Techniques of bacterial taxonomy
Yuhua Xin
China General Microbiological Culture Collection Center (CGMCC)
Institute of Microbiology
Chinese Academy of Sciences
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).
Bacterial Taxonomy
• 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
Polyphasic 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
The Species Concept in Microbiology
• 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
Some Phenotypic Characteristics of Taxonomic Value
Some Phenotypic Characteristics of Taxonomic Value
Other Chemical characterization： Peptidoglycan, Polyamines, techoic acids,
mycolic acids, Lipopolysaccharides
Phenotypic characteristics
Morphology
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
Scanning electron micrograph
• Cell shape and size – supported by photographs
• Characteristic features (eg. stalks, prosthecae, budding or
branching, cell aggregates )
• Spore formation
5.0 μm
• Location of flagella
5.0 μm
• Motility (form, speed)
• Intracellular structures
• Colony shape and size
• Cellular pigments
500 nm
5.0 μm
200 nm
 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)
Phenotypic characteristics
physiological and biochemical data
• 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.
Traditional methods
Nitrate Reduction
Carbohydrate Fermentation
VP test
MR test
Urease detection
Traditional methods
Nitrate reduction
Citrate utilization
Indole prudution
Antibiotic sensitivity
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).
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
 Disadvantages of traditional methods
 Need experience
 Complicated process
 Labor-consuming
 Time-consuming
How to perform and interpret the miniaturized,
multi-test technique for bacterial identification?
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

…….
suspension
reagents
incubation chamber
eg. API 20E
reaction strip
Isolate
Prepare
Positive
Read
Incubate
Negative
15
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.
simple, straightforward procedure
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
• Commercial systems are very accurate for the more
common species and provide quick test results in a costeffective manner.
• The MicroStation System has extensive applications also
for microbial community analysis in soil, water and other
environments.
The importance of growth phenotypes
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.
Challenges in phenotypic identification
1. As the number of newly described taxa increased substantially, a problem with
commercial systems is the construction of databases
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Species validly published
600
Challenges in phenotypic identification
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.
Phenotypic characteristics
Chemical characterization
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
Fig. 1. Nature Reviews Molecular Cell Biology 2002,3, 836-847
• 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 .
Benzoquinones
Ubiquinone (Q-n)
Naphthoquinones
Menaquinone (MK-n)
n
Plastoquinone
Demethylmenaquinone (DMK)
(DMMK-)
Rhodoquinone (RQ)
Thermoaquinone
Caldariellaquinone
Chlorobiumquinone (CK )
Sulfolobusquinone
Epoxyubiquinone
Methionaquinone
Partially hydrogenated
menaquinone (MK-n(Hm))
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)
Ubiquinone ( Coenzyme Q, Co Q or Q )
Menaquinone: MK-n
---Ubiquinones: aerobic Gram-negative, α-, β-, γ- group
of Proteobacteria ＆rodshaped acetic acid bacteria;
--- Most Gram-positive bacteria and anaerobic Gramnegative bacteria,
---Rhodoquinone (RQ): photosynthetic bacteria,
--- Archeae: dementhylmenaquinone (DMK),
---the number of isoprenoid units in side-chain
arevariable (Q-7-Q-14).
--- Actinomycetes: partially hydrogenated MK,
--- A variable number of isoprenoid residues: MK-5-15.
