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Techniques of bacterial taxonomy

China General Microbiological Culture Collection Center (CGMCC)

Institute of Microbiology

Chinese Academy of Sciences

Yuhua Xin

Hug et al. present a new view of the tree of life, revealing the existence of two extraordinarily

diverse and poorly characterized prokaryotic lineages: CPR bacteria (blue) and DPANN archaea

(purple).

• Microbiol taxonomy is a science of study and grouping of microorganisms.

• Bacterial Taxonomy concludes three separate but interrelated areas

– Classification

• Arrangement of organisms into groups (taxa) on the basis of similarities or relationships.

– Identification

• Process of characterizing organisms to determine an isolate as a member of an established taxon or a previously unidentified species.

– Nomenclature

• Assignment of a specific name according to international rules (International Code of Nomenclature of Bacteria[Sneath,1992]).

Bacterial Taxonomy

• Bacterial taxonomy incorporates multiple methods for

identification and description of new species

• The polyphasic approach to taxonomy uses three

methods

1) Phenotypic analysis

2) Genotypic analysis

3) Phylogenetic analysis

Bacterial Taxonomy

– Phenotypic characteristics• Morphological data• physiological and biochemical data• Chemotaxonomic characteristics

Fatty acid analysis The patterns of polar lipids present in the membrans Composition of cell wall

– Genotypic characteristics• DNA-DNA Hybridization • the guanine (G)+ cytosine (C) content (% GC). • Multilocus Sequence Typing (MLST)• DNA profiling

– Phylogenetic Analysis• 16S rRNA gene sequence analysis• Multi-gene sequence analysis• Whole-genome sequence analysis

Polyphasic Taxonomy

• No universally accepted concept of species for

prokaryotes

• Current definition of prokaryotic species

– Collection of strains sharing a high degree of

similarity in several independent traits

• Most important traits include： 70% or greater DNA-DNA

hybridization and 98.5 % or greater 16S rRNA gene

sequence identity

The Species Concept in Microbiology

Some Phenotypic Characteristics of Taxonomic Value

Some Phenotypic Characteristics of Taxonomic Value

Other Chemical characterization： Peptidoglycan, Polyamines, techoic acids,

mycolic acids, Lipopolysaccharides

Morphology

Phenotypic characteristics

Microscopic morphology

Cell morphology: rod, coccus, or spirillum

Cell arrangement: diplococcus, streptococcus,

tetrad, sarcina, irregular clusters (Micrococcus or

staphycoccus)

Special cell structures: flagellum, cilia, spore, capsule

cocci

• Morphology

• Morphological criteria

• Cell shape and size – supported by photographs

• Characteristic features (eg. stalks, prosthecae, budding or

branching, cell aggregates )

• Spore formation

• Location of flagella

• Motility (form, speed)

• Intracellular structures

• Colony shape and size

• Cellular pigments

5.0 μm

5.0 μm

5.0 μm

500 nm 200 nm

Scanning electron micrograph

Colony morphology :

Colonies can exhibit macroscopic

differences

colour, size,

shape, margin or edge,

surface feature etc.

Slant culture morphology

• Morphology

Staining

• Gram stain (the reaction may alter as the cells age)

• Acid-fast staining (strains containing mycolic acids)

• Sudan Black staining (stains containing lipophilic cellular

inclusions, eg. polyhydroxybutyric acid)

• Others (eg. spore staining, capsule staining)

physiological and biochemical data

Phenotypic characteristics

• Physiology and biochemistry

• The growth tolerance (eg. pH, temperature, NaCl

concentration)

• Enzyme activity, substrate utilization, antibiotics resistance,

etc. Fast methods: API and Biology test plates.

Note:

• To test with identical media and conditions or at least

comparable.

• To compare with type strain of type species of appropriate

genera.

• To analyze including strains of the most closely related taxa

rather than using the previously published data.

Nitrate Reduction Carbohydrate Fermentation Urease detection

Traditional methods

VP test MR test

Traditional methods

Nitrate reduction Citrate utilization Indole prudution

the ability of a microorganism to

withstand the effects of an antibiotic

on agar plates (Whether bacteria are

susceptible, intermediate, or resistant

depends on the amount of antibiotic

and the diameter of zone of inhibition).

