Download Microbial Evolution and Diversity

PART
V
Microbial Evolution
and Diversity
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The objectives of this chapter are to:
N Provide information on how bacteria are named and what is
meant by a validly named species.
N Discuss the classification of Bacteria and Archaea and the recent
move toward an evolutionarily based, phylogenetic classification.
N Describe the ways in which the Bacteria and Archaea are identified
in the laboratory.
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17
Taxonomy of Bacteria and
Archaea
It’s just astounding to see how constant, how conserved, certain
sequence motifs—proteins, genes—have been over enormous
expanses of time. You can see sequence patterns that have persisted probably for over three billion years. That’s far longer than
mountain ranges last, than continents retain their shape.
—Carl Woese, 1997 (in Perry and Staley, Microbiology)
T
his part of the book discusses the variety of microorganisms
that exist on Earth and what is known about their characteristics and evolution. Most of the material pertains to the Bacteria
and Archaea because there is a special chapter dedicated to eukaryotic
microorganisms. Therefore, this first chapter discusses how the Bacteria and Archaea are named and classified and is followed by several
chapters (Chapters 18–22) that discuss the properties and diversity of
the Bacteria and Archaea.
When scientists encounter a large number of related items—such as
the chemical elements, plants, or animals—they characterize, name, and
organize them into groups. Thousands of species of plants, animals, and
bacteria have been named, and many more will be named in the future
as more are discovered. Not even the most brilliant biologist knows all
of the species. Organizing the species into groups of similar types aids
the scientist not only in remembering them but also in comparing them
to their closest relatives, some of which the scientist would know very
well. In addition, biologists are interested in evolution, because this is
the process through which organisms became diverse. Unraveling the
route of evolution leads to an understanding of how one species is related to another. As discussed in subsequent text of this chapter, evolutionary relationships are assessed by molecular phylogeny, the analysis
of gene and protein sequences to determine the relatedness among
organisms. To date, approximately 5,000 bacterial and archaeal species
have been named and, based on their characteristics, placed
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486
Chapter Seventeen
within the existing framework of other known species.
The branch of bacteriology that is responsible for characterizing and naming organisms and organizing them
into groups is called taxonomy or systematics.
Taxonomy can be separated into three major areas
of activity. One is nomenclature, which is the naming of
bacteria. The second is classification, which entails the
ordering of bacteria into groups based on common properties. In identification, the third area, an unknown bacterium, for example, from a clinical or soil sample, is
characterized to determine its species. This chapter covers all three of these areas.
17.1 Nomenclature
Bacteriologists throughout the world have agreed on a
set of rules for naming Bacteria and Archaea. These rules,
called the “International Code for the Nomenclature of
Bacteria” (1992), state what a scientist must do to describe a new species or other taxon (taxa, pl.), which is
a unit of classification, such as a species, genus, or family. Each bacterium is placed in a genus and given a
species name in the same manner as are plants and animals. For example, humans are Homo sapiens (genus
name first, followed by species), and a common intestinal bacterium is named Escherichia coli. This binomial
system of names follows that proposed for plants and
animals by the Swedish taxonomist Carl von Linné (Linnaeus; 1707–1778).
According to the rules of bacterial nomenclature, the
root for the name of a species or other taxon can be derived from any language, but it must be given a Latin ending so that the genus and species names agree in gender.
For example, consider the species name Staphylococcus
aureus. The first letter in the genus name is capitalized,
the species name is lowercase, and they are both italicized to indicate that they are Latinized. When writing
species names in longhand, as for a laboratory notebook,
they should be underlined to denote that they are italicized. The genus name Staphylococcus is derived from
the Greek Staphyl from staphyle, which means a “bunch
of grapes,” and coccus, from the Greek, meaning “a
berry.” The o (“oh”) between the two words is a joining
vowel used to connect two Greek words together. The
figurative meaning of the genus name is “a cluster of
cocci,” which describes the overall morphology of members of the genus. The species name aureus is from the
Latin and means “golden,” the pigmentation of members of this species. The -us ending of the genus and
species names is the Latin masculine ending for a noun
(Staphylococcus in this case) and its adjective (aureus).
Successively higher taxonomic categories are family, order, class, phylum, and domain (Table 17.1)
TABLE 17.1
Hierarchical classification
of the bacterium
Spirochaeta plicatilis
Taxon
Name
Domain
Phylum
Class
Order
Family
Genus
Species
Bacteria
Spirochaetes (vernacular name: spirochetes)
Spirochaetes
Spirochaetales
Spirochaetaceae
Spirochaeta
plicatilis
The International Journal of Systematic and Evolutionary
Microbiology (IJSEM) is a journal devoted to the taxonomy of bacteria that is published by the Society for General Microbiology. IJSEM publishes papers that describe
and name new bacterial taxa and contains an updated
listing of all new bacteria whose names have been
validly published. Thus, although bacterial species may
be described in other scientific journals, they are not considered validly published until they have been included
on a validation list in IJSEM.
IJSEM also provides a forum to debate specific controversies in nomenclature by allowing a scientist to
challenge the current nomenclature of an organism or
group of organisms. Such challenges, if accepted by peer
review, are then published as a question in the IJSEM.
The question is then evaluated by the Judicial Commission of the International Union of Microbiological Societies, which subsequently publishes a ruling in the journal. One typical example of a problem considered by the
Judicial Commission was the question about Yersinia
pestis, the causative agent of bubonic plague. Scientific
evidence indicates that Y. pestis is really just a subspecies
of Yersinia pseudotuberculosis, a species name that has
precedence over Y. pestis because of its earlier publication. Because of the potential confusion and possible
public health issues that could arise by renaming Y.
pestis, Y. pseudotuberculosis subspecies pestis, the Judicial Commission ruled against renaming the bacterium
despite its scientific justification.
SECTION HIGHLIGHTS
Nomenclature is concerned with naming organisms. For Bacteria and Archaea, specific
rules must be followed in order to name and
describe new species. Organisms that are
placed on the approved or validated lists are
officially recognized species.
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Taxonomy of Bacteria and Archaea
17.2 Classification
Classification is that part of taxonomy concerned with
the grouping of bacteria into taxa based on common
characteristics. The earliest classifications did not consider microorganisms. There were two kingdoms of life:
Plants and Animals. In 1868, Ernst Haeckel, a German
scientist, proposed a third kingdom specifically for microorganisms. Approximately a century later, in 1969,
Robert Whittaker proposed a five-kingdom system of
classification. His classification included Plants (Plantae), Animals (Animalia), Fungi, Protista, and Monera. In
this system, the eukaryotic microorganisms were placed
in the Protista kingdom and the fungi had their own special kingdom. The bacteria and archaea were placed, as
prokaryotes, in the kingdom Monera. Organisms were
separated from one another on the basis of nutrition and
cell structure. Therefore, plants are photosynthetic eukaryotes, fungi are heterotrophs that use dissolved nutrients, and animals are heterotrophs that ingest their
food. The five-kingdom classification remained popular
until recently. Then, in 1990, Carl Woese and colleagues
proposed an entirely new classification, the Tree of Life
(see Chapter 1). Unlike all previous classifications this
new classification uses a molecular phylogenic approach. The Tree of Life is based on the sequence analysis of a common macromolecule that all organisms share,
the RNA in the small subunit of the ribosome (see Chapter 4). This RNA was used to separate all organisms on
Earth into three different domains, the Bacteria, the Archaea, and the Eukarya. Viruses, which are not organisms
because they are not cellular (see Chapter 1), cannot be
classified by this system.
Classification systems can be either artificial or natural. Artificial systems of classification are based on expressed characteristics of the organisms, or the phenotype
of the organism. In contrast, natural or phylogenetic
systems are based on the purported evolution of the organism. Until recently, all bacterial classifications were
artificial because there was no meaningful basis for determining their evolution. In contrast, plants and animals have a fairly extensive fossil record on which to
base an evolutionary classification system. Although fossils of microorganisms do exist (see Chapter 1), the simple structures of microorganisms do not permit their
identification into a taxonomic group by morphological
criteria. Furthermore, the stable morphology of and ontology of plants and animals have been useful in developing natural systems of classification. This type of information is rarely available for microorganisms.
When considering bacterial classification, it is important to keep in mind that bacteria have been evolving on
Earth for the past 3.5 to 4 billion years. Therefore, it should
not seem surprising that two separate domains of prokary-
487
otic organisms exist—the Bacteria and the Archaea—versus only one domain for eukaryotes, Eukarya. In addition,
the Eukarya may have evolved more recently as the result
of symbiotic events between different early prokaryotic
forms of life (see Chapter 1). Because of the long period of
evolution of bacteria and archaea, the various groups
within these domains exhibit considerable diversity, particularly metabolic and physiological. In contrast, the
metabolic diversity of the Eukarya is limited, especially
with respect to energy generation. The vast diversity of
metabolic types of prokaryotes is discussed more fully in
Chapter 5 and in Chapters 18–22. This chapter first discusses the traditional system of classification and then covers what is being done to make it phylogenetic.
Artificial versus Phylogenetic Classifications
Conventional artificial taxonomy uses phenotypic tests
to determine differences between strains and species.
These tests are typically weighted so that characteristics
that are considered to be more important are given
higher priority. For example, in traditional taxonomy,
the Gram stain has been given more weight in determining the classification of an organism than whether the
organism uses glucose as a carbon source. Therefore, all
gram-positive strains would be ascribed to one family
or genus, and, within that group, certain species or
strains would use glucose and others would not.
Most bacteriologists favor a phylogenetic system for
the classification of bacteria, and with the advent of molecular phylogeny, this hope is now being realized. The
current accepted treatise that contains a complete listing
of prokaryotic species and their classification is Bergey’s
Manual of Systematic Bacteriology (2001–2008), published
by Springer, and its more condensed edition, Bergey’s
Manual of Determinative Bacteriology (1994). In addition
to containing a complete classification of bacteria and
archaea, the more comprehensive version of Bergey’s
Manual contains a description of all known validly described bacterial species. Therefore, it is the “encyclopedia” of the bacteria and archaea that is widely used by
bacteriologists (Box 17.1). Bergey’s Manual of Systematic
Bacteriology is now in its second edition. The first edition
was based on an artificial classification because too little phylogenetic information was available. However,
the second edition is based on the Tree of Life that uses
a phylogenetic framework as discussed in subsequent
text of this chapter.
Phenotypic Properties and Artificial
Classifications
Phenotypic properties are those that are expressed by an
organism, and they have always played a major role in
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Copyright 2007 Sinauer Associates Inc.
488
Chapter Seventeen
BOX 17.1
Milestones
Bergey’s Manual Trust
David Bergey was a professor of
bacteriology at the University of
Pennsylvania in the early 1900s. As
a taxonomist he was a member of a
committee of the Society of
American Bacteriologists (SAB—
now called the American Society for
Microbiology), which was interested
in formulating a classification of the
bacteria that could be used for
identification of species. In 1923, he
and four others published the first
edition of Bergey’s Manual of
Determinative Bacteriology. This was
followed by new editions every few
years. Royalties collected by the
publication activities of the committee were held in SAB. When David
Bergey and his co-editor, Robert
Breed, requested money from the
account to be used for preparation
of the fifth edition, the leadership in
SAB refused. After a long and bitter
fight, the SAB relented and turned
the total proceeds over to Bergey,
who promptly put the money into a
nonprofit trust with a board of
trustees to oversee the publication
of manuals on bacterial systematics.
The Trust, now named in his honor
as Bergey’s Manual Trust, is responsible for the publication of Bergey’s
Manual of Determinative
Bacteriology, which is now in its
ninth edition, as well as other taxonomic books such as Bergey’s
Manual of Systematic Bacteriology.
The Trust is headquartered at
University of Georgia and has a
nine-member international board of
trustees, as well as associate members from many countries.
microbial taxonomy. Indeed, early classifications had to
be based entirely on phenotypic properties because they
were the only properties that could be studied.