Respiratory quinones profiles of bacteria
Taxon
Main Quinone System
Proteobacteria
α-Subcalss
Agrobacterium
Rhodomicrobium vannielii
Rhodopseudomonas acidophila
Q-10
Q-10+RQ-10
Q-10+RQ-10+MK-10
β- Subclass
Alcaligenes
Brachymonas, Zoogloea
Rhodocyclus, Rubrivivax
Q-8
Q-8+RQ-8
Q-8+ MK-8
γ- Subclass
Acinetobacter, Pseudomonas
Azotobacter
Chromatiaceae
Ectothiorhodospiraceae
Enterobacteriaceae
Vibrio
Q-9
Q-8,
Q-8+MK-8
Q-7+MK-7, Q-8+ MK-8
Q-8+MK-8 +DMK-8
Q-8+MK-8( +DMK-8)
δ- Subclass
Desulfobulbus
Desulfococcus
Desulfovibrio
MK-5(H2)
MK-7
MK-6, MK-6(H2)
Respiratory quinones profiles of the bacteria
Taxon Gram (+) bacteria
Main Quinone system
Low G+C content group
Bacillus
Enterococcus
MK-7
MK-8 , DMK-9
High G+C content group
Arthrobacter
Aureobacterium
Corynebacterium
Kribbella
Streptomyces
MK-9(H2)
MK-11+MK12
MK-8(H2), MK-9(H2)
MK-9(H4)
MK-9(H6), MK-9(H8)
Cyanobacteria
Nostoc
PQ-9+K1
Spirosoma group
Spirosoma
MK-7
Bacteroides/Flavobacteria group
Bacteroides
Capnocytophaga
Flavobacterium/Cytophaga
Sphingobacterium
MK-10+MK-11
MK-6
MK-6+MK-7
MK-7
Others
Chlorobium
Chloroflexus
Deinobacter
MK-7+CK
MK-10+MK-8
MK-8
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)
Fatty
acids
Glycerol
backbone
Inositol
head group
PI (phosphatidylinositol)
Glycolipids:
PI (phosphatidylinositol),
PIMs (phosphatidylinositol mannosides) etc.
Aminolipids ( free amino groups):
PE (phosphatidylethanolamine),
PS (phosphatidylserine),
PME (phosphatidylmethylethanoamine),
PE (phosphatidylethanolamino)
polar lipids for taxonomy
（Phosphatidyl ethanolamine, PE）
（Phosphatidyl choline, PC）
（Lechevallier, 1980）
（ Phosphatidyl methy ethanolamine, PME）
（Phosphatidyl glycerol, PG）
（Phospholipids of unknown
structure containing glucosamine,
GluNu）
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.
Bacterial Cell Wall
• 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.
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
DNA-DNA hybridization
•
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
G+C content
• 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.
Methods
• paper chromatography method
• thermal denaturation temperature method
• HPLC method
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
Multilocus Sequence Typing (MLST)
– 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
Phylogenetics
“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)
Phylogenetic Analysis
• 16S rRNA gene sequence analysis
• Multi-gene sequence analysis
• Whole-genome sequence analysis
Arcticibacter Hh36T(JX949238)
100
Arcticibacter svalbardensis MN12-7T (AQPN01000042)
87
Pedobacter tournemirensis TF5-37.2-LB10T (GU198945)
Pedobacter xinjiangensis 12157T (EU734803)
51
Pedobacter zeaxanthinifaciens TDMA-5T (AB264126)
Pedobacter lentus DS-40T (EF446146)
Pedobacter daechungensis Dae 13T (AB267722)
100
66
Pedobacter terricola DS-45T (EF446147)
Pedobacter koreensis WPCB189T (DQ092871)
Pedobacter insulae DS-39T (EF100697)
100
Pedobacter boryungensis BR-9T (HM640986)
Pedobacter westerhofensis WB3.3-22T (AM491369)
79
68
Pedobacter caeni LMG 22862T (AJ786798)
83
95
0.01
Pedobacter africanus DSM 12126T (AJ438171)
Pedobacter duraquae WB2.1-25T (AM491368)
Pedobacter steynii WB2.3-45T (AM491372)
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
Phylogenetic Analysis——16S rRNA gene
• 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——Tree Building methods
• Phenetic Methods—
Distance based
– UPGMA
– Minimum Evolution
– Neighbor Joining
– Bayesian analyses
• Cladistic Methods—
Character Based
– Maximum likelihood
– Maximum Parsimony
Phylogenetic Analysis——Tree Building methods
Phylogenetic Analysis——Tree Building methods
EzTaxon-e
RAxML
Multilocus sequence analysis——MLSA
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.
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
• 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.
Richter M & Rosselló-Móra R. Proc Natl Acad Sci U S A, 2009, 106(45): 19126-19131.
Nomenclature
– Nomenclature
• Assignment of a specific name according to
international rules (International Code of
Nomenclature of Bacteria[Sneath,1992]).
http://www.bacterio.net/
Taxonomy References
• 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 Taxonomy
http://www.ncbi.nlm.nih.gov/Taxonomy/
TOBA
http://www.taxonomicoutline.org/
Bergey’s Taxonomy
http://www.bergeys.org/outlines.html
List of Prokaryotic Names with Standing in Nomenclature
http://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/
Some National Microbial Culture Collections