Antibiotic sensitivity

Serological analysis

• Proteins and polysaccharides of some bacteria can function as

identifying markers

– Generally molecules on surface structures

• e.g., Cell wall, glycocalyx, flagella, pili

– Detection is based upon the

specific interaction between

antibodies and these

antigens

• e.g., Rapid detection of

Streptococcus pyogenes

How to perform and interpret the miniaturized,

multi-test technique for bacterial identification?

Disadvantages of traditional methods

Need experience

Complicated process

Labor-consuming

Time-consuming

Rapid Tests

• Commercial modifications of traditional biochemical tests

• APITM system

• Biolog Microbial ID System

• The API identification system is numerical taxonomy according to

the microbial physiological and biochemical characteristics.

• The API tests (kit) can identify a wide range of microorganisms.

• Have standardized and extensive databases of characteristic

biochemical reactions of microorganisms.

commercial products for bacteria identification

The API identification system is numerical taxonomy according to the microbial physiological and biochemical characteristics.

• API 50 CH – Performance of carbohydrate

metabolism tests

• API ZYM® – Semiquantification of enzymatic

activities

API 20E – 11 biochemical tests and enzymatic activities, 9 Fermentation/Oxidation

…….

15

eg. API 20E

Isolate Prepare Incubate Read

suspension

reaction strip

reagents

incubation chamber

Positive Negative

Bacteria, Yeast and Fungi Identification

The Biolog Microbial ID System can rapidly identify over 2,500 species of aerobic and anaerobic bacteria, yeasts and fungi.

Tetrazolium redox dyes are used to colorimetrically indicate utilization of the

carbon sources or resistance to inhibitory chemicals.

1. Isolate pure culture on agar media

2. Prepare inoculum at specified cell density

3. Inoculate the Biolog MicroPlate

4. Incubate the plate, observe and enter the reaction

pattern to obtain ID result

simple, straightforward procedure

• Commercial systems are very accurate for the more

common species and provide quick test results in a cost-

effective manner.

• The MicroStation System has extensive applications also

for microbial community analysis in soil, water and other

environments.

Cornerstone of microbial taxonomy

• Bacterial identification

• Microbial ecology

• Evolution

• Cultivate more unknown bacteria

The importance of growth phenotypes

• With the publication of the first edition of the Bergey's Manual of Determinative Bacteriology in 1923, microbiologists began to systematically describe and define bacterial species based on lists of phenotypes, primarily growth related.

• 1926, L.E. den Dooren de Jong showed that bacteria could be

readily distinguished by growth assays on agar media with

several hundred C- and N-sources.

The importance of growth phenotypes

1. As the number of newly described taxa increased substantially, a problem with

commercial systems is the construction of databases

Challenges in phenotypic identification

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2. Phenotypic properties do not accurately reflect the entire extent of the

genomic complexity of a given species

3. Phenotypic properties can be unstable at times and expression can be

dependent upon changes in environmental conditions, e.g., growth

substrate, temperature, and pH levels

4. Can be used only for organisms that can be cultivated in vitro

M.J. Janda & S.L. (2002), J. Clin. Microbiol.

Challenges in phenotypic identification

Chemical characterization

Phenotypic characteristics

Fatty Acid Analyses

– Relies on variation in type and

proportion of fatty acids present in

membrane lipids for specific prokaryotic

groups

– Requires rigid standardization because

FAME profiles can vary as a function of

temperature, growth phase, and growth

medium

MIDI Sherlock® Microbial Identification System

Procedure of fatty acids analysis

Cultivation of bacteria

The growth temperature and growth media effect the fatty acids compositions,

culture conditions must be standardized for all strains and strains were collected at the same logarithm growth period, when comparing the fatty acid composition within a group of bacteria

Preparation of fatty-acid methyl esters (FAMEs)

Fatty acids Saponification /Methylation FAMES

Identification of fatty acids

GC analysis and identified by MIDI system

Respiratory quinones

• Respiratory quinones:a group of non protein, lipid-soluble electron carriers in the respiratory electron-transport system.

• Function:promote the transfer of electrons between the proteins of the electron-transport chain.

• Distribution:in both anaerobic and aerobic organisms within the Bacteria and Archaea.

• Type:divided into two basic structural classes, benzoquinones and naphthoquinones .

