Download Bacillus taxonomy in the genomic era finds

Infection, Genetics and Evolution 11 (2011) 789–797
Contents lists available at ScienceDirect
Infection, Genetics and Evolution
journal homepage: www.elsevier.com/locate/meegid
Review
Bacillus taxonomy in the genomic era finds phenotypes to be essential though
often misleading
Heather Maughan a,1,*, Geraldine Van der Auwera b,c,1
a
Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, ON, Canada M5S 3B2
Department of Microbiology & Molecular Genetics, Harvard Medical School, MA, USA
c
Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Louvain-la-Neuve, Belgium
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 14 September 2010
Received in revised form 31 January 2011
Accepted 1 February 2011
Available online 18 February 2011
Bacillus is a diverse bacterial genus characterized by cells growing aerobically and forming dormant
endospores. Although Bacillus species were some of the first bacteria ever characterized, their
relationships to one another remain enigmatic. The recent deluge of environmental sequencing projects
has further complicated our view of Bacillus taxonomy and diversity. In this review we discuss the
current state of Bacillus taxonomy and focus on two examples that highlight the ecological diversity
found within identical 16S rDNA-based clusters: the identification of ecologically distinct clusters of B.
simplex in Evolution Canyons and the demarcation of species in the industrially and medically important
B. cereus group. These examples highlight the difficulties of purely 16S rDNA-based taxonomy,
emphasizing the need to interpret the massive amounts of molecular data from environmental
sequencing projects in a bacterial ecology framework. Such interpretations are likely to reveal ecological
diversity within Bacillus that extends beyond that previously imaginable, providing a true picture of
Bacillus ecology and evolution.
! 2011 Elsevier B.V. All rights reserved.
Keywords:
Bacillus
Taxonomy
16S rDNA
Bacillus cereus sensu lato
Phylogenetics
Bacterial species
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . .
Historical overview of Bacillus taxonomy .
Modern Bacillus taxonomy . . . . . . . . . . . .
Limitations of 16S taxonomy . . . . . . . . . .
4.1.
B. simplex . . . . . . . . . . . . . . . . . . . . .
4.2.
B. cereus group . . . . . . . . . . . . . . . .
Reconciling molecules and ecology. . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
The genus Bacillus is comprised of low G+C Gram positive
bacteria (Kingdom Bacteria; Phylum Firmicutes; Class Bacilli;
Order Bacillales; Family Bacillaceae) and is most closely related to
the genera Listeria, Streptococcus, and Staphylococcus (Ciccarelli
et al., 2006; Wu et al., 2009). Bacillus species are ubiquitous in
* Corresponding author. Tel.: +1 416 946 7121.
E-mail address: [email protected] (H. Maughan).
1
These authors contributed equally to this work.
1567-1348/$ – see front matter ! 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2011.02.001
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789
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nature, having been isolated from environments as diverse as
freshwater, saline water, soil, plants, animals, and air (Pignatelli
et al., 2009). The phenotypic diversity encompassed by members of
the Bacillus genus is spectacular: high temperatures, extreme
salinity, acidic conditions and the immune systems of many
animals pose little challenge to some members (Holt, 1986). Some
species use unusual terminal electron acceptors like arsenic or
selenium (Switzer Blum et al., 1998), have increased growth in the
presence of activated charcoal (Fujita et al., 1996), and are capable
of colonizing environments as peculiar as paintings (Taubel et al.,
2003). The metabolic breadth of Bacillus has been harnessed by
industry for the production of molecules such as riboflavin,
790
H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797
Streptavidin, b-lactamase, and a diversity of insect and nematode
toxins (de Maagd et al., 2003; Zeigler and Perkins, 2009). At least
two Bacillus species, B. cereus and B. anthracis, infect humans
causing food-borne illness and anthrax, respectively. These
examples illustrate the usefulness of some Bacillus species and
the danger of others. However, as we discuss in this review,
resolving Bacillus isolates into neatly defined species with welldefined boundaries is not straightforward.