In classifications, it is important to select phenotypic
characteristics that allow one to group organisms together and others that enable one to distinguish organisms from one another. Furthermore, it is important that
the identifying characteristics selected be easy to determine. Two examples of simple phenotypic characteristics that have been widely used in artificial classification
schemes are the Gram stain and cell shape. It turns out
that each of these has some utility in classifications. For
example, the Gram stain tells something about the nature of the cell wall (see Chapter 4). Furthermore, the
Gram stain happens to be important phylogenetically
because two of the phylogenetic groups of Bacteria are
gram-positive (i.e., Firmicutes and Actinobacteria) and the
other 20 or more are gram-negative. However, there are
some drawbacks to phenotypic properties. For example,
it is noteworthy that some species of gram-positive bacteria stain as gram-negative bacteria. Likewise, the mycoplasmas, which stain as gram-negative organisms,
have been found to be members of the Firmicutes
through 16S rRNA analyses (see Chapter 20). The rea-
David Bergey. Courtesy of the National
Library of Medicine.
son the mycoplasmas stain as gram-negative is that they
lack cell walls altogether. Therefore, they have apparently evolved from a group of gram-positive bacteria
that lost their peptidoglycan wall during evolution.
In contrast to the gram-positive bacteria, gram-negative bacteria and archaea fall into many different phylogenetic groups, including peptidoglycan-containing
types and non–peptidoglycan-containing types that are
bacteria as well as archaea. Therefore, gram-negative organisms are very diverse phylogenetically.
At one time, some bacteriologists proposed that the
simplest, and purportedly the most stable, cell shape—
the sphere—must have been the shape of the earliest bacteria. They then developed an evolutionary scheme based
on this theory, in which all of the coccus-shaped bacteria
were included in the same phylogenetic group. The validity of this classification has not been borne out by molecular phylogeny. For example, there are both gram-negative as well as gram-positive cocci. Some cocci are
photosynthetic Proteobacteria, others are nonphotosynthetic, some are highly resistant to ultraviolet (UV) light
(Deinococcus), some are Archaea, and others are Bacteria.
Nonetheless, cell shape is important for some groups.
For example, the spirochetes phylum contains all of the
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Copyright 2007 Sinauer Associates Inc.
Taxonomy of Bacteria and Archaea
helically shaped bacteria with periplasmic flagella (see
Table 17.1 and Chapter 22). Because of their morphology, these bacteria were correctly classified with one another in the order Spirochaetales and now in the phylum
Spirochaetes long before phylogenetic data confirmed the
grouping. Apart from this group, overall cell shape has
little meaning at higher taxonomic levels. Nonetheless,
it can still be significant at the species, genus, and even
family levels.
Other phenotypic properties have also proved useful
in both artificial and phylogenetic classifications. For example, because of their unique ability to produce
methane gas, the methanogenic microorganisms have
always been classified together in artificial classifications. Likewise, from a phylogenetic standpoint, all the
methanogenic organisms are members of the Euryarchaeota of the Archaea.
Of course, phenotypic properties have a special significance not found in gene sequence analyses in that these
features provide information about what the organism is
capable of doing. One cannot directly conclude from a sequence that an organism is or is not a methanogen, for example, unless that particular feature has been tested for
and determined. Thus, phenotypic tests provide valuable
information about the capabilities of the organism that
may help explain its role in the environment in which it
lives.
Numerical Taxonomy
When a large number of similar bacteria are being compared, computers are very useful in the analysis of the
data. This aspect of taxonomy, which has been used in
artificial classifications, is referred to as numerical taxonomy. Numerical taxonomy is most useful at the
species and strain level, where phylogenetic relatedness
has already been established by ribosomal RNA (rRNA)
sequencing and DNA–DNA reassociation.
In numerical taxonomy all characteristics are given equal
weight. Therefore, metabolism of a particular carbon
source is considered to be as important as the Gram stain
or the presence of a flagellum. In characterizing strains
in this manner, a large number of characteristics are determined, and the similarity between strains is then compared by a similarity coefficient. Each strain is compared
with every other strain. The similarity coefficient, SAB,
between two strains, A and B, is defined as follows:
S AB =
a
a+b+c
where a represents the number of properties shared in
common by strains A and B; b represents the number of
properties positive for A and negative for B; and c rep-
489
resents the number of properties positive for B and negative for A.
Characteristics for which both strains A and B are negative are considered irrelevant because there would be
many such features that would have no bearing on their
similarity. For example, endospore formation is an uncommon characteristic for bacteria. It is not significant
to incorporate this characteristic when comparing two
species within a genus that do not produce endospores.
It would, however, be of value in comparing endosporeforming organisms to closely related organisms. It
should be noted that the similarity coefficient can be
used to relate not only phenotypic features of one organism to another, but also to relate the sequence similarity of macromolecules of different organisms.
In numerical taxonomy, it is best to have as many tests
of phenotypic characters as possible. Typically at least
50 independent characters are used, and many strains are
usually compared simultaneously. Ideally, each characteristic should represent a single and separate gene. The
same gene should not be assessed more than once, and,
therefore, overlapping phenotypic tests must be
avoided. Typically, SAB values are greater than or equal
to 70% within a species and greater than 50% within a
genus. An example of a numerical analysis is shown in
Box 17.2.
In artificial classifications, bacteria are grouped into
a hierarchy based on phenotypic properties. For example, the chemoautotrophic bacteria that obtain energy
from the oxidation of inorganic nitrogen compounds,
such as ammonia and nitrite, would be classified in the
group of nitrifying bacteria. This group would be regarded as an order. One subgroup, termed the family,
would contain ammonia oxidizers and another would
contain nitrite oxidizers. Within each of these groups,
features such as cell shape would be further used to define differences among the various genera and species.
However, this artificial classification does not take into
account the evolutionary relatedness among the members of the nitrifying bacteria.
Phylogenetic Classification and Molecular
Phylogeny
An important article published by Zuckerkandl and
Pauling in 1962 suggested that the evolution of organisms might be recorded in the sequences of their macromolecules. Subsequent research in the late 1960s and
1970s has supported this concept, resulting in a revolution in bacterial taxonomy. In particular, molecules such
as rRNA and some proteins have changed at a very slow
rate during evolution; therefore, their sequences provide
important clues to the relatedness among the various
bacterial taxa and their relatedness to plants and animals
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Copyright 2007 Sinauer Associates Inc.
490
Chapter Seventeen
BOX 17.2
Methods & Techniques
Determination of Similarity Coefficients
In this example, eight strains, A through H, are compared to one another by ten phenotypic tests. The results are
shown in the first table:
Table 1 Results of phenotypic tests for eight strains, A–H
Strains Tested
Tests
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9
10
+
–
+
+
+
+
–
+
+
–
–
+
+
–
–
+
+
–
–
+
+
+
–
–
–
–
+
–
+
+
+
–
+
–
+
+
+
+
+
–
+
+
–
+
+
–
+
–
+
+
+
–
+
+
+
–
–
+
+
–
–
+
+
–
–
+
–
+
–
+
+
+
+
+
–
–
+
–
+
+
Similarity coefficients are then determined by comparing the results of the tests for each of the strains against one
another, using the formula given in the text. The results are shown in Table 2.
Table 2 Similarity coefficients (× 100 to give percent similarity) for the eight strains
Strains
A
B
C
D
A
B
C
D
E
F
G
H
100
20
20
75
40
86
33
40
100
43
33
38
10
67
50
100
33
71
22
25
71
100
40
63
33
40
E
F
G
H
100
44
20
75
100
22
44
100
33
100
The information from this matrix is then used to group the strains into similar types, as shown in the following matrix:
Table 3 Similarity matrix of grouped strains
Strain
A
F
D
E
H
C
G
B
A
F
D
E
H
C
G
B
100
86
75
40
40
20
33
20
100
63
40
44
22
22
10
100
40
40
33
33
33
100
75
71
20
38
100
71
33
50
100
25
43
100
67
100
According to these tests, strains A, F, and D are very similar to one another and probably compose a single species.
Likewise, E, C, and H are very similar and appear to be a separate species. Strains B and G may also be a different
species, although more tests should probably be performed to substantiate this. As mentioned earlier in the chapter,
phenotypic data such as this can provide an indication of relatedness at the species level, but if new species are being
described, DNA/DNA reassociation tests should be performed.
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Copyright 2007 Sinauer Associates Inc.
Taxonomy of Bacteria and Archaea
as well. The result is that a major breakthrough has occurred in the classification of prokaryotic organisms,
which has rapidly become phylogenetic through the
study of molecular phylogeny as discussed in subsequent text. The fact that the two domains Bacteria and
Archaea exist was not at all appreciated until molecular
phylogenetic studies were performed. Bacteriologists are
now trying to determine when the split occurred between the Bacteria, Archaea, and Eukarya and what the
nature was of their last common ancestor.
By the 1970s, data had accumulated indicating that
a true phylogenetic classification of bacteria was possible. What made it possible was, first of all, acceptance of
the evidence that some of the macromolecules of organisms were highly conserved, that is, changed very slowly
during evolution, and that their sequences held the key
to unlocking the relatedness of bacteria to one another
and to plants and animals. Second, sequencing techniques were developed and improved so that it became
easy to conduct sequence analyses of rRNA and other
macromolecules.
In this section, emphasis will be given to rRNA sequencing, in particular the RNA of the small subunit of
the ribosome (see Chapter 4), 16S rRNA (or 18S rRNA
of Eukarya), as it is the most common conserved molecule used to study the phylogeny of microorganisms
(Figure 17.1). In actual practice, the 16S rRNA gene, or
16S rDNA, is sequenced because the polymerase chain
reaction (PCR) procedures are simple, and both strands
of the DNA can be used to confirm the actual sequence.
Several reasons justify the choice of rRNA as an evolutionary marker. First, the ribosome is found in all cellular organisms (see Chapter 4) and therefore allows one
to compare all organisms. Second, the function of the ribosome as the structure responsible for protein synthesis
holds true for all classes of life. Therefore, it is possible to
compare the phylogeny of all organisms by analysis of
a single structure with an important cellular function.
The third advantage of using the ribosome in phylogeny
is that ribosomes are highly conserved and therefore have
changed very slowly over many millions of years. This
is true because it is a very complex structure that carries
out a specific function—protein synthesis (see Chapter
11). Keep in mind that the 16S rRNA is interacting in a
three-dimensional structure with protein and other rRNA
molecules as well messenger RNA (mRNA). Therefore,
a high rate of evolutionary change in ribosome structure
has been selected against during evolution. Mutant organisms with dysfunctional ribosomes would be unable
to compete with existing types and have therefore not
survived. In this manner, evolution has selected against
major changes in the ribosome. Nonetheless, incremental modifications have occurred over the billions of years
of biological evolution and these differences are used to
construct evolutionary trees.
491
Ribosomal RNAs are not the only macromolecules
that have been considered in determining relatedness
at higher taxonomic levels. Proteins such as cytochrome
c and ribulose bisphosphate carboxylase are just two
examples of other molecules that have been used.
However, not all organisms synthesize these macromolecules. Furthermore, not all cytochrome c–like molecules found in different organisms have the same
physiological function. Because these other molecules
are not universally distributed among all organisms,
they cannot be used to compare distantly related taxa.
However, the sequences of highly conserved proteins,
such as cytochromes and nitrogenase, are very useful
in constructing phylogenies of their origin and evolution within disparate microbial groups. It should be
noted that because proteins are difficult and expensive
to sequence, their sequences are normally deduced
from their gene sequences.
Within the biological world, ribosomes share many
similarities, indicating the conservative nature of the
structure. Prokaryotic ribosomes contain three types of
RNA: 5S, 16S, and 23S. Both 5S and 16S rRNA have been
used to determine relatedness among organisms. Because the 16S molecule is larger (with about 1,500 bases),
it contains more information (see Figure 17.1) than the
smaller 5S molecule with only about 120 bases (Figure
17.2). Less work has been done on the 23S molecule because it is longer (about 3,000 nucleotides) and therefore
not as easy to study. Therefore, scientists interested in
the classification and evolution of bacteria have concentrated on 16S rRNA. The method of evaluation that provides the most information is sequence determination,
especially for the complementary DNA of 16S rRNA,
that is, the double-stranded 16S rRNA gene or 16S
rDNA. It has been found that some regions of these molecules are more highly conserved than others. The more
highly conserved regions permit the comparison of distantly related organisms (Figure 17.3), and the more
variable domains are used for comparing more closely
related organisms. Figure 17.3 shows the “stem-loop”
secondary structure of a model 16S rRNA that allows for
comparison among all organisms. The stem and loop
portions, designated by the blue color, contain the most
highly variable regions. For example, the blue area designated with a red star has been expanded for three
species—E. coli (a bacterium), Methanococcus vannielii (an
archaean), and Saccharomyces cerevisiae (a eukaryote)—
to illustrate the variation among these three species. The
regions that are unique to a given taxon are termed signature sequences. They can be used for the design of
specific hybridization probes for identification (see later
section on Identification).