Fig. 1. Nature Reviews Molecular Cell Biology 2002,3, 836-847

Ubiquinone (Q-n)

Plastoquinone

Rhodoquinone (RQ)

n

Sulfolobusquinone

Caldariellaquinone

Epoxyubiquinone

Menaquinone (MK-n)

Demethylmenaquinone (DMK) (DMMK-)

Thermoaquinone

Chlorobiumquinone (CK )

Methionaquinone

Partially hydrogenated menaquinone (MK-n(Hm))

Benzoquinones Naphthoquinones

Menaquinone: MK-n

--- Most Gram-positive bacteria and anaerobic Gram-negative bacteria,

--- Archeae: dementhylmenaquinone (DMK),

--- Actinomycetes: partially hydrogenated MK,

--- A variable number of isoprenoid residues: MK-5-15.

Ubiquinone ( Coenzyme Q, Co Q or Q )

---Ubiquinones: aerobic Gram-negative, α-, β-, γ- group of Proteobacteria＆rodshaped acetic acid bacteria;

---Rhodoquinone (RQ): photosynthetic bacteria,

---the number of isoprenoid units in side-chain arevariable (Q-7-Q-14).

Ubiquinone and Menaquinone

Ubiquinone and menaquinone are abbreviated as Q-n and MK-n(Hm);

n is the number of isoprene units and m is the number of hydrogen atoms substituting unsaturated bond.

Ubiquinone: Q-n Menaquinone: MK-n(Hm)

Respiratory quinones profiles of bacteria

Taxon Main Quinone System

Proteobacteria

α-SubcalssAgrobacteriumRhodomicrobium vannieliiRhodopseudomonas acidophila

Q-10Q-10+RQ-10Q-10+RQ-10+MK-10

β- SubclassAlcaligenesBrachymonas, ZoogloeaRhodocyclus, Rubrivivax

Q-8Q-8+RQ-8Q-8+ MK-8

γ- SubclassAcinetobacter, PseudomonasAzotobacterChromatiaceaeEctothiorhodospiraceaeEnterobacteriaceaeVibrio

Q-9Q-8,Q-8+MK-8Q-7+MK-7, Q-8+ MK-8Q-8+MK-8 +DMK-8Q-8+MK-8( +DMK-8)

δ- SubclassDesulfobulbusDesulfococcusDesulfovibrio

MK-5(H2)MK-7MK-6, MK-6(H2)

Taxon Gram (+) bacteria Main Quinone system

Low G+C content groupBacillusEnterococcus

MK-7MK-8 , DMK-9

High G+C content groupArthrobacterAureobacteriumCorynebacteriumKribbellaStreptomyces

MK-9(H2)MK-11+MK12MK-8(H2), MK-9(H2)MK-9(H4)MK-9(H6), MK-9(H8)

CyanobacteriaNostoc PQ-9+K1

Spirosoma group Spirosoma MK-7

Bacteroides/Flavobacteria group BacteroidesCapnocytophagaFlavobacterium/CytophagaSphingobacterium

MK-10+MK-11MK-6MK-6+MK-7MK-7

OthersChlorobiumChloroflexusDeinobacter

MK-7+CKMK-10+MK-8MK-8

Respiratory quinones profiles of the bacteria

Identification of quinones (HPLC)

Polar lipids analysis

There is a vast diversity of polar lipids now known to be present in prokaryotes.

Phospholipids form an essential component of the cell membrane.

Be related to permeability of the membrane and regulation at the membrane.

bacause they possess not only a hydrophobic region but also a hydrophilic region on the molecule. They show a distinctive amphipathic characteristics.

The polar lipids known to occur in bacteria:

---phospholipids,

---glycolipids,

---glycophospholipids,

---aminophospholipids,

---amino acid derived lipids,

---capnines,

---sphingolipids,

---sulfur-containing lipids. Structure of PC (phosphatidylcholine )

The mainly kinds of polar lipids in bacterial cell

Phospholipids: PC (phosphatidylcholine),

PE (phosphatidylethanolamine),

PI (phosphatidylinositol),

PG (phosphatidyglycerol),

PS (phosphatidylserine),

PME (phosphatidylmethylethanolamine),

PIMs (phosphatidylinositol mannosides),

DPG (diphosphatidylglycerol),

PB (phosphatidylbutanediol),

PA (phosphatidic acid, phosphatidate)

Glycolipids:

PI (phosphatidylinositol),

PIMs (phosphatidylinositol mannosides) etc.