The classification of strains and species in an appropriate
manner is vital for the responsible production of industrially
important molecules and the effective control of insect and animal
pathogens. Moreover, the exponential growth of genomic databases from environmental sequencing projects will require a firm
ecological and evolutionary framework in which to classify Bacillus
sequences and appropriately define new species. The need for
efficient classification has been made apparent by recent research
suggesting that Bacillus species boundaries defined by gene
sequence data are not always in agreement with boundaries
defined by ecology; we discuss this issue below in the context of B.
cereus and its close relatives. Bacillus is not unique in this respect,
as classifications of other genera have similar issues. Thus, the
current challenge, in bacterial taxonomy in general and Bacillus
taxonomy in particular, is to find a systematic way to reconcile
molecular data with ecology for a valid and biologically meaningful
classification (Achtman and Wagner, 2008; Cohan, 2002; Fox et al.,
1992; Koeppel et al., 2008). We highlight these issues in Bacillus
and discuss the implications for the levels of ecological diversity
likely present, based on environmental sequencing projects.
One lineage that suitably illustrates the disagreement between
molecular and phenotypic/ecological methods of classification in
Bacillus is the B. cereus group. This group, also called B. cereus sensu
lato, contains six very closely related species according to current
taxonomy: B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B.
pseudomycoides, and B. weihenstephanensis. Over the past century
or so, these six species were described as individual species of the
Bacillus genus using pathogenic host range, colony morphology
and metabolic properties as distinguishing criteria, along with
motility, resistance to penicillin and sensitivity to gamma phage.
However, molecular methods have since shown that the species
boundaries between members of this group are difficult to define,
forcing us to rethink our current descriptions of these and other
Bacillus species.
2. Historical overview of Bacillus taxonomy
Bacillus species such as B. anthracis and B. subtilis were among
the first species of bacteria ever described in a systematic manner
in the late 19th century by premier German biologists Ferdinand
Cohn and Robert Koch (Cohn, 1962; Koch, 1962). Cohn was first to
discover sporulation in B. subtilis (Cohn, 1962) and Koch famously
proved the germ theory of disease using B. anthracis (Koch, 1962),
the causative agent of anthrax and early biological warfare agent.
Cohn and Koch classified bacteria based on cell shape (sphericals,
short rods, threads, and spirals). The use of such crude
morphological criteria, imposed by the technological limitations
of the time, led to an overly simplistic and often misleading
classification that failed to reflect relationships of common
ancestry between organisms. For example, Escherichia coli and
Pseudomonas aeruginosa were classified as species of Bacillus by
virtue of their rod-shaped cells (Daegelen et al., 2009; Hugh and
Leifson, 1964). Furthermore, use of the ability to form spores as a
defining criterion resulted in the inclusion in the Bacillus genus of a
considerable diversity of bacteria that have since been reassigned
into different genera (Ash et al., 1991, 1993; Shida et al., 1996).
In the 20th century, the study of bacteria blossomed from
simple microscopic observations to the detailed study of cell
structure and physiology. In addition to providing the first glimpse
of molecular biology, these studies enabled the characterization of
any bacterial culture using a series of biochemical, physiological,
and morphological tests, e.g., catalase enzyme production, carbon
source utilization, lipid composition, and the presence and
positioning of flagella. These characterizations led to a formalized
classification in reference books such as Bergey’s manual (Holt,
1986), which microbiologists could use as a key to identify novel
isolates of interest. Using such criteria Bacillus was defined as
Gram-positive, aerobic, motile, spore-forming rods that produce
catalase; additional tests were used to further characterize species
within the Bacillus genus.
Although biochemical testing was a very useful tool that
yielded invaluable details about function and ecology, it suffered
from two major drawbacks. In practice, despite being relatively
easy to perform, the biochemical tests were difficult to standardize
between laboratories and could only be performed on bacteria that
would grow under ordinary laboratory conditions. This method of
classification would therefore be inapplicable to fastidious or
uncultured organisms, which are estimated to represent a huge
proportion of the true bacterial diversity to be found in nature. But
most importantly, the functional nature of such tests meant that
they yielded little information concerning the genetic basis for the
abilities being probed, and potentially misleading information in
terms of evolutionary relatedness. Indeed, between any two
isolates, shared abilities could be due to evolutionary convergence
or, in the case of shared genetic material, horizontal gene transfer.