It appears that an analysis of 16S rDNA sequences
provides important information on the evolution of
prokaryotes. However, before concluding that the se-
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Copyright 2007 Sinauer Associates Inc.
492
Chapter Seventeen
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G
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U
G
C
A
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G
G
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A
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U
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G
A
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G
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G
A
A
U
U
A
CG
U
U
A
G 800
A
A
A UGA GA A U G
C
G
U
G
U
A
G
C
1050 C
U
A
G [ m2G ]
C
A
A
A
G
G
C 750
G
C
C
G
G A C U U U U G AA G
U A
U
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U
G
U
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G
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G
G
G
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A
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GG
650 U
C
C
U
C A
U
A
UA
U
A C
G
C
C
1000
U
UGCA UCUGA CU GGCA A G C
A
U
A
C
A A CCUGGGA
A
U
U
GC
G
C
C
C
C
U
G
C
U
A
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U C GGGCCCC
C
G
C G
U A
GU GU A GA C U GA U U GU U U GG
C
G
C
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A
C G
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U
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U
A
A
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G
G
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C
G
A
G
A A
U
600
G
A
G
U
A
U
A
G
AC
U
C
G A
U
C C UA C
C
G
A
G
GC
U
G
A
U
A
G
C
C
G
C
G
A
G
G
G
G
G
850
A
G
C
C
C
G
UA
G
U
G
A
G
C CG
U
U
U
GU CGA CU
A
U
GCC
A
A
U
A U AC
C
A CC
G
A
U A UU G
C
A
A
C
C
G
G
G
A
G
A
C
U
m7
A
A
A
G
C
U
U
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U
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U G
A
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U
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A
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A
A
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U
A
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A
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G
1250
G
U
G
A
A
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A
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A
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C A A
C
G
C
A
U
5
A
C
G
950 U
C
U
U
A A A G
G
mC 2 U
C
U
G
mG
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U
A
A
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C
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A
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U
U
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G UA
CA
G
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y
A
U
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GGCC G
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C
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A 900
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A A G
A
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U GG
A
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U
A
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450
AA
U
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G G
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G
A A
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A CG G C
U
U UG A CG GGGCCCG A CA A
G
C
U
C A
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C
G
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A 500 C
A
CG U A CU
C
A
CU
A
U 550 A A
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A C UGU UCCGGGC UG
GA
G
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GUA C
G
A
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A
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A 10
C
A 1350
G
A
A
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U
1400 C
C
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U
U A U G U GG C
U CCGG
G
U
U
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A
G 400
G
C
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4
U
U
AGAA
G
C
G G
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G
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A
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C
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G
m3U
G
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A
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A GCCUG A UGC A G
A
G
C 1500 U A A C C G U A G G
G
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C
6
U
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C
A
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C
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C
5'
mC
U
A C G UA
U
G CGGGU
A
G
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A
G
2
A A G
A
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A
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A
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U
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GUC A CG GU CA G GA A GA A GC
G
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300 A
C
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GGU
CGG UGA CA GUC UUUCUUCG
A
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C
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A
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C AU
U
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A
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A
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A
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A
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G
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A
A
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U
A
A
G
A
C
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A
G
C
U GA
C
A
U
A
G
C
A
250
G
C
G
C
G
U
G
1450 U
U
U
U
U
C
G
GC
G
A
G
G
C
G
C
G
C
G
U
G
A
U
A
G
C
A
U
150
U
A
G
C
C
G
AUA
ACUACUGG
A G G G GG
G GG CCUCUU G
A
C
A
U
CGCC
CGA UGGC A
U CC GGGGA G
A
A
U
A
AG
UA A
U
200 A
A
A
A
C
C
C
G
A
U
G
C
G
A
A C
Canonical base pair (AU, GC)
GU base pair
GA base pair
Non-canonical base pair
Figure 17.1 16S rRNA
Secondary structure of the 16S rRNA molecule from the small
subunit of the ribosome from the bacterium Escherichia coli. The
bases are numbered from 1 at the 5’ end to 1,542 at the 3’ end.
Every tenth nucleotide is marked with a tick mark, and every
fiftieth nucleotide is numbered. Tertiary interactions with strong
comparative data are connected by solid lines. From the
Comparative RNA Web Site, www.rna.icmb.utexas.edu (courtesy
of Robin Gutell).
This material cannot be copied, disseminated, or used in any way without the express written permission of the publisher.
Copyright 2007 Sinauer Associates Inc.
E. coli
5′
3′
10
G
UGCCUGGCGGCC
40
H. sapiens
CAUG
C
C
C
C
C
G
A
A
UCA
G
U C
A
30
C
C G A 50
GA
CCA
U
C
U
G
G
A
G
A
C A
U
G 60
20
G
G C
C
C
G G
U
C
UA G GA
C
G 70
C
A C GG A C C G U C A AGG C
G G
A
120
110
A
U U
G G
A C
G U
A A
100
G G
80
C U
G G
U U
A G
C
G
C G
C G
C U
90 U C
CG UC
U
U
C
G
U
A
A
U
UC
G
C C
C G
C GA
G C G GA
C
C
U
A
A
A
G
A
CG
U
A
C
G
C
C G
G
A
U
A C
AU C G C
G
C
GUCU A CGGC
G
C
U C G G A U G U C G UG C
U
G
U
G G
C G
C UU
A A
U G
G — C
A U
A
A
G C
G
U
G U
G U
G
A
U G
C
G
C UA
U
U
G G
C G
C
G
A
A
G
493
Taxonomy of Bacteria and Archaea
Figure 17.2 5S rRNA
Comparison of the secondary structures of 5S
rRNA molecules from the bacterium Escherichia
coli and the eukaryote Homo sapiens. Note how
similar the overall secondary structure is between
these two molecules even though they represent
many millions of years of divergence from one
another.
5′
3′
Canonical base pair (AU, GC)
GU base pair
GA base pair
Non-canonical base pair (UU, AC)
(A)
(B)
A U AC
A
C
U
U
U
UG
U
A
G
A
C
A
U
U
CA U
G
U
A
G
G
A
G
CGU
U
G
A A G
A
C
G
C
450
A
C
G G
G
G
C
G
A
C
G
G
A
A
A
C
G C
G
U
A G
U
C
UAA
A
U
G
A
G GGUU
GUA C
C
U
U A U G U GG C
U CCGG
A
G
C
AGAA
G
C
G
Identical in 98% or
more of all organisms
Conserved only
in the Bacteria
Conserved only
in the Archaea
Conserved only
in the Eukarya
Conserved within
each domain, variable
among domains
Regions that vary
structurally among
domains
E. coli
A
A CA
A
G
C
U
U
GU
U
G
G
A
A
A
G
C
G
U
A
A
A
U
U
C
U
A G
U
AG
C
U
A GCA UGGGC
C
A C G U A C U C G U GA
A
C
U
U
G
M. vannielii
A A UA
A
C
U
C
U
GU
A
UA
A
A
CA
C
G
U
A
G
G
A
G
A
G
A
U
G
C
A
A U
A
A
U
G
A
CGGGUCUUGUA UUG
S. cerevisiae
5'
U
U
C
A
A
A CCCGGGA CA U A GCAA UA A
A
U
3'
G
Figure 17.3 Conservation and variation in small
subunit rRNA
(A) This diagram shows conserved and variable regions of the
small subunit rRNA (16S in prokaryotes or 18S in eukaryotes).
Each dot and triangle represents a position that holds a
nucleotide in 95% of all organisms sequenced, although the
actual nucleotide present (A, U, C, or G) varies among species.
(B) The starred region from (A) as it appears in a bacterium
(Escherichia coli), an archaean (Methanococcus vannielii), and a
eukaryote (Saccharomyces cerevisiae). This region includes
important signature sequences for the Bacteria and Archaea.
Figure by Jamie Cannone, courtesy of Robin Gutell; data from
the Comparative RNA Web Site: www.rna.icmb.utexas.edu.
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Copyright 2007 Sinauer Associates Inc.
494
Chapter Seventeen
quence of bases in rDNA accurately reflects the phylogeny of organisms, it is important to find totally separate and independent evolutionary markers to confirm
the classification. Some work has been performed with
sequencing of ATP synthases, elongation factors, RNA
polymerases, and other conserved macromolecules. The
outcome of this research, which represents one of the
most exciting areas of biology, is leading to the development of a complete phylogenetic classification of the
Bacteria, Archaea, and other microorganisms. The 16S
rDNA–based phylogeny will face its most stringent test
as additional genomes are sequenced and compared.
Before we discuss the use of 16S rDNA sequences in
arriving at the current phylogeny of Bacteria and Archaea,
it is first necessary to consider phylogenetic trees.
Like a family tree, a phylogenetic tree contains the tree of descendants of a biological
family or group. However, whereas the family tree traces
the genealogy of a family of humans, phylogenetic trees
trace the lineage of a variety of different species. Thus,
phylogenetic trees reflect the purported evolutionary relationships among a group of species, usually through the
use of some molecular attribute they possess, such as
the sequence of their rRNA. In this particular section we
will discuss molecular phylogeny based on a comparison
of the 16S rRNA sequences of organisms.
Phylogenetic trees have two features—branches and
nodes (Figure 17.4). Each node represents an individual
species. External nodes (usually drawn to the extreme
right of the tree) represent living species, and internal
(A) Unrooted trees
(B) Rooted trees
A
B
C
A
A
C
C
A
B
C
PHYLOGENETIC TREES
In both types of
trees, internal
nodes represent
ancestor species…
…external nodes
(here, A through E)
represent extant,
known species…
… and branch
lengths represent
the evolutionary
distance or degree
of relatedness
between species.
A
A
B
B
C
C
D
Internal
nodes
Branch
E
External
nodes
D
Internal
nodes
E
Branch
External
nodes
Figure 17.4 Phylogenetic trees
Two different formats of phylogenetic trees used to show
relatedness among genes or species.
B
B
B
C
A
Figure 17.5 Unrooted and rooted trees
Representations of the possible relatedness between three
species: A, B, and C. (A) A single unrooted tree (shown in
both formats; see Figure 17.4). (B) Three possible rooted
trees (in one format).
nodes represent ancestors. A branch is a length that represents the distance between or degree of separation of
the species (nodes).
Trees may be rooted or unrooted. Figure 17.5A shows
unrooted trees containing three different species: A, B,
and C. Unrooted trees typically compare one feature of
a group of related organisms, such as the sequence of
their 16S rRNA gene. There is only one shape to this particular tree with three species.
In contrast, rooted trees provide more information.
Typically, rooted trees use the same gene from a distantly
related organism for the root. This allows one to compare the relatedness of the more closely related species—
A, B and C—to one another. A rooted tree containing
three species has three possible shapes (Figure 17.5B).
In the examples shown, A and B are more closely related
to one another in the uppermost example, whereas A
and C and B and C, respectively, are more closely related
in the two lower examples.
Alternatively, an additional gene can be used to compare the species. When using this approach, a different
gene such as the sequence of a macromolecule that underwent a gene duplication event prior to when the taxa
that are being studied, diverged from one another. As
an example, a comparison of the elongation factors Tu
and G (see Chapter 13) was used to root the 16S rRNA
Tree of Life (see Figure 1.6).
For bacterial phylogeny, trees are constructed from
information based on the sequence of subunits in macro-
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Copyright 2007 Sinauer Associates Inc.
Taxonomy of Bacteria and Archaea
molecules. As mentioned previously, rRNA, in particular the small subunit rRNA molecule, 16S rRNA, has
been selected as the molecule of choice because of its
conserved nature and length.