Aminolipids ( free amino groups):

PE (phosphatidylethanolamine),

PS (phosphatidylserine),

PME (phosphatidylmethylethanoamine),

PE (phosphatidylethanolamino)

PI (phosphatidylinositol)

Fatty acids

Glycerol backbone

Inositolhead group

（Phosphatidyl ethanolamine, PE） （ Phosphatidyl methy ethanolamine, PME）

（Phosphatidyl choline, PC） （Phosphatidyl glycerol, PG）

（Phospholipids of unknown structure containing glucosamine,

GluNu）

polar lipids for taxonomy （Lechevallier, 1980）

Common polar lipids appeared in bacteria

α-, β-, γ- group of Proteobacteria: generally posses three major phospholipids: PG, PE and DPG.

Firmicutes and Actinobacteria: complex mixtures of polar lipids.

Bacillus, Rhodococcus, Nocardia, Mycobacterium, Planococcus and Sporosarcina contain PE.

Corynebacterium, Micrococcus and Staphlococcus do not contain PE.

• Peptidoglycan Types

• Amino acid

– Diamino acid

meso-DAP, LL-DAP, Lysine, Ornithine, OH-lysine, OH-ornithine, OH-DAP, DAB, Lanthionine, Diaminopimelic acid

– Composition

– Sequence

• Acyl type

• Cell wall sugar (family, genus, species)

arabinose, galactose, xylose, madurose(3-O-methyl-D-

galactose) etc. Actinomycetes in the 3-O-methylrhamnose,

2-O-methylrhamnose, etc.

Bacterial Cell Wall

IJSEM, 2002, 52(3): 1049-1070

References:

1. Schleifer, K. H. and Kandler, O. (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Reviews. 36, 407-477 .

2. Minikin, D. E. et al. (1984 ) An intergrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids J. Microbiol Methods. 2, 233-241.

3. Tindall, B. J., Rossello-Mora, R., Busse, H,-J., Ludwig, W. and Kampfer, P. (2010) Note on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60, 249-266.

4. Komagta, K. and Suzuki K.-I. (1987) Lipid and cell-wall analysis in bacterial systematics Methods Miceobiol 19, 161-203.

5. Michael Goodfellow and Anthony G. O’ Donnell Chemical Methods in Prokaryotic Systematics.

Genetic-based characterization

• Several methods of genotypic analysis are available and

used

• DNA-DNA Hybridization

• the guanine (G)+ cytosine (C) content (% GC).

• Multilocus Sequence Typing (MLST)

• DNA profiling

Genotypic Methods

• Genomes of two organisms are hybridized to

examine proportion of similarities in their gene

sequences

– Provides rough index of similarity between two

organisms

– Useful complement to SSU rRNA gene sequencing

– Useful for differentiating very similar organisms

– Hybridization values 70% or higher suggest strains

belong to the same species

DNA-DNA hybridization

• G+C content- percentage of Guanine (G) and Cytosine (C) base

pairs in the genome;

• One of the required characteristics of the minimum list of data for a description of a new species;

– Vary between 20 and 75% among Bacteria and Archaea;

– Generally accepted that if GC content of two strains differ by ~ 5% they are unlikely to be closely related.

G+C content

• paper chromatography method

• thermal denaturation temperature method

• HPLC method

Methods

Reference

• Marmur, J. & Doty, P. (1962). Determination of the base composition of

deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5,

109-118.

• Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise

measurement of the G+C content of deoxyribonucleic acid by high-performance

liquid chromatography. Int J Syst Bacteriol 39, 159-167.

• De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of

DNA hybridization from renaturation rates. Eur J Biochem 12, 133-142.

• DNA Profiling

– Several methods can be used to generate DNA

fragment patterns for analysis of genotypic similarity

among strains, including

• Ribotyping

• RFLP, AFLP

• AP-PCR, RAPD

• ARDRA, rep-PCR

– Method in which several different

“housekeeping genes” from an

organism are sequenced (~450-bp)

– Has sufficient resolving power to

distinguish between very closely

related strains

Multilocus Sequence Typing (MLST)

“Nothing in Biology makes sense except in the light of evolution” (T. Dobzhansky, 1900-1975)

“Nothing in evolution makes sense except in the light of phylogenetics” (many phylogenetists)

Phylogenetics

Phylogenetic Analysis

• 16S rRNA gene sequence analysis

• Multi-gene sequence analysis

• Whole-genome sequence analysis

Arcticibacter Hh36T(JX949238)

Arcticibacter svalbardensis MN12-7T (AQPN01000042)

Pedobacter tournemirensis TF5-37.2-LB10T (GU198945)

Pedobacter xinjiangensis 12157T (EU734803)