Conversely, shared disabilities could be due to independent gene
loss events or differential regulation. Therefore, phenotypic
similarities cannot be taken with certainty to indicate close
evolutionary relatedness.
During the heyday of phenotype-based taxonomy, the DNA of
many isolates was also compared using pairwise whole genome
hybridization techniques. These techniques were based on
comparing hybridization kinetics for DNA of a single strain vs.
DNA of a pair of divergent strains; DNA of a single strain will
hybridize quickly whereas divergent DNA will take longer to
hybridize, the more divergent the longer, or in the case of unique
regions of DNA, will not hybridize at all. Although hybridization
techniques enabled the quantification of genetic distance between
different isolates, they were only useful for very closely related
species. The contribution of molecular genetics to bacterial
taxonomy really flourished when American biologist Carl Woese
showed that the gene sequence encoding the ubiquitous 16S rRNA
gene of the ribosomal subunit could be used to reconstruct
phylogeny on an unprecedented scale (Olsen and Woese, 1993;
Woese et al., 1990), finally making it possible to classify bacterial
species in an evolutionary framework. 16S rDNA sequencing
seemed at the time to be the perfect solution since it is relatively
inexpensive, fully reproducible between laboratories, does not
require culturing of the bacteria and it is a universal molecule. The
general outcome of Woese’s work was an evolution-based view of
organismal taxonomy that divided prokaryotes into eubacteria and
archaea and joined these with eukaryotes to result in the three
domains of life (Woese et al., 1990). It also enabled a fairly detailed
mapping of phylogeny within eubacterial genera, including a
considerable amount of remapping and re-assigning species to
different genera than what the original identifications made using
classical criteria had dictated.
16S rDNA-based taxonomy seemed like a clear way forward,
except in the case of closely related species groups such as Bacillus,
where insufficient divergence in 16S rDNA prevented the
resolution of strain and species relationships. Subsequent use of
housekeeping genes that are essential and thus not lost from
genomes, but that evolve more quickly than 16S rDNA, has proven
to be useful for taxonomic classification (Palys et al., 2000, 1997).
H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797
791
Fig. 1. Evolutionary relationships of 59 Bacillus species inferred using (A) Maximum Likelihood analysis of the 16S rDNA locus and (B) rearrangement of branches in A to
minimize the number of evolutionary changes in the following 11 phenotypes: maximum growth temperature, minimum growth temperature, Voges–Proskauer test, ability
to grow anaerobically, acid production from glucose, acid production from arabinose, acid production from mannitol, hydrolysis of starch, flagella present, spore shape, and
sporangium swelling. Numbers on the molecular tree indicate support from 1000 bootstrap pseudoreplicates in FastTree (Price et al., 2009, 2010). Tree branch
rearrangements were done in Mesquite (Maddison and Maddison, 2009) using the ‘‘Search for a Better Tree’’ function. Listeria monocytogenes was chosen as the outgroup
because it is a well-supported sister group of Bacillus (Ciccarelli et al., 2006; Wu et al., 2009). Phenotypic information was obtained from the following sources: (Ajithkumar
et al., 2002; Arfman et al., 1992; Boone et al., 1995; Boyer et al., 1973; Combet-Blanc et al., 1995; De Clerk et al., 2004; Fujita et al., 1996; Gordon et al., 1977; Heyrman et al.,
2003; Holt, 1986; Kanso et al., 2002; Kuhnigk et al., 1995; Lechner et al., 1998; Li et al., 2002; Logan et al., 2000, 2002; Nagel and Andreesen, 1991; Nakamura, 1989, 1998;
Nakamura et al., 1988, 1999; Nielsen et al., 1995; Palmisano et al., 2001; Pettersson et al., 2000, 1996; Pichinoty et al., 1983; Priest et al., 1987, 1988; Reva et al., 2002; Roberts
et al., 1994, 1996; Spanka and Fritze, 1993; Switzer Blum et al., 1998; Venkateswaran et al., 2003; Yoon et al., 2001, 2003; Yumoto et al., 2003; Zhou et al., 2008).