SEQUENCING 16S rDNA
If one isolates a new bacterium and wishes to determine its phylogenetic position among the known bacteria, it is necessary to determine the sequence of its 16S rRNA gene. This can be
accomplished in a number of ways. One of the most
common ways is to use polymerase chain reaction (PCR)
to amplify the 16S rDNA from genomic DNA from the
bacterium (see Chapter 16). The amplified 16S rDNA
may be sequenced directly or ligated into a cloning vector and cloned into E. coli. This latter step allows a large
quantity of 16S rDNA to be produced through growth
prior to sequence analysis. The entire 16S rDNA can be
sequenced using a standard set of oligonucleotide
primers and standard sequencing techniques.
ALIGNMENT WITH KNOWN SEQUENCES The next
step is to incorporate the determined linear DNA sequence into an alignment with the sequences of other
known organisms. Two internet databases are of special
importance in this regard. One is called the Ribosome
Database Project, located at Michigan State University.
It contains the 16S rRNA sequences for those bacteria
that have been sequenced. Sequences from this database can be retrieved electronically via the Internet
(http://rdp.cme.msu.edu/). Another frequently used
database for sequence analysis is the National Center for
Biotechnology Information (NCBI) (www.ncbi.nlm.
nih.gov/Genbank), which contains a comprehensive
listing for 16S rRNA genes as well as other genes.
By using a series of computer programs and careful
manual examination, one can align and compare the sequence with those retrieved from the database.
PHYLOGENETIC ANALYSIS Having the sequence is
only half of the story. It is next necessary to compare the
sequence of the unknown bacterium to that of other bacteria from the database. The determination of the evolutionary relatedness among organisms can be accomplished by one of a number of phylogenetic methods.
There are several types of analysis that can be used. Distance matrix methods are one type of approach. In distance matrix methods, the evolutionary distances, based
on the number of nucleic acid or amino acid monomers
that differ in a sequence, are determined among the
strains being compared. A second approach is to use
maximum parsimony methods. In maximum parsimony, the goal is to find the simplest or most parsimonious phylogenetic tree that could explain the relatedness between different sequences. In both approaches,
495
the sequence of nucleic acid subunits among different
strains of bacteria or other genes is compared.
Figure 17.6 illustrates the use of two different methods to create a tree from an aligned hypothetical sequence region of rRNA from four different strains
(shown in Figure 17.6A). The first approach is a distance
matrix method called the “unweighted pair group
method with arithmetic mean,” or UPGMA (see Figure
17.6B). This is one of the simplest analytical methods that
can be used. Figure 17.6C shows an analysis of the same
sequences by maximum parsimony.
In the UPGMA method, a distance matrix is set up to
compare the differences in sequence or “distance,” d, between each of the four strains. There are four nucleotide
differences between the sequence of organism a and the
sequence of organism b; therefore, dab is determined to
be 4. Likewise, dac is 5, dbc is 5, and so forth. In this instance, the shortest distance, 2, is between strains c and
d. From these two strains, which show the closest relationship to one another, a simple tree is constructed that
shows c and d connected by a node that is half the distance between the two, that is, 1 unit. This is expressed
in the actual tree as a horizontal branch length of one
unit from each of the organisms to a common ancestral
node (see Figure 17.6B).
The next step is to construct another matrix in which
c and d are considered as a single composite unit (cd) and
compared with a and b. From this matrix, the two most
similar organisms are a and b, and the length of this
branch is calculated as the distance, dab/2, which is equal
to 2 units. From this an intermediate second tree is
formed. Finally, a is different from (cd) by (dac + dad)/2,
or (5 + 6)/2 = 5.5. Likewise, b is different from (cd) by (dbc
+ dbd)/2 = 4.5. To determine the connection between the
composite branch cd and ab, the distance is calculated as
the average of d(cd)(ab), or 5.5 + 4.5/2, which is 5.0. This
value is then divided by 2 to give the average distance
between the two composites, cd and ab. Using this reasoning, a final tree (see Figure 17.6B) is produced showing the relationship among the four different strains.
The UPGMA method is the simplest of the distance
methods used. More sophisticated distance methods include transformed distance and neighbor-joining methods, which will not be discussed here.
As mentioned earlier, in maximum parsimony the
goal is to identify the simplest tree that could explain the
difference between two different sequences or species.
This approach has its philosophical basis in Occam’s razor, commonly used in the sciences, which states that the
likely solution to a problem is the simplest one. In this case,
to explain the evolutionary difference between two
species, one looks at the tree that has the fewest changes
(mutational events) that could explain their differences.
This is accomplished with a computer that, in theory, con-
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Copyright 2007 Sinauer Associates Inc.
496
Chapter Seventeen
Figure 17.6 Phylogenetic analysis
(A) 16S rRNA sequences
Phylogenetic analysis of four different strains—a, b, c,
and d—using a hypothetical region of their 16S rRNA
that contains nine bases (A). (B) The UPGMA method
of determining a phylogenetic tree. (C) The maximum parsimony method (see text for details).
This table shows the sequence of a nine-base
region of the 16S rRNAs of the four strains.
Organism
(strain)
1
2
3
4
a
b
c
d
G
G
G
G
C
A
A
A
G
C
A
A
G
G
A
A
Site number
5
6
A
C
U
G
C
C
C
C
7
8
9
A
A
U
U
A
A
A
A
A
G
A
G
(B) UPGMA method
1 Construct a distance matrix showing
the relatedness (as distance, d) between
strains to determine the most closely
related strains—in this case, c and d.
First matrix
b
c
d
a
b
c
dab = 4
dac = 5
dad = 6
—
—
—
dcd = 2
dbc = 5
dbd = 4
(cd)
a
b
c
where
d
d(cd)a = 11/2
d(cd)b = 9/2
dcd
=1
2
1
3 Construct a second matrix to
assess the distance between
the first paired group (cd) and
the remaining strains (a and b).
Second matrix
Beginning tree
2 Diagram the relatedness
between these strains.
Second tree
4 Since a and b are close to
one another, determine and
diagram their relatedness.
c
a
d
a
—
dab = 4
where
b
dab
2
=2
2
Final tree
5 Determine the relatedness between
cd and a and b, and diagram this.
c
d
a
where d(cd)(ab) =
(11/2 + 9/2 )/2
= 2.5
2
b
2.5
siders all possible trees and then identifies the simplest
one (the one with the fewest assumed mutational events).
As for all phylogenetic analyses, the alignment of the sequences must be accurate.
In maximum parsimony it is important to recognize
sites in the sequences that are useful for a comparison between organisms. These are termed informative sites.
These sites are then used to determine the most parsimonious tree. For example, Site 1 in the example given (see
Figure 17.6C) is not informative because all the bases are
identical. Site 2 is not informative either, because three of
the strains have A and one has C, suggesting that a single mutational event has occurred. Site 3 is not informative, because Trees 1 and 2, which have two changes,
are equally parsimonious, and Tree 3 differs from Tree 2
only in the inferred ancestor. Site 5 is not informative because all trees constructed from the information at this
site differ from one another by three mutations. In contrast, Site 4 is informative, because one tree (Tree 1) is
more parsimonious than the other two. Sites 6 and 8 are
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497
(C) Maximum parsimony method
1 Identify the informative sites in the sequences. Here,
construction of trees for sites 3, 4, 5, 7, and 9 shows that
only sites 4, 7, and 9 are informative, because one tree
is more parsimonious at these sites than are the others.
Following a maximum
parsimony analysis, this
proves to be the most
parsimonious tree.
Site 3
Three possible trees
a
c
b
G
*
d
C
b
G
A
G
*
Site 5
Site 4
G
A
A
G
A
G
C
G
*
A
A
*
C
A
C
G
A
*
Site 7
*
U
A
G
A
G
A
C
A
*
Site 9
U
A
U
G
A
A
U
*
*
G
A
G
G
Tree 1
a
*
*
A
c
d
A
b
G
A
*
A
A
C
G
G
G
*
A
*
A
U
G
A
*
*
G
*
G
U
C
A
A
A
*
G
A
U
A
A
A
*
G
G
Tree 2
a
*
A
d
c
Tree 3
*
C
A
*
A
Two changes in
trees 1 and 2;
three changes
in tree 3
*
G
A
G
*
A
One change
in tree 1; two
changes in
other trees
*
G
G
*
*
U
U
Three changes
in all trees
*
U
A
A
*
U
One change
in tree 1; two
changes in
other trees
*
G
A
*
G
A
A
One change
in tree 2; two
changes in
other trees
Informative sites are at positions 4, 7, and 9.
Mutations (changes) in trees at informative sites:
Tree 1
1+1+2=4
Tree 2
2+2+1=5
Tree 3
2+2+2=6
2 Determine the most
parsimonious tree by
analyzing each of the
informative sites.
Therefore, tree 1 is the most parsimonious tree.
not informative because all bases are identical, but both
Sites 7 and 9 are informative. Site 7 favors Tree 1, whereas
Site 9 favors Tree 2. Thus, for this set of data, Tree 1 is favored two of three times, Tree 2 is favored one of three
times, and Tree 3 is not favored at any time. Adding the
changes at those three sites gives the following data: Tree
1 is the most parsimonious because a total of only four
changes ( 1 + 1 + 2 = 4) would explain its phylogeny,
whereas in Tree 2, five changes are required ( 2 + 2 + 1 =
5), and in Tree 3 six changes are required (2 + 2 + 2 = 6).
Note that the two trees that were constructed from the
distance matrix and maximum parsimony methods are
identical in shape. Both trees indicate that two of the
strains, a and b, are more closely related to one another
than they are to c and d. Likewise, c and d are more
closely related to one another than they are to a and b. As
you can imagine, some rather sophisticated computer
programs have been developed to handle the immense
amount of information inherent in longer sequences such
as 16S rDNA, which contains about 1,500 bp. Moreover,
when such large sets of data are being analyzed, it is often not possible to determine that a proposed tree is, in
fact, the true tree. Indeed, all trees should be considered
as hypotheses until additional information has been analyzed. For example, the inclusion of a sequence from a
newly discovered, closely related organism may alter the
shape of a tree when it is included in the analysis.
To help support the reliability of a given tree, other
techniques are used. For example, in “bootstrap” analyses, random portions of the sequence are selected by the
computer, and the trees formed from them are compared
with the proposed tree to assess the statistical significance of the proposed tree. In this statistical approach,
some 100 or 1,000 different bootstrap comparisons might
be made and provided as evidence that the proposed
tree is indeed the most parsimonious one. Bootstrap
analyses are applicable for all phylogenetic treatment
procedures.
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Chapter Seventeen
As mentioned previously, other analytical methods
can be used to analyze sequence information and construct phylogenetic trees. A common one used by microbiologists is the maximum likelihood method, which
involves selecting trees that have the greatest likelihood
of accounting for the observed data. This is accomplished by assigning a probability to the mutation of any
one base to any other base at each possible sequence position. From this, all possible topological trees are constructed. By integrating the probabilities for each mutation over each tree, a degree of improbability for a tree
is assessed. The least improbable tree is chosen as the
“true” tree.
A final note on studying microbial phylogeny: It should
be emphasized that many other genes besides 16S rDNA
can be used for phylogenetic analysis. An appropriate
gene must have the degree of conservation necessary for
the analysis desired and must have a homolog in the other
organisms of interest. A homologous gene is a gene that
shares a common ancestry.
Speciation
The process by which organisms evolve is termed speciation. As with plants and animals, bacteria evolve in
habitats. However, unlike plants and animals, bacteria
can evolve very quickly because of their rapid growth
rates, high population sizes, and haploid genomes that
allow for the rapid expression of favorable mutations
through natural selection. Thus, a lineage of bacteria is
determined in large part through vertical inheritance,
the process by which the parental genotype is transferred to the progeny cells following DNA replication
and asexual reproduction.
However, bacteria can also acquire genetic material
from other different organisms through their various genetic exchange mechanisms: conjugation, transformation, or transduction (see Chapter 15). This phenomenon is referred to as horizontal (or lateral) gene transfer
(HGT) to distinguish it from vertical inheritance. As a
result, prokaryotic organisms may undergo dramatic
changes in their population structure in a relatively short
period. For example, we know that multiple-drug resistance can be rapidly acquired by a bacterial species that
is sensitive to antibiotics if it is exposed to antibiotics in
the presence of other antibiotic-resistant bacteria.
