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Chapter 10:
ID & Classification
of Prokaryotes
Important Point:
Principles of Taxonomy
 “Taxonomy is the science that studies organisms
in order to arrange them into groups; those
organisms with similar properties are grouped
together and separated from those that are
different.”
 “Taxonomy can be viewed as three separate but
interrelated areas:
 Identification--the process of characterizing
organisms
 Classification--the process of arranging
organisms into similar or related groups,
primarily to provide easy identification and
study
 Nomenclature--the system of assigning
names to organisms.”
 We’ll consider each in turn.
Taxonomy: Identification
 We’ll spend quite a bit of time in this chapter and in
lab on methods of bacterial identification.
 In practical terms, bacterial identification usually is
done for reasons of health, either in the course of
treating infectious disease or to prevent disease
 e.g., to identify fecally contaminated water.
 e.g., to identify organisms associated with food
spoilage or contamination.
 e.g., to identify pathogens in the clinical
microbiology laboratory.
 It is important to keep in mind that the appropriate
tests to employ will depend on the organisms likely
present plus time, skills of the tester, and budgetary
concerns.
 In the clinic patient symptoms help to define what
organisms of interest are likely present.
Taxonomy: Classification
 The goal of classification is to determine genetic
similarity, though in the past genetic similarity had to
be inferred based on phenotypic similarity.
 As a consequence of the sequencing of organism
genomes, the science of classification is very much in
flux (classifications are changing often) as
phenotype-based classification is replaced by
genotype-based classification.
 All is not happy even with genotype-based
classification, however, since horizontal gene transfer
greatly complicates estimations of genetic similarity,
with different parts of genomes displaying different
levels of genetic similarity between organisms.
 Note that though classification can be considered
strictly in terms of genetic similarity, traditionally in
genetics similarity is considered to be an indication of
“similarity by decent,” a.k.a., of blood relatedness,
a.k.a., of evolutionary relatedness.
Taxonomic Rank
Strain
O157:H7
One difficulty is deciding how different two isolates must be before one
describes them as different species rather than as different strains.
Taxonomic Rank
Note that not all groupings of microorganisms
have taxonomic meaning: e.g., lactic acid
bacteria, endospore-formers, or enteric
pathogens. These are examples of classifying
“mistakes” due to phenotypic similarity that
results from convergent evolution.
Strain
O157:H7
One difficulty is deciding how different two isolates must be before one
describes them as different species rather than as different strains.
Taxonomy: Nomenclature
Nomenclature: “Bacterial
names may originate from
any language, but they
must be given a Latin
suffix.” (i.e., they must be
Latinized).
Strain
O157:H7
Recall Binomial Nomenclature.
Three-Domain Classification
Line lengths are based on genetic
distances, i.e., longer lines reflect
greater differences.
Three-Domain Classification
Note that majority of genetic diversity is among singlecelled organisms
Methods of Identification
Note existence of other methods of visualization beyond Gram stain...
Note Phenotype- vs. Genotypebased classification.
Phenotypic Characteristics
 Microscopy: Gram stain, acid-fast stain, cell
morphology, cell arrangements.
 Colony morphology: size, color, border shape, etc.
 Growth on Differential media: blood agar, MacConkey
agar, etc.
 Growth on Selective/Differential media: MacConkey
agar, EMB agar, etc.
 Biochemical tests: “Most biochemical tests rely on a
pH indicator or chemical reaction that results in a
color change [or gas production] when a [specific]
compound is degraded.”
 Keep in mind that to do well in lab it is important to gain
some reasonable understanding of how the various
biochemical tests work.
 Note that “because each test often requires an
incubation period… it [is] too time-consuming to
proceed one [phenotypic characterization] step at a
time.”
Gas Generation: Catalase Test
Durham Tube: Gas Production
Color Change: Urease Test
Commercial Biochemical Test Kits
Dichotomous Key
Don’t worry about the
specific details of this
key but do keep in
mind how it is
organized.
Typing of Strains
Biovar, Biotype (syn)
Serovar, Serotype (syn)
Typing via RFLP analysis
Ribotype (RFLP of rRNA)
Also Phage Type
Restriction Fragment Length
Polymorphism (RFLP) Analysis
1 2 3 4 5
Genomic DNA is
isolated, cut using
specific restriction
enzymes, run out on a
gel, and then Southern
Blotted with specific
DNA probes.
Can you tell me which
lanes contain bacteria
that are clonally
related?
What is an RFLP? (fig. 9.9)
What is an RFLP? (fig. 9.9)
Different strains have
recognition sites in
different places due to
genotypic differences.
Note resulting
differences in
restriction fragment
length.
Southern Blotting (Fig. 9.8)
RFLP generation.
Gels separate DNA
by size.
This is a post-probe
detection blot.
Ribotyping
Clonally related
strains should have
same RFLP type (i.e.,
same band pattern).
These are two strains
that have been
differentiated based
on RFLP analysis.
Why care about clonal relatedness? Is a contamination
problem due to fault in your aseptic technique? Or is there
just a particularly invasive bug making the rounds?
Phages are
viruses of
bacteria.
Phage Typing
This is a “Soft-Agar
Overlay.”
The clear
spots are due
to bacteria
killing (lysis).
“Phage” = “Bacteriophage”
The disks are
impregnated
with specific
antibiotics.
Antibiogram
The streaks
make up a
bacterial
“lawn”.
These two strains have different
antibiotic-resistance patterns.
Numerical Taxonomy
 Numerical Taxonomy is a method of classification
that pools phenotypic characteristics.
 The key to numerical taxonomy is that no one trait
(e.g., ability to ferment glucose) is elevated in
importance above any other trait (e.g., Gram
staining characteristics).
 Instead, all traits are weighted equally.
 Classification in numerical taxonomy is expressed
in terms of a Similarity Coefficient found between
two compared strains. It is equal to:
 [# pos both strains]/[# pos one or both strains]
 by “pos” I mean that the characteristic is
present, e.g., ferments glucose or has pili, etc.
 Greater similarity coefficient, closer relatedness is
inferred. Greater than ~70% and inference is that
two compared bacteria are of the same species.
Numerical Taxonomy
Similarity Coefficients
Rearranged into two distinct species...
G-C Content (DNA Base Ratio)
dsDNA is
distinguished
from ssDNA
based on UV
absorbance.
Different
species have
different DNA
melting temps.
G-C Content (DNA Base Ratio)
Helix has higher
stability with
greater fraction GC vs. A-T pairing.
Higher melting
points therefore
correspond to
greater G-C content
DNA-DNA Hybridization
DNA-DNA Hybridization
Similarity is in terms of
nucleotide sequence between
the two compared organisms.
16S rRNA Sequence Comparison
16S rRNA
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