Pedobacter zeaxanthinifaciens TDMA-5T (AB264126)

Pedobacter lentus DS-40T (EF446146)

Pedobacter daechungensis Dae 13T (AB267722)

Pedobacter terricola DS-45T (EF446147)

Pedobacter koreensis WPCB189T (DQ092871)

Pedobacter insulae DS-39T (EF100697)

Pedobacter boryungensis BR-9T (HM640986)

Pedobacter westerhofensis WB3.3-22T (AM491369)

Pedobacter africanus DSM 12126T (AJ438171)

Pedobacter duraquae WB2.1-25T (AM491368)

Pedobacter caeni LMG 22862T (AJ786798)

Pedobacter steynii WB2.3-45T (AM491372)

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16S rRNA gene sequence analysis

• The most widely used molecular clocks are small

subunit ribosomal RNA (SSU rRNA) genes

– Found in all domains of life

• 16S rRNA in prokaryotes and 18S rRNA in eukaryotes

– Functionally constant

– Sufficiently conserved (change slowly)

– Sufficient length

• Carl Woese

– Pioneered the use of SSU rRNA for

phylogenetic studies in 1970s

– Established the presence of three

domains of life:

• Bacteria, Archaea, and Eukarya

– Provided a unified phylogenetic

framework for Bacteria

• 16S rRNA gene sequences are useful in taxonomy; serve

as “gold standard” for the identification and description of

new species

– Proposed that a bacterium should be considered a new

species if its 16S rRNA gene sequence differs by more than

3% from any named strain, and a new genus if it differs by

more than 5%

– Less than 98.5% 16S similarity indicates different species, but

greater than 98.5% does not indicate the same species.

Phylogenetic Analysis——16S rRNA gene

• Phenetic Methods—

Distance based

– UPGMA

– Minimum Evolution

– Neighbor Joining

– Bayesian analyses

Phylogenetic Analysis——Tree Building methods

• Cladistic Methods—

Character Based

– Maximum likelihood

– Maximum Parsimony

Phylogenetic Analysis——Tree Building methods

RAxML

EzTaxon-e

Phylogenetic Analysis——Tree Building methods

MLSA is a method for the genotypic characterization of a diverse group of prokaryotes by comparing

sequences of multiple housekeeping genes. Multiple genes provide more informative nucleotide sites

and buffers against the distorting effects of recombination of one of the loci. The best approach is to

concatenate the sequences of at least 12 genes from a set of strains and to use the concatenated

sequences to reconstruct a phylogenetic tree which can identify deeply branching clusters and help to

delineate genotypic clustering within a genus or species.

Multilocus sequence analysis——MLSA

Schleifer, Karl Heinz. Syst Appl Microbiol 32.8 (2009): 533-542.

Multilocus sequence analysis——MLSA

Maiden, et al., Nat Rev Micro. 2013, 11(10): 728-736.

• Whole-genome sequence analyses are becoming

more common

— ANI (average nucleotide identity) has been demonstrated to

correlate with DDH, where the range of ~95–96% similarity

may reflect the current boundary of 70% DDH similarity (Goris

et al., 2007). ANI may substitute for DDH analyses in the near

future.

Whole-genome sequence analyses

Richter M & Rosselló-Móra R. Proc Natl Acad Sci U S A, 2009, 106(45): 19126-19131.

– Nomenclature

• Assignment of a specific name according to international rules (International Code of Nomenclature of Bacteria[Sneath,1992]).

Nomenclature

http://www.bacterio.net/

• Major references in bacterial diversity

– Bergey’s Manual of Systematic Bacteriology (Springer)

Bergey’s Manual of Determinative Bacteriology

Bergey’s Manual of Systematic Bacteriology

– The Prokaryotes (Springer)

Taxonomy References

• NCBI Taxonomyhttp://www.ncbi.nlm.nih.gov/Taxonomy/

• TOBAhttp://www.taxonomicoutline.org/

• Bergey’s Taxonomyhttp://www.bergeys.org/outlines.html

• List of Prokaryotic Names with Standing in Nomenclaturehttp://www.bacterio.cict.fr/index.html

• Bacterial Nomenclature Up-to-Date http://www.dsmz.de/microorganisms/bacterial_nomenclature.php

• The International Code of Nomenclature of Prokaryotes: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=icnb

• EzTaxon-e Database

http://eztaxon-e.ezbiocloud.net/

Taxonomy References

Some National Microbial Culture Collections