Consider the situation of a bacterium that is a member of the normal microbiota of the intestinal tract that
is exposed to an antibiotic to which it is sensitive. The
bacterium may either perish or—if a gene is available in
the environment that confers resistance and the bacterium has the capability—acquire the resistance gene
through an HGT process and survive. This example of
a strong selective pressure likely explains how it is pos-
sible for sensitive bacteria to quickly become resistant to
an antibiotic. This scenario applies equally to other environmental pressures that confront bacteria, such as exposure to potentially toxic hydrocarbons that are used
as an energy source by other species in the environment.
Many of the known examples of rapid genetic change
occur through the acquisition of plasmids from related
organisms. Thus, in the preceding examples, some plasmids are known that carry multiple antibiotic resistant
genes and others are known that carry hydrocarbondegrading genes.
In addition, we do know that genes can be acquired
from distantly related organisms. For example, it has
been recently reported that some members of the Proteobacteria and other phyla of the Bacteria have been found
to contain a gene responsible for bacteriorhodopsin synthesis, which was only known previously from members
of the Archaea. Thus, this example appears to represent
the transfer of genetic material across two different domains as well as phyla within the Bacteria. Another example is the bacterium, Agrobacterium tumefaciens, which
naturally transfers genetic material to plants (Chapters
16 and 19).
From an analysis of genomes that have been sequenced, genes that have been derived from other organisms have been identified in some prokaryotic
genomes (Figure 17.7). The transfers between phyla appear to be relatively rare events. In addition, it should
be noted that each species has hundreds of core genes
that can be used to compare its relatedness to other organisms. If HGT occurred too extensively, it could confuse phylogenetic classifications based on vertical inheritance to such an extent that it would render them utterly
useless. Fortunately, this does not appear to pose a major problem for 16S rRNA gene trees.
SECTION HIGHLIGHTS
Both phenotypic and genotypic properties
have been used to describe microorganisms,
and both are important in describing and
naming new prokaryotic species. Artificial taxonomy entails the use of phenotypic tests,
whereas a taxonomy based on evolutionary
processes relies on molecular phylogeny. Molecular phylogeny uses 16S rRNA gene sequence and protein sequence analyses, which
have become very important in the classification of Bacteria and Archaea. Several different
methods, such as distance, parsimony, and
maximum likelihood methods, are used to
construct phylogenetic trees.
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499
Pseudomonas aeruginosa
Escherichia coli
Mycobacterium tuberculosis
Bacillus halodurans
Vibrio cholerae
Bacillus subtilis
Synechocystis PCC6803
Deinococcus radiodurans
Xylella fastidiosa
Pasteurella multocida
Lactococcus lactis
Archaeoglobus fulgidus
Neisseria meningitidis Z2491
Neisseria meningitidis MC58
Halobacterium NRC-1
Thermotoga maritima
Mycobacterium leprae
Pyrococcus abyssi
Pyrococcus horikoshii
Methanobacterium thermautotrophicum
Aeropyrum pernix
Campylobacter jejuni
Haemophilus influenzae
Helicobacter pylori 26695
Aquifex aeolicus
Thermoplasma acidophilum
Methanococcus jannaschii
Treponema pallidum
Borrelia burgdorferi
Rickettsia prowazekii
Mycoplasma pneumoniae
Ureaplasma urealyticum
Buchnera aphidicola
Mycoplasma genitalium
0
1
2
3
4
Megabases of protein-coding DNA
5
6
Figure 17.7 Horizontal gene transfer
Analyses of sequenced bacterial genomes indicate that a significant proportion of
their genes can be traced to other phylogenetic groups, indicating the importance of
horizontal gene transfer (HGT) in bacterial speciation. This diagram shows the proportion of DNA that was acquired by HGT (in red) in some microbial genomes.
Courtesy of Jeffrey Lawrence.
17.3 Taxonomic Units
The basic taxonomic unit is the species, although as mentioned earlier, some species have subspecies categories
as well. The categories above the species are (sequentially) genus, family, order, class, phylum, and domain
(see Table 17.1). It should be noted that uncertainties exist in bacteriology about the meaning of the higher taxonomic categories such as kingdom because oftentimes
the phylogenetic markers that have been used (primarily 16S rDNA sequences) cannot definitively resolve the
earliest branching points in the Tree of Life. Thus, although we know that each of these major branches is
equivalent to the plant and animal “kingdoms,” how the
microbial “kingdoms” or phyla are related to one another is only poorly understood.
Each colony or culture of an organism represents an
individual strain or clone in which all of the cells are descended from one single organism. In a somewhat different sense of meaning, a strain can also refer to a mutant of a species that has changed characteristics (for
example, lacks a particular gene). The strain, however,
is not considered a formal taxonomic unit, and Latin
names are therefore not ascribed to strains; they have
only informal designations, such as E. coli strain K12.
There can also be varieties within species that exhibit
differences. These are called biovars (i.e., biological
varieties). For example, a serological variety such as E.
coli O157:H7 is a pathogenic serovar that causes hemolytic uremic syndrome and can be lethal to children
who become infected by eating contaminated food. Likewise, pathogenic varieties are termed pathovars, ecolog-
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Chapter Seventeen
ical types ecovars, and so forth. Now let us look at the
individual taxa beginning with the species to see what
features are typical at each taxonomic level.
The Species
The definition of a bacterial species differs from that of
plants and animals. In mammals, the classical species
is defined as a group of individuals (males and females)
that exhibit evident morphological similarities and produce fertile progeny through sexual reproduction. Indeed, the production of progeny in many animals such
as mammals requires sexual reproduction.
Although gene exchange occurs in prokaryotic organisms, it is not essential for reproduction. Most bacterial
reproduction is asexual and occurs by simple binary
transverse fission or budding. In prokaryotic organisms,
sexuality is uncommon and different from that of eukaryotes. Eukaryotes produce haploid gametes in meiosis (see Chapter 1). During sexual reproduction, the haploid gametes (egg and sperm) from the male and female
fuse to form the diploid zygote. In bacterial conjugation,
DNA from one cell is transferred during replication to a
receptor cell; however, only partial diploidy occurs. Genetic material can also be transferred by other mechanisms such as transformation and transduction (see
Chapter 15). These transfers are not always restricted to
members of the same species.
A bacterial species comprises a group of organisms
that share many phenotypic properties and a common
evolutionary history and are therefore much more
closely related to one another than to other species. This
definition, which is very subjective, has been interpreted
differently by bacteriologists in describing species. For
example, at one extreme some taxonomists are called
lumpers because they group (or “lump”) fairly diverse
organisms into a single species or genus. An example of
a lumper is F. Drouet, who has proposed reducing the
number of cyanobacteria from 2,000 species to only 62!
At the opposite pole are splitters. These are taxonomists
who consider even the slightest differences sufficient for
a new species. For example, many years ago it was proposed that the genus Salmonella be “split” into hundreds
of different species, a separate species for each of the
hundreds of different serotypes (or serovars) that are recognized based on specific cell-surface antigens of their
lipopolysaccharides and flagella. However, the views of
lumpers and splitters illustrated here are considered
to be extreme and are not accepted by the majority of
microbiologists.
In fact, more recently, a less arbitrary, quantitative basis has been proposed to define a bacterial species.
Agreement was reached by a group of prominent bacterial taxonomists to define a bacterial species based on
genomic similarity between strains. Accordingly, a
species is defined as follows: two strains of the same
species must have a similar mole percent guanine plus
cytosine content (mol % G + C) and must exhibit 70% or
greater DNA–DNA reassociation. The procedures used
to determine these features are described here.
MOLE PERCENT GUANINE PLUS CYTOSINE (MOL %
G + C) The mol % G + C refers to the proportion of
guanine and cytosine to total bases (guanine, cytosine,
adenine, and thymine) in the DNA. Recall that because
G and C are paired in the double-stranded DNA molecule by hydrogen bonds, as are A and T, they occur in
equal concentrations. The formula is given as:
mol % G + C =
moles (G + C)
× 100
moles (G + C + A + T)
Several methods can be used to determine the mol % G +
C, sometimes also called the “GC ratio” of a bacterium.
All of them require that the DNA be first isolated from a
bacterium and purified. Thus, it is necessary to lyse the
cells to release the cytoplasmic constituents including
DNA, and the DNA must then be purified to remove proteins and other cellular material. Cell lysis is typically accomplished by treatment with lysozyme and detergents,
and the DNA is precipitated with ethanol. When the DNA
has been sufficiently purified, it can be analyzed chemically to determine the content of each of the bases. Several
different procedures can be used to determine the GC ratio of the purified DNA. We will describe two of them here.
A common procedure to determine GC ratios is by
thermal denaturation. The principle behind this method
is that the hydrogen bonds between the double strands
can be broken by heating dissolved DNA. As the hydrogen bonds are broken and the two strands separate, the
absorbance of the DNA increases. This procedure, called
“melting the DNA,” is conducted with a spectrophotometer set at 260 nm, a wavelength at which DNA absorbs
strongly. The hydrogen bonding of the GC base pair is
stronger than the AT pair in the double-stranded DNA
molecule. Therefore, a higher temperature is required to
melt DNA that has a high content of GC pairs, that is, a
high GC ratio. Figure 17.8 shows a graph of the melting
of a double-stranded DNA molecule. This process is accomplished by gradually increasing the temperature of a
solution of the DNA in an appropriate buffer (ionic
strength is important). As the temperature is increased,
the melting process begins and continues until the double-stranded DNA molecule is completely converted to
the single-stranded form. The absorbance increases during this melting process. The midpoint temperature (Tm)
is directly related to the GC ratio of the DNA. Thus, the
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Taxonomy of Bacteria and Archaea
UV absorbance (at 260 nm)
0.4
Prokaryotes
Bacteria
Archaea
0.3
For each DNA, the midpoint of
the melting process occurs…
0.2
Humans
Plants
Algae
Fungi
0.1
…at a characteristic
midpoint temperature,
Tm, in this case 90°C.
Tm
80
85
90
95
Protozoa
0
100
Figure 17.8 DNA melting curve
Melting curve for a double-stranded molecule of DNA. As
the temperature is increased during the experiment, the
double-stranded DNA is converted to the single-stranded
form and the UV absorbance of the solution increases. The
midpoint temperature, Tm, can be calculated from the
curve. This process is reversible if the temperature of the
solution is slowly decreased to allow the single strands to
reanneal. The Tm of this species, Escherichia coli, can be used
to determine its mol % G + C content (see Figure 17.9).
GC ratio can be read from a chart showing the relationship between Tm and GC content (Figure 17.9). Once the
DNA has been melted, it will reanneal if the temperature is slowly lowered. Thus, the process shown in Figure 17.8 is reversible. However, if the solution is cooled
rapidly, hybrid formation does not recur, and the molecules of DNA are left in the single-stranded state.
100
80
Mycobacterium phlei
Pseudomonas
60
40
Serratia
E. coli
Bacillus subtilis
Cytophaga
20
0
60
70
80
90
100
110
Tm (°C)
Figure 17.9 Tm and DNA base composition
10
20
30
40 50 60
Mol % G + C
70
80
90
100
Figure 17.10 DNA base composition range
Temperature
Mol % G + C
Eukaryotes
Animals
Graph showing the direct relationship between mol % G +
C and midpoint temperature (Tm) of purified DNA in thermal denaturation experiments.
Range of mol % G + C content among various groups of
organisms. Note the broad range of GC ratios for bacteria,
archaea, and the lower eukaryotes in comparison to plants
and animals.
Another method is to use the “readout” from a
genome sequence, which contains all of the genetic information of a species.
Figure 17.10 shows the range of GC ratios in various
groups of organisms. On this basis alone, one can see
that bacteria, which have GC ratios ranging from approximately 20 to greater than 70, are truly a very diverse group. In contrast, higher organisms such as animals have a very restricted GC ratio range.
The GC ratio provides only the relative amount of
guanine and cytosine compared to total bases in the
DNA of an organism and says nothing about the inherent characteristics of the organisms or what genes are
present. Indeed, two very different organisms can have similar or even identical GC ratios. For example, the DNA of
Streptococcus pneumoniae and humans have the same mol
% G + C content.