Although such approaches are useful for single isolates studied
intensively in the laboratory, 16S rDNA remains the gold standard
for environmental sequencing projects due to its ubiquity and ease
of amplification from divergent species (e.g., Pignatelli et al., 2009).
To illustrate how taxonomic relationships may differ between
phenotypic and 16S rDNA-based inferences, we inferred the
relationships of 59 Bacillus species using 16S rDNA sequences, and
subsequently altered the topology to minimize changes in 11
phenotypes (see Fig. 1 legend for details). These phenotypes were
chosen based on their early use to classify Bacillus in Bergey’s
manual, and subsequent species descriptions were used for
additional phenotype information (see Fig. 1 legend for references). As shown in Fig. 1, relationships between species are
drastically different when phenotypic information is used to refine
the tree topology. For example, the clade containing the wellstudied B. subtilis subsp. subtilis (blue font in Fig. 1) has high
support in the molecular tree, but when phenotypic information is
used to rearrange branches, most species in this clade are
dispersed throughout the tree. However, many of the closest
relatives of B. subtilis subsp. subtilis in the molecular tree (B.
mojavensis, B. pumilus, B. vallismortis, and B. atrophaeus) maintain
each other as close relatives in the phenotypic tree. The retention
of closely related groups is also seen with the B. cereus group,
where most of these species are close relatives in both trees. This
indicates that when strains/species have low 16S rDNA divergence,
many of their phenotypes are likely to be similar, but the converse
is not true: strains/species with similar phenotypes are not
necessarily closely related at the 16S rDNA level. This concept is
not novel; although there are disadvantages to using only the 16S
rDNA locus for phylogenetic reconstruction (Forney et al., 2004),
the underlying idea that molecular data should be the basis of
modern bacterial taxonomy has become largely uncontroversial.
3. Modern Bacillus taxonomy
What have 16S rDNA data told us about Bacillus? Sequencing
16S rDNA from environmental samples has given us a renewed
appreciation of the diversity of Bacillus. To evaluate this diversity
we inferred a new Bacillus tree using 16S rDNA sequences available
in the Ribosomal Database Project (RDP; Cole et al., 2009); these
sequences correspond to both cultured, well-studied Bacillus
species as well as 16S rDNA sequences obtained by direct
sequencing of environmental samples. 7510 sequences were
aligned and their relationships inferred with Maximum Likelihood
in FastTree using 16S rDNA sequences from the closely related
genus Listeria for an outgroup (Price et al., 2009, 2010). Sequence
identity cutoffs were implemented in TreeChopper (http://microbiomeutil.sourceforge.net/). We found that using the conventional
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H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797
previous phenotype-based classifications. In the next section we
present two examples.
4. Limitations of 16S taxonomy
16S rDNA-based taxonomy suffers from the major drawback
that ecology does not inform species designations and cutoff values
are essentially arbitrary. We present two examples where
consideration of ecologically important phenotypes leads to a
significantly improved species grouping than obtained using only
16S rDNA sequencing. In the first example we discuss how B.
simplex lineages with the same 16S rDNA sequence correspond to
divergent ecotypes. In the second example, we discuss how some
very closely related lineages of bacteria in the B. cereus group are
designated as different species despite having the same 16S rDNA,
for historical reasons and partly because their ecologies have
repercussions on human affairs. Although 16S-rDNA based
taxonomy has failed to identify ecologically important clusters
in these species groups, molecular techniques focused on more
rapidly evolving portions of the genome have identified such
clusters, or ecotypes, in B. simplex. This has proven to be more
difficult in the B. cereus group.
