.content suggest close relatedness.

G+C contents of microorganismsG+C contents of microorganismsOrganism % G + C

Actinomyces 59–73

Bacillus 32–62

Clostridium 21–54

Escherichia 48–52

Micrococcus 64–75

Neisseria 47–54

Proteus 38-41

Pseudomonas 58-70

Salmonella 50-53

Staphylococcus 30-38

Aspergillus niger 52

Nucleic Acid Hybridization

By mixing ssDNA from two different species By mixing ssDNA from two different species and determining the percentage of the DNA and determining the percentage of the DNA that can form dsDNA hybridsthat can form dsDNA hybrids

The greater the percent hybridization, the The greater the percent hybridization, the closer the speciescloser the species

Nucleic Acid Hybridization Heat mixture of dsDNA.Heat mixture of dsDNA. cool and hold at a temperature cool and hold at a temperature

about 25°C.about 25°C. The base sequences will The base sequences will

reassociate to form stable reassociate to form stable dsDNA. dsDNA.

Whereas noncomplementary Whereas noncomplementary strands will remain single strands will remain single ..

IIncubation of the mixture at 30 ncubation of the mixture at 30 to 50°C to 50°C will allow hybrids of will allow hybrids of more diverse more diverse ssDNAs to form, ssDNAs to form, at 10 to 15°C at 10 to 15°C permits permits hybrid hybrid formation only with almost formation only with almost identical strands.identical strands.

Nucleic Acid Hybridization Nitrocellulose filters with bound nonradioactive Nitrocellulose filters with bound nonradioactive

DNA strands are incubated with single-DNA strands are incubated with single-stranded DNA fragments made radioactive stranded DNA fragments made radioactive with 32P, 3H, or 14C. with 32P, 3H, or 14C.

Radioactive hybridize with the membrane-Radioactive hybridize with the membrane-bound ss-DNA.bound ss-DNA.

Wash to remove any nonhybridized ssDNA Wash to remove any nonhybridized ssDNA Measure the radioactivity. Measure the radioactivity. The quantity of radioactivity bound to the filter The quantity of radioactivity bound to the filter

reflects the amount of hybridization and thus reflects the amount of hybridization and thus the similarity of the DNA sequences. the similarity of the DNA sequences.

Nucleic Acid Hybridization The degree of similarity is expressed as the % The degree of similarity is expressed as the %

DNA radioactivity retained on the filter DNA radioactivity retained on the filter compared with the % of homologous DNA compared with the % of homologous DNA radioactivity bound under the same conditionsradioactivity bound under the same conditions

Two strains whose DNAs show 70% Two strains whose DNAs show 70% relatedness and less than a 5% difference in relatedness and less than a 5% difference in Tm are considered members of the same Tm are considered members of the same species.species.

Very different DNA molecules will not form a Very different DNA molecules will not form a stable, detectable hybrid. stable, detectable hybrid.

More distantly related organisms are compared More distantly related organisms are compared by carrying out DNA-RNA hybridization.by carrying out DNA-RNA hybridization.

DNA Chip technology Quick method to detect a pathogenic Quick method to detect a pathogenic

microorganisms in a host by identifying a microorganisms in a host by identifying a gene that is unique to that pathogen.gene that is unique to that pathogen.

Protocol:Protocol: DNA chip is made up of DNA Probes.DNA chip is made up of DNA Probes. A DNA sample should be collected from A DNA sample should be collected from

unknown organism is labelled with a unknown organism is labelled with a fluorescent dye and added to the chip.fluorescent dye and added to the chip.

Hybbridization between probe DNA and Hybbridization between probe DNA and sample DNA is detected by fluorescence.sample DNA is detected by fluorescence.

DNA chip Technology

Nucleic Acid Sequencing Genome structures directly compared only by Genome structures directly compared only by

sequencing DNA and RNA.sequencing DNA and RNA. The 5S and 16S rRNAs isolated from the 50S The 5S and 16S rRNAs isolated from the 50S

and 30S subunits, respectively.and 30S subunits, respectively. Purified, radioactive 16S rRNA is treated with Purified, radioactive 16S rRNA is treated with

the enzyme T1 ribonuclease. the enzyme T1 ribonuclease. The fragments are separated and sequenced. The fragments are separated and sequenced. 16S rRNA fragments from different 16S rRNA fragments from different

procaryotes are aligned and compared using a procaryotes are aligned and compared using a computer, and association coefficients (computer, and association coefficients (Sab Sab values) are calculated. values) are calculated.

Nucleic Acid Sequencing

Complete Complete rRNAs sequencingrRNAs sequencing First, RNA is isolated and purified. First, RNA is isolated and purified. Then, revers transcriptase is used to make Then, revers transcriptase is used to make

cDNA. cDNA. Next, the PCR amplifies the cDNA. Next, the PCR amplifies the cDNA. Finally, the cDNA is sequenced and the Finally, the cDNA is sequenced and the

rRNA sequence deduced from the results.rRNA sequence deduced from the results.

Numerical TaxonomyNumerical Taxonomy Peter H. A. Sneath and Robert Sokal have defined Peter H. A. Sneath and Robert Sokal have defined

Numerical taxonomy: “the grouping by numerical Numerical taxonomy: “the grouping by numerical methods on the basis of their character states.” methods on the basis of their character states.”

Information about organisms is converted into Information about organisms is converted into numerical form and compared by means of a numerical form and compared by means of a computer. computer.

Each characteristics given equal weight.Each characteristics given equal weight. At least 50 and preferably several hundred At least 50 and preferably several hundred

characteristics compared. characteristics compared. Determine the presence or absence of selected Determine the presence or absence of selected

characters in the group of organisms.characters in the group of organisms. Calculate simple matching coefficient (Calculate simple matching coefficient (SSM),SSM),

Jaccard coefficient.Jaccard coefficient.

Numerical TaxonomyNumerical Taxonomy

Numerical Taxonomy The simple matching coefficients, or other The simple matching coefficients, or other

association coefficients are then arranged to association coefficients are then arranged to form a similarity matrix.form a similarity matrix.

The results of numerical taxonomic analysis The results of numerical taxonomic analysis are often summarized with a treelike are often summarized with a treelike diagram called a dendrogram.diagram called a dendrogram.

Organisms with great similarity are grouped Organisms with great similarity are grouped together and separated from dissimilar together and separated from dissimilar organisms organisms such groups of organisms are such groups of organisms are called called phenons.phenons.

Phenons formed at about 80% similarity Phenons formed at about 80% similarity often are equivalent to species.often are equivalent to species.

Numerical Taxonomy

Clustering and Dendrograms in Numerical Taxonomy. (a) A small similarity matrix that compares six strains of bacteria. The degree of similarity ranges from none (0.0) to complete similarity (1.0). (b) The bacteria have been rearranged and joined to form clusters of similar strains. For example, strains 1 and 2 are the most similar. The cluster of 1 plus 2 is fairly similar to strain 3, but not at all to strain 4. (c) A dendrogram showing the results of the analysis in part b. Strains 1 and 2 are members of a 90-phenon, and strains 1–3 form an 80-phenon. While strains 1–3 may be members of a single species, it is quite unlikely that strains 4–6 belong to the same species as 1–3.