DNA–DNA REASSOCIATION OR HYBRIDIZATION Although the determination of GC ratio is useful in bacterial taxonomy, it does not tell us anything about the
linear arrangement of the bases in the DNA. It is the
arrangement of the DNA subunits that codes for specific
genes and proteins and therefore determines the features
of an organism. DNA–DNA reassociation or hybridization is one method used to compare the linear order of
bases in two different organisms (Box 17.3). It is important to recognize that the = 70% level of reassociation
used for the species definition does not indicate that the
two DNAs are 70% homologous or identical.
In DNA–DNA reassociation, the actual order of bases
in the DNA is not determined, but rather the extent of
reannealing between the DNAs of two different strains
is assessed. Ideally one would like to know the actual
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Chapter Seventeen
BOX 17.3
i
Methods & Techniques
DNA–DNA Reassociation
DNA–DNA reassociation can be
performed by using a variety of different methods. In all approaches,
it is necessary to begin with purified DNA from the two organisms
that are being compared. The DNA
is first cut into smaller segments
(i.e., sheared ) and then denatured
by melting. DNA from the two different strains are mixed and
allowed to cool together to allow
reannealing to occur. This reannealing will occur both between DNA
Unlabeled DNA
strands of the same species and
between strands of the comparison
species. The degree of reannealing
depends on how similar the DNAs
are to one another. If two strains
are very similar, their DNAs will
reanneal to a high degree. In contrast, if two strains are very different, then the extent of reannealing
will be much less. One way to perform DNA–DNA reassociation is to
radiolabel the DNA by growing the
bacterium with tritiated thymidine
Radiolabeled DNA
or 14C-labeled thymidine (other
DNA bases or 32P labeling can be
used as well). If the bacterium takes
up this labeled substrate and incorporates it into DNA, then the DNA
becomes labeled. Alternatively, the
DNA can be purified from the bacterium and labeled enzymatically in
the laboratory.
After the DNA has been labeled
and purified, it is sheared to an
appropriate length by sonication. It
is then ready for the hybridization
1 Mix radiolabeled single-stranded
DNA (obtained by labeling doublestranded DNA, then shearing and
denaturing it) with large amounts
of unlabeled single-stranded DNA
segments from (in this control
experiment) the same strain.
2 Heat the solution,
then slowly cool.
3 Reannealing occurs between
complementary segments.
4 Treat the solution with S-1 endonuclease to
digest any remaining single-stranded segments.
5 Collect the double-stranded segments
on a membrane filter, and measure the
amount of radiolabel; this reveals the
degree of reassociation (similarity
between strains).
DNA–DNA reassociation. In this example, which is a control experiment (the radiolabeled
sample is reannealed with unlabeled DNA from the same strain), the degree of reassociation is highest and treated as 100%. If a different strain is reannealed with the radiolabeled DNA, it will show a lower degree of reannealing (compared with the 100% attributed to the control), indicative of the similarity between the two strains being tested.
Strains with reannealing values of 70% or greater are considered to be the same species.
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Taxonomy of Bacteria and Archaea
BOX 17.3
503
Continued
experiments. First, single-stranded
DNA is prepared. This is accomplished by heating the isolated DNA
molecules to render them singlestranded and then cooling them
rapidly to prevent reannealing.
First, let’s look at the control
assay for the DNA reassociation
experiment. In this case, a small
amount of sheared radiolabeled
DNA is rendered single-stranded.
This is then mixed with a much larger amount of unlabeled DNA
obtained from the same bacterial
strain. These are heated together
and cooled slowly to allow the two
single-stranded groups to reanneal
to form hybrid double strands.
Because the amount of labeled
DNA relative to the unlabeled DNA
is small, there is a very low probability that it will reanneal with other
labeled strands. Most of the reassociations will occur between unlabeled strands, and most of the
remainder will be between the
labeled and unlabeled strands. The
single-stranded fragments that did
not reanneal are removed by
enzyme digestion using S-1
endonuclease, which specifically
degrades only single-stranded DNA,
and the double-stranded fragments
are collected on a membrane filter
or in a column. The amount of
radioactivity remaining on the filter
or on the column, after washing to
remove low-molecular-weight
material, represents the amount of
hybrid formation between the
labeled and unlabeled DNA for this
identical strain. This is the control
reaction, and the amount of radiolabel (the extent of hybridization) is
considered to be 100%.
To determine the extent of reassociation between the strain
described and an unknown strain,
similar experiments need to be performed. In this instance, unlabeled
single-stranded DNA from the
unknown strain is prepared and
mixed with the known strain for
which we have labeled the DNA. As
sequence of genes of a species. Indeed, sequencing entire bacterial genomes has become quite common (see
Chapter 16). It is worthwhile noting that one could consider that the actual DNA base sequence of a strain is the
ultimate definition of a strain—analogous to the chemical formula for a compound. However, it is important
to recognize that, unlike chemical compounds, bacterial
strains and species are not static; they continue to evolve.
Interestingly, the bacterial species definition appears
to be much broader than that used for animals and
plants when one considers DNA–DNA reassociation
and other molecular criteria. For example, the bacterial
species E. coli can be compared to its host mammalian
species using a variety of molecular features, including
the range in GC ratio, 16S rDNA sequence (versus 18S
rDNA sequence), and DNA–DNA reassociation (Table
17.2). Therefore, although there is essentially no variation in the range in GC ratio for the human species, it is
about 4 mol % within the E. coli species. Indeed, there
is less variation in GC ratio in the Primate order than
indicated previously, those strains
that show 70% or greater reassociation or hybrid formation with the
labeled strain (determined by the
amount of hybrid DNA that is radiolabeled compared with the same
strain control of 100% shown in the
figure) are considered to be the
same species. Anything less is considered a different species.
The temperature and salt concentration at which the DNA–DNA
reannealing occurs will influence
the degree of reassociation
between single strands. Scientists
conducting DNA–DNA reassociation experiments typically use a
reannealing temperature that is
25°C lower than the average midpoint temperature (Tm) of the DNAs
being compared.
This temperature is sufficiently
high that only those sequences
that are most complementary will
reanneal. Therefore, this is considered a stringent condition for reannealing.
there is in the species E. coli. Likewise, the 16S rDNA sequences of E. coli possess more than 15 substitutions,
whereas the difference between 18S rDNA of the mouse
(order Rodentia) and the human is less than 16. Finally,
DNA–DNA reassociation data, which are used to define
the bacterial species at 70% or greater, indicate that humans are much more highly similar to one another in
comparison with E. coli. Therefore, it is evident that the
typical bacterial species is equivalent to a genus or family of mammals based on molecular divergence, indicating that a bacterial species is defined much differently
from its eukaryotic counterparts. This difference is further evidenced by the biological species definition for
animals, which requires that within a species, mating between sexes produces fertile progeny.
The Genus
All species belong to a genus, the next higher taxonomic
unit. When DNA–DNA reassociation is performed
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Chapter Seventeen
TABLE 17.2
Comparison of E. coli and its host speciesa
Property
Comparison
Mole % G + C
Among E. coli
Among Homo sapiens
Among all primates
Between H. sapiens and mouse
Between H. sapiens and chimpanzee
Between H. sapiens and lemurs
a
48–52
42
42
—
—
—
16S or 18S rRNA
Substitutions
DNA/DNA
Reassociation
>15 bases
—
—
16 bases
—
—
>70%
—
—
—
98.6%
>70%
Adapted from J. T. Staley, ASM News, 1999.
within a genus, some species of the genus may show little or no significant reassociation with other species.
This does not indicate that they are unrelated to one another, only that this technique is too specific to identify outlying members of the same genus. Therefore,
DNA–DNA hybridization has limited utility for determining whether a species is a member of a known bacterial genus.
The definition of the genus is based on one or more
prominent phenotypic characteristics that permit it to be
distinguished from its closest relatives. Oftentimes some
striking physiological or morphological feature is present that permits the genus to be differentiated from
closely related taxa. For example, the genus Nitrosomonas
is a group of rod-shaped bacteria that grow as chemoautotrophs, gaining energy from the oxidation of ammonia. Other ammonia oxidizers with coccus-shaped and
helical cells are placed in other genera. Of course, all
strains of each of these genera need to be more closely
related to one another phylogenetically than to strains
of other genera in a phylogenetic classification. Ideally
then, the genus makes up a monophyletic lineage (i.e.,
one in which all are members of the same phylogenetic
cluster or clade).
thesis are sufficient to proclaim a separate genus, even
though two groups are otherwise very closely related.
As mentioned previously, the taxonomy of prokaryotes is undergoing major changes. Although according
to classical taxonomy each genus belongs to a family of
similar genera, relatedness at the familial and higher
level is often uncertain for bacteria. Bacteriologists have
been reluctant to ascribe organisms to formal Latinized
families and orders. However, as more becomes known
about bacterial phylogeny, it is increasingly apparent
that higher taxonomic levels do have meaning and can
be distinguished from one another by comparing the sequences of certain macromolecules, as is reflected in the
new edition of Bergey’s Manual of Systematic Bacteriology.
SECTION HIGHLIGHTS
Bacteria and Archaea are classified in a hierarchical structure from the domain level to the
phylum, class, order, family, genus, and finally
species. Species are described based on both
phenotypic and genotypic properties including GC ratio, 16S rRNA sequence, and
DNA–DNA hybridization.
Higher Taxa
Odd bedfellows are sometimes found in phylogenetic
trees; therefore, photosynthetic and nonphotosynthetic
members of some closely related groups have been reported. Of course, loss of a key gene or two may result
in converting a formerly photosynthetic organism to one
that is not photosynthetic. Consequently, although the
plant and animal kingdoms are differentiated on the basis of whether or not they are photosynthetic, both features have been reported in two closely related bacterial genera. However, because phenotype is so important
at the genus level, important features such as photosyn-
17.4 Major Groups of Archaea and
Bacteria
Bacteriologists have begun to construct classifications
using phylogenetic information from rRNA analyses. As
mentioned in Chapter 1, some prokaryotes are very different from others, a revelation that came through an
analysis of rRNA (Box 17.4). Ribosomal RNA data allow
the division of all organisms on Earth, prokaryotic and
eukaryotic, into three domains: Bacteria, Archaea, and Eukarya (see Figure 1.6). These domains can also be dis-
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Taxonomy of Bacteria and Archaea
BOX 17.4
505
Research Highlights
The Discovery of Archaea
Clearly, one of the most exciting
developments in bacterial classification of the twentieth century was
the discovery that there are two
major groups called Domains,
named the Bacteria and the
Archaea. The appreciation of the
difference between the Bacteria
and the Archaea was the culmination of years of research by microbiologists throughout the world.
However, the final piece of evidence that convinced microbiologists of this dichotomy was the discovery that these organisms had
very different 16S rRNAs. This
research was performed in Carl
Woese’s laboratory at the
University of Illinois. At that time,
rRNA sequencing was not done
routinely in laboratories. Instead,
16S rRNA was purified and digested
by ribonucleases that cut between
specific nucleotide pairs. The
oligonucleotide fragments
produced were subjected to
two-dimensional electrophoresis. The pattern of
spots on a two-dimensional
chromatogram represented
the various rRNA oligonucleotides that were typical of
each species. Studies from
Woese’s laboratory demonstrated that the patterns
were very different for
Bacteria and Archaea. Indeed,
their studies of 18S rRNA
from eukaryotic organisms
indicated that the Archaea
are as different from Bacteria
as they are from eukaryotes.
The seminal findings of this
work have forever changed
the way microbiologists view
taxonomy and phylogeny.
tinguished from one another by phenotypic testing. For
example, consider the cell envelope composition of the
organisms. Peptidoglycan is found only in Bacteria, although two groups—the mycoplasmas and the Planctomycetales—lack it. Furthermore, the lipids of the Bacteria and Eukarya are ester-linked, whereas they are
ether-linked in the Archaea (see Chapters 4 and 18).
At this time, 28 different phylogenetic groups, referred to here as phyla, are known. Included are 24
phyla of Bacteria and four phyla of Archaea. Each of these
phyla has specific signature sequences in their ribosomes
that are distinctive to them. The prokaryotic phyla are
listed here along with a brief description of their major
features. They are treated in more detail in subsequent
chapters (Chapters 18 through 22), and the groups in
each chapter are indicated below. It is noteworthy that
many new phyla of the Bacteria, in particular, have been
discovered in natural environments using clone library
approaches, but have not yet been isolated in pure culture (Box 17.5). Thus, at least ten to 15 additional major
prokaryotic groups are very poorly understood.