Fig. 2. Bacillus phylogeny based on Maximum Likelihood analysis of 16S sequences
listed as Bacillus (or Listeria for outgroup) in the Ribosomal Database Project as of
July 2009. The inner most ring shows the branches indicating evolutionary
relatedness and divergence. These branches are extended with dashes to the
species names (black ring fanning out towards outside of circle). Colors indicate
the following: blue: B. cereus group; yellow: B. subtilis; green: uncultured; aqua:
Listeria outgroup. No color indicates a previously described Bacillus species
outside the B. subtilis and B. cereus groups. Species names are as assigned in the
RDP database. Numbers refer to the species groups discussed in the main text (1: B.
subtilis group; 2: B. cereus group; 3: uncultured or previously described Bacillus
species).
cutoff of 97% identity (Stackebrandt and Goebel, 1994) predicts the
existence of 116 species of Bacillus.
Fig. 2 shows the evolutionary relationships of the 7510 16S
rDNA sequences. Culture-associated sequences designated as B.
subtilis (yellow labels and clade 1 in Fig. 2) or belonging to the B.
cereus group (blue labels and clade 2 in Fig. 2) dominate the 16S
rDNA phylogeny, which is easily explained by the extensive
sampling and sequencing efforts that focus on these groups. The
interesting observation here is that despite the disproportionately
large amount of sampling, the 16S rDNA diversity within these
groups is low, as evidenced by the shallow branch lengths that
populate them. This marks a striking contrast to the generally
much longer branch lengths extending to 16S rDNA sequences
from uncultured environmental samples not belonging to B. subtilis
or B. cereus (green labels and clade 3 in Fig. 2), and described
species represented by only one or a few sequences (no label and
clade 3 in Fig. 2). Crucially, these longer branch lengths underscore
the tremendous diversity present outside of the two well-studied
groups, B. subtilis and B. cereus, greatly extending previous
descriptions of this diversity (Priest, 1993; Zeigler and Perkins,
2009). Furthermore, although most sequences from uncultured
samples are clustered together with other previously described
Bacillus species that are not members of the subtilis or cereus
groups, over one hundred are actually interspersed within the B.
subtilis and B. cereus groups. This strongly suggests that although
these groups are thought to be very well-studied, there may be
some fundamental aspects of their diversity that still defy our
understanding.
Although 16S rDNA sequencing has been exceptionally
powerful for taxonomical classification in an evolutionary
framework, it has also highlighted some major issues with
4.1. B. simplex
Nevo and colleagues have spent years measuring ecological
divergence between strains of B. simplex living in different habitats
within three ‘‘Evolution Canyons’’ (EC) in Israel (Sikorski and Nevo,
2005, 2007). The north facing slope, valley bottom, and south
facing slope within each EC differ in their soil characteristics and
the UV and sunlight intensities they receive, but these environmental parameters are comparable between the corresponding
areas of the ECs (Fig. 3). This consistently replicated pattern of
divergent ecological conditions within a relatively small geographic space has provided an excellent system in which to address
ecological divergence and sympatric speciation. Hundreds of
strains were isolated from each of the three canyon environments
and characterized at the genetic and phenotypic levels. Although
all strains were identical at the 16S rRNA gene, distinct genetic
profiles could be distinguished using Random Amplified Polymorphic DNA-PCR fingerprinting techniques and sequencing of
housekeeping genes. Phenotypic profiles were then drawn by
measuring UV survival, mutation rates, and growth at high
temperature for each strain. Comparison of phenotypic and
genetic profiles showed that strains from similar ecologies were
more similar to each other than were strains from adjacent
geographical areas. For example, strains living on the hotter south
facing slopes of each of the three canyons were more similar to
each other, regardless of canyon of origin, than to strains on the
opposite slopes of their respective canyons of origin (Fig. 3).
Simulations predicting ecologically distinct groups of strains
were used with sequence data from housekeeping genes for a
subset of strains (Koeppel et al., 2008). These simulations predicted
ecologically distinct lineages that corresponded nicely with the
phenotype data, indicating that lineages with different ecologies
are diverging at the genetic level. Because ecologically distinct
populations are not subject to each other’s selective sweeps,
divergence can occur at the genetic level (Cohan, 2002; Koeppel
et al., 2008). Taken together, these results show that even though
all B. simplex strains share the same 16S rDNA sequence, they are