The second edition of Bergey’s Manual of Systematic
Bacteriology has been largely followed in organizing the
Carl Woese. Courtesy of Jason Lindsey.
bacterial groups treated in this book. The Archaea are described in Chapter 18 and the Bacteria in Chapters 19–22
(Table 17.3).
Domain: Archaea
The Archaea are divided into the following four phylogenetic groups or phyla.
CRENARCHAEOTA This phylum contains the most
thermophilic organisms known. Some of these organisms grow at temperatures higher than the boiling point
of water. Most rely on sulfur metabolism either as an energy source or as an electron sink. For example, some oxidize reduced sulfur compounds aerobically to produce
sulfuric acid. Others reduce elemental sulfur and use it
as an electron acceptor to form hydrogen sulfide. Some
are iron and manganese reducers. Not all organisms are
thermophilic. They are also significant in deep-sea environments as well as in polar seas.
The methanogens (methane producers) are noted for their ability to produce methane gas
EURYARCHEOTA
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506
Chapter Seventeen
Research Highlights
BOX 17.5
Novel Phyla Discovered by Molecular Analyses of Natural Habitats
S1
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the various 16S rDNA types
retrieved from a natural sample can
provide important information
about the diversity of prokaryotes
that occur in that environment.
Using these approaches, it has
recently been determined that
more than 50 major phyla of
Bacteria exist, yet isolates have
been obtained of only 24.
I
interest from the DNA. For phylogenetic (i.e., diversity) information,
16S rDNA primers for the Bacteria
or Archaea or universal primers that
amplify both groups are commonly
used. The segments retrieved, typically about 500 bp, can then be
sequenced to identify the phylogenetic groups. Although PCR
approaches are not quantitative,
Nitrospir
One of the major advances in
exploring the diversity of microorganisms in natural environments
has been the application of molecular approaches developed in
Norman Pace’s laboratory. In the
most recent variation of this
approach, DNA is extracted from
the environment of interest. Then
PCR is used to amplify genes of
W
S6
TM
7
Aq
a
eri
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Fu
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ter
bac
teo
Pro
11
OP
Archaea
A phylogenetic tree of 16S rDNA sequences of Bacteria, based on pure cultures and
clonal libraries from natural samples. Note the existence of many phyla (shown in
outline rather than as solid black lines) that have not yet been cultivated. Courtesy
of Phil Hugenholz and ASM Publications (Hugenholz, P., B. M. Goebel and N. R.
Pace. 1998. J. Bacteriol. 180: 4765–4774).
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Taxonomy of Bacteria and Archaea
TABLE 17.3
507
Overall organization for treatment
of Bacteria and Archaea
Bacterial Group
This Book
Bergey’s Manual of
Systematic Bacteriologya
Archaea
Proteobacteria
Gram-positive bacteria
Phototrophic bacteria
Other bacterial phyla
Chapter 18
Chapter 19
Chapter 20
Chapter 21
Chapter 22
Volume 1
Volume 2
Volumes 3 and 5
Volumes 1, 2, and 3
Volume 4
a
For more complete treatment of organization of taxa see Bergey’s Manual of Systematic
Bacteriology (2nd ed.).
from simple carbon sources. Some use carbon dioxide
and hydrogen gas, whereas others use methanol or
acetic acid. These archaea are anaerobes, some of which
grow at the lowest oxidation–reduction potentials of
all prokaryotes. Some of these archaea fix carbon dioxide, but they use neither the Calvin cycle nor the reductive tricarboxylic acid (TCA) cycle. Some of the Euryarcheota are hyperthermophilic. Extreme halophiles
make up another phenotypic subgroup. These extremely
halophilic archaea grow only in saturated salt-brine solutions. They lyse when placed in distilled water.
It should be noted that there is an overlap between
the extreme halophiles and methanogens. Thus, some
species of methanogens grow in high-salt environments.
NANOARCHEOTA This recently discovered group of
the Archaea comprises obligate parasites of other members of the Archaea. They are among the smallest of organisms, hence their name.
These archaeal microorganisms have
been found in hot springs, but no strains have yet been
isolated in pure culture, so little is known about their phenotypic properties.
KORARCHEOTA
Domain: Bacteria
The Bacteria are divided into a number of phyla, which
are described in the following text.
PROTEOBACTERIA The Proteobacteria comprise a very
large and diverse group of organisms. All four of the
major bacterial nutritional types are represented
within this group. Some of these organisms are photosynthetic (treated in Chapter 21), whereas some are
heterotrophic and others are chemolithotrophic. The
chemolithotrophic bacteria include the nitrifiers, the
thiobacilli, the filamentous sulfur oxidizers (Beggiatoa
and related genera), and many species that grow as hydrogen autotrophs. Carbon dioxide fixation, when
present, is via the Calvin cycle in all members of this
phylum.
This phylogenetic group contains many of the wellknown gram-negative heterotrophic bacteria such as
Pseudomonas, the enteric bacteria including E. coli, Vibrio
and luminescent bacteria, and the more morphologically
unusual bacteria such as the prosthecate bacteria. In addition, many symbiotic genera such as Agrobacterium,
Rickettsia, and Rhizobium are members of this group.
The gram-negative bacterial sulfate reducers such as
Desulfovibrio are also found in this group. Also included
in this phylum are the exotic myxobacteria that form
fruiting structures as well as unicellular, nongliding
forms such as the bacterial predator Bdellovibrio.
Finally, the mitochondria present in almost all eukaryotes evolved from this group of bacteria.
FIRMICUTES The bacteria in this group are all grampositive, although the Mycoplasma group lacks a cell wall
altogether and therefore stains as gram-negative. All
other members of the group contain large amounts of
peptidoglycan in their cell wall structure.
The Firmicutes are unicellular organisms that have a
low mol % G + C content. Most are cocci or rods, and
some produce endospores. Bacillus are aerobic or facultative spore formers, whereas Clostridium species are anaerobic fermenters. Some are sulfate reducers. One group,
the heliobacteria, are photosynthetic, and members produce a unique form of bacteriochlorophyll, Bchl g.
ACTINOBACTERIA These gram-positive bacteria range
in shape from unicellular organisms to branching, filamentous, mycelial organisms. Most are common soil organisms, some of which produce specialized dissemination stages called conidiospores, which enable them to
survive during dry periods.
This group contains the genus Chloroflexus, a green gliding bacterium that is metabolically
versatile. Members can grow as heterotrophs or photo-
CHLOROFLEXI
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508
Chapter Seventeen
synthetically. Carbon dioxide is not fixed by either the
Calvin cycle or the reductive TCA cycle but by a special
pathway known only for this group of bacteria.
PLANCTOMYCETES The Planctomycetes group of Bacteria are budding, unicellular, or filamentous bacteria.
These bacteria lack peptidoglycan.
CHLOROBI The green sulfur bacteria are anoxygenic
photosynthetic bacteria. Some are unicellular forms, and
others produce networks of cells. None are motile by flagella or gliding motility. Some have gas vacuoles. They
use the reductive TCA cycle rather than the Calvin
cycle to fix carbon dioxide.
CHLAMYDIAE
CYANOBACTERIA The cyanobacteria are the only bacteria that carry out oxygenic photosynthesis. This is a diverse group of bacteria ranging from unicellular to multicellular filamentous and colonial types. Some grow in
association with higher plants and animals. All cyanobacteria use the Calvin cycle for carbon dioxide fixation. The
chloroplast found in all eukaryotic photosynthetic organisms evolved from this group of bacteria.
The Chlamydiae are a group of obligately intracellular parasites and pathogens whose closest relatives are the Planctomycetes. They also lack peptidoglycan.
VERRUCOMICROBIA These bacteria are unusual in that
some members have bacterial tubulin genes. Very few
representatives of this phylum have been isolated in
pure culture, although they comprise up to 3% of the microbiota from soils.
This is a newly discovered phylum.
Some isolates are marine, others are intestinal symbionts
of mammals.
LENTISPHAERA
The spirochetes are morphologically
distinct from other bacteria. Their flexible cells are helical. All are motile due to a special flagellum-like structure, the axial filament, not found in other bacteria.
SPIROCHAETES
These bacteria are hydrogen autotrophs.
This phylogenetic group contains the most thermophilic
member of the Bacteria known and makes up one of the
deepest branches of the Bacteria.
AQUIFICAE
These are anaerobic bacteria, some of
which live in the gastrointestinal tracts of animals. Some
are cellulose degraders.
FIBROBACTERES
This is a fermentative genus that contains some of the most thermophilic members of the Bacteria known. The term “toga” refers to the outer extracellular material that surrounds the cells. They grow at
temperatures from 55°C to 90°C. Their cell lipids are unusual.
THERMOTOGAE
THERMOMICROBIA The genus Thermomicrobium contains small, rod-shaped thermophiles that grow as heterotrophs in hot springs with an optimal temperature
for growth of 70°C to 75°C. The cell wall contains very
low amounts of diaminopimelic acid.
This is a group of thermophilic sulfur-reducing bacteria.
THERMODESULFOBACTERIA
These bacteria are commonly found
in soils and sediments, but few strains have been cultivated. In addition to the aerobic genus Acidobacterium, this
phylum contains homoacetogenic bacteria, Holophaga, and
iron-reducing bacteria in the genus Geothrix.
ACIDOBACTERIA
These obligately anaerobic bacteria are
commonly found in the oral cavities and intestinal tracts
of animals.
FUSOBACTERIA
DICTYOGLOMI Species of this group are thermophilic,
obligately anaerobic fermentative bacteria.
DEINOCOCCUS–THERMUS This is a very small group
of organisms currently represented by very few genera. The genus Deinococcus contains gram-positive bacteria. However, they differ from other gram-positive bacteria in showing strong resistance to gamma radiation
and UV light. Thermus contains thermophilic, rodshaped bacteria. Ornithine is the diamino acid in the cell
walls of both Thermus and Deinococcus.
17.5 Identification
BACTEROIDETES This is a diverse group containing
heterotrophic aerobes and anaerobes. Some are gliding
heterotrophic bacteria that have a low DNA base composition (about 30 to 40 mol % G + C).
The final area of taxonomy is identification. Bacteriologists are often confronted with determining to which
species a newly isolated organism belongs. Clinical microbiologists need to know whether a specific patho-
SECTION HIGHLIGHTS
Currently four phyla have been described in
the domain Archaea and 24 have been described in the domain Bacteria.
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Copyright 2007 Sinauer Associates Inc.
Taxonomy of Bacteria and Archaea
genic bacterium is present so they can properly diagnose
a disease. Food microbiologists need to determine
whether Salmonella, Listeria, or other potentially pathogenic bacteria are present in foods. Dairy microbiologists
need to keep their important lactic acid bacteria in culture to produce a uniform variety of cheese. Brewers and
wine makers need to keep their cultures pure to inoculate the proper strains for quality control of their fermentations. Analysts at water treatment plants need to make
sure that their chlorination treatment is effective in
killing coliform bacteria in the treated water and distribution systems. Microbial ecologists need to identify
bacteria that are responsible for important processes
such as pesticide breakdown and nitrogen fixation.
The process of identification first assumes that the
bacterium of interest is one that has already been described and named. This is the usual case for most clinical specimens or specimens from known fermentations.
However, microbial ecologists often find that the organism they are interested in is new. It is estimated that less
than 1% of prokaryotic species have been isolated, studied in the laboratory, and named. Therefore, it is not always possible to identify a bacterium that has been isolated from an environmental sample.
Phenotypic Tests
Phenotypic tests based on readily determined characteristics are often used to identify a species. Most are simple to perform and inexpensive. Furthermore, the
amount of time and equipment required for conducting
genotypic tests such as DNA–DNA reassociation preclude the use of these tests in routine diagnosis. By performing a battery of some 10 to 20 simple phenotypic
tests, it is often possible to determine the genus, and perhaps even the species, of a clinically important bacterium, although some taxa are much more difficult to
identify than others.
Traditional methods for identification require growing the organism in question in pure culture and performing a number of phenotypic tests. For example, if
one wishes to identify a rod-shaped bacterium, the first
test would be a Gram stain. If the organism is determined
to be a gram-negative rod, the next questions to ask include the following: Is it motile? Is it an obligate aerobe? Is it fermentative? Does it have catalase? Can it grow
using acetate as a sole carbon source? The answers to
these questions will direct the investigator toward the
next step in identification. Conversely, if the organism
is a gram-positive rod, then a completely different set of
tests would need to be performed such as a test for endospore formation.
These tests take time to perform, and the appropriate
tests for one genus of bacteria differ from that of oth-
509
ers. Therefore, the results of one set of tests will determine which tests will need to be performed for further
clarification of the taxon. Sometimes several weeks
might be required to conduct all the tests needed to identify a strain. Rapid tests are extremely helpful, especially
in the medical, food, and water-testing areas. Fortunately, standardized, routine tests can be performed for
most clinically important bacteria that allow for their
rapid identification. Several companies have now produced commercial kits that are helpful in assisting in the
identification of unknowns (see Figure 30.4).
An increasingly popular approach to identification of
bacterial unknowns involves characterization of their
fatty acids. The fatty acids are found in membrane lipids
and are readily extracted from the cells and analyzed.
Different species of Bacteria produce different types and
ratios of fatty acids. The Archaea, of course, do not produce fatty acids, so this procedure is not of value for their
identification. However, they do produce characteristic
lipids that are useful taxonomically, especially for the
halobacteria.
The fatty acid analysis procedure involves hydrolyzing a small quantity of cell material (about 40 mg is all
that is needed) and saponifying it in sodium hydroxide.
This is acidified with hydrochloric acid in methanol so
that the fatty acids can be methylated to form methyl esters. The fatty acid methylated esters (FAME) are then
extracted with an organic solvent and injected into a gas
chromatograph. The resulting chromatogram (Figure
17.11) can be used to identify the fatty acids that are indicative of a species. Commercial firms have developed
databases of fatty acid profiles that can be used for the
identification of species. The advantage of this procedure is that many samples can be analyzed quickly and
without great effort. However, all organisms must be
grown under controlled conditions of temperature and
length of incubation and on the same medium.
Nucleic Acid Probes and Fluorescent Antisera
One exciting current area of research and commercial
application involves the development of DNA or RNA
“probes” that are specific for the signature sequences
of rRNA or some other appropriate gene such as an enzyme that is characteristic of a species of interest. The
probes are labeled in some manner so that the hybridization can be visualized. This is accomplished either by
making the probes radioactive or by tagging them to a
fluorescent dye or an enzyme that gives a colorimetric
reaction.
By the proper selection or design of probes, it is possible to identify an organism to a domain, genus, or
species by demonstrating specific hybridization to the
probe (see Chapter 25).
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510
Chapter Seventeen
10.024
1.601
10.322
Solvent
3500
13.477
18:1 omega 7 cis,
omega 9 trans,
omega 12 trans
16:1
omega
7 cis
16:0
Quantity of fatty acid
3000
2500
4.147
10:0
2000
16:0
2O H
4.668
12:0
1500
19:0 cyclo
omega 8
12.450
15.354
1
5
10
15
20
Retention time (minutes)
Figure 17.11 Fatty acid analysis
Fatty acid methyl ester (FAME) chromatogram of an
unknown species, showing chromatographic column retention times and peak heights. Note: 10:0, 12:0, 16:0, and 19:0
indicate saturated fatty acids with 10, 12, 16, and 19 carbons;
16:1 and 18:1, monounsaturated 16-carbon and 18-carbon
fatty acids; omega number, the position of the double bond
relative to the omega end—that is, the hydrocarbon end (not
the carboxyl end)—of the fatty acid chain; cis and trans, the
configuration of the double bond. For example, omega 7 cis
Potential applications for probe technology are considerable. A number of commercial firms are already
marketing probes to identify pathogenic bacteria from
clinical samples. A goal of this technology is to enable
the rapid identification of organisms directly from clinical or environmental samples without actually growing them in culture first. Fluorescent antiserum tests
are also useful. For example, Legionella spp., the
causative agents of Legionnaires’ disease, are very difficult to cultivate. However, good fluorescent antisera
are available for the identification of these species directly from clinical samples or even from environmental samples (Figure 17.12).
Another approach in the identification of species is
through the use of multiple locus sequence typing
(MLST). In this approach, several core genes (typically
seven or eight) of an organism are sequenced and used
to compare the organism to known species. In the clin-
indicates a cis double bond between the seventh and eighth
carbons from the omega end of the fatty acid. Also, 2OH
indicates a hydroxyl group at the second carbon from the
omega end; cyclo omega 8, a cyclo-carbon at the eighth position from the omega end. The 18:1 omega 7 cis, omega 9
trans, and omega 12 trans peak results from either one fatty
acid or a mixture of fatty acids with double bonds at the
three positions indicated (the chromatographic column does
not separate these three fatty acids). Courtesy of MIDI
(Microbial Identification, Incorporated, Delaware).
ical setting, if the organism has been isolated from a
patient, its MLST “type” can be compared to that of a
large database to determine whether it belongs to a
known pathogenic species. The MLST approach is rapidly gaining acceptance as a means of identification of
pathogenic strains and species.
Culture Collections
Unlike plants and animals, many bacteria and archaea
can be easily grown in pure culture and preserved by
freeze-drying (lyophilization), handled in small test
tubes and vials, and readily sent anywhere in the world.
As lyophils, many of these organisms remain viable for
10 to 20 years or more and can be revived and studied by
anyone anywhere. Cultures can also be frozen at –80°C
in vials containing a suspension medium amended with
15% glycerol. These remain viable for many years. Thus,
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Taxonomy of Bacteria and Archaea
511
Figure17.12 Legionella
fluorescence
Legionella species are difficult to
grow, but can be identified to
species by fluorescence
microscopy using tagged and
specifically labeled antisera.
In this image the bacteria are
yellow-green. ©Michael
Abbey/Visuals Unlimited.
unlike plants, for which an herbarium is used to preserve
the specimens collected of an original species, bacteria
are preserved as type cultures that are clones of the original viable type strain of a species. The type strain of a
species is the one on which the species definition has been based.
Through culture collections type strains are made available to professional microbiologists throughout the
world. Many countries maintain national collections of
microorganisms, such as the American Type Culture Collection (ATCC) in the United States (www.atcc.org). If a
microbiologist from India wishes to determine if he or
she has a new species, the original type strain can be
obtained from a culture collection and used to conduct
DNA–DNA reassociation assays and other tests to compare it with his or her isolates.
Because of the importance of biological materials to
science and industry, culture collections have become biological resource centers. Thus, strains that have been
patented are also deposited in culture collections so that
they are accessible. Clones of genomes that have been
sequenced are also deposited as are viruses and other
biological materials.
SECTION HIGHLIGHTS
A number of tests are used to identify bacteria
that have been isolated from clinical specimens and natural sources. Some tests are phenotypic, whereas others use molecular sequence information.
SUMMARY
• Bacterial taxonomy or systematics consists of three
areas: nomenclature, classification, and identification. Nomenclature is the naming of an organism.
An International Code for the Nomenclature of
Bacteria has been published containing the rules for
naming Bacteria and Archaea.
• Classification is the organization of Bacteria and
Archaea into groups of similar species. Bacteria and
Archaea are classified in increasing hierarchical rank
from species, genus, family, order, class, phylum,
and domain. Artificial classifications are not based
on the evolution of organisms but on expressed features or the phenotype of an organism that includes
properties such as cell shape and nutritional patterns. Phylogenetic classifications are based on the
evolution of a group of organisms. Bacterial phylogeny is now based on the sequence information
from the highly conserved macromolecule, 16S
rRNA, as well as the sequences of other genes and
proteins.
• Phylogenetic trees, with branches and nodes, can
be constructed based on the sequence of macromolecules such as rRNA. The length of the branch represents the inferred difference (number of changes)
between organisms. External nodes represent
extant species, whereas internal nodes represent
ancestor species. Rooted trees are based on a comparison of related species and an out-group.
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Chapter Seventeen
• Speciation is the process whereby organisms evolve.
Typically genetic material is transferred from the
parental bacterium to the progeny through vertical
inheritance. Horizontal gene transfer (HGT) occurs
in which genetic material is transferred among bacteria that may or may not be closely related.
• A bacterial species is a group of similar strains that
show at least 70% DNA–DNA hybridization.
Organisms of the same species will have similar if
not identical DNA mol % G + C content. Organisms
that have the same GC ratio are not necessarily similar. Based on molecular criteria, such as DNA–DNA
reassociation, bacterial species are much more
broadly defined than are plant and animal species.
• Identification is the process whereby unknown
cultures can be compared to existing species to
determine if they are sufficiently similar to be
members of the same species.
• Type strains of all species must be deposited in at
least two different types of culture collections,
repositories where strains are preserved by
lyophilization and deep freezing. The culture collections provide cultures to microbiologists worldwide so they can compare unidentified strains to
the official type strains.
i Find more at www.sinauer.com/microbial-life
REVIEW QUESTIONS
1. Is it important to name and classify bacteria?
2. What procedure(s) are necessary to identify a
bacterial isolate as a species?
3. Differentiate between an artificial and a phylogenetic classification.
4. In what ways does the classification of Bacteria
differ from that of eukaryotic organisms?
5. How do the Archaea differ from Bacteria? From
eukaryotes?
6. How is DNA melted and reannealed, and why is
this useful in bacterial taxonomy?
7. How would you go about identifying a bacterium that you isolated from a soil habitat?
8. Why is morphology of little use in bacterial classification? Is it of any use?
9. What is weighting and should phenotypic features be weighted in a bacterial classification
scheme?
10. Distinguish between lumpers and splitters.
11. If you were working in a clinical laboratory, outline the types of procedures you would use to
identify isolates. Why do you recommend using
the procedures you suggest?
12. Why is rRNA of use in bacterial classification?
13. Compare the information obtained from determining the DNA base composition (GC ratio)
with that obtained by DNA reassociation
experiments.
SUGGESTED READING
Boone, D., R. Castenholz and G. Garrity, eds. 2001. Bergey’s
Manual of Systematic Bacteriology. 2nd ed., Vol. 1. New
York: Springer-Verlag.
Brenner, D. J., N. R. Krieg, J. T. Staley and G. Garrity, eds.
2005. Bergey’s Manual of Systematic Bacteriology. 2nd ed.,
Vol. 2. New York: Springer-Verlag.
Gerhardt, P., ed. 1993. Methods for General and Molecular
Microbiology. Washington, DC: ASM Press.
Graur, D. and W. H. Li. 2000. Fundamentals of Molecular
Evolution. 2nd ed. Sunderland, MA: Sinauer Associates,
Inc.
Hall, B. G. 2004. Phylogenetic Trees Made Easy. 2nd ed.
Sunderland, MA: Sinauer Associates, Inc.
COMPUTER INTERNET RESOURCES
American Type Culture Collection: http://www.atcc.org/.
This site has a listing of all the bacterial strains deposited in the American Type Culture Collection, as well as
growth media and conditions.
Bergey’s Manual Trust. Headquarters at the University of
Georgia. This website has information on the current
classification of Bacteria and Archaea:
http://www.bergeys.org/.
National Center for Biotechnology Information (NCBI):
http://www.ncbi.nlm.nih.gov/. This center contains
information that allows for comparison of genes from
different organisms through BLAST (Basic Local
Alignment Search Tool), Genbank, which contains a
huge collection of gene sequences that can be used for
comparative analyses and a section on taxonomy.
Ribosome Database Project (RDP), Michigan State University:
http://rdp.cme.msu.edu/. This site has information on
the 16S rRNA sequences of more than 250,000 bacterial
and archaeal sequences. The database allows one to conduct phylogenetic analyses of unknown strains whose
16S rRNA sequence has been determined and to compare the sequence with those already reported.
Comparative RNA Web Site: www.rna.icmb.utexas.edu. A
remarkable collection of RNA sequence information
presented with secondary structure models, conservation diagrams, and more. Published as Cannone, J. J.,
et al. 2002. “The Comparative RNA Web Site: An online
database of comparative sequence and structure information for ribosomal, intron, and other RNAs.” BioMed
Central Bioinformatics 3: 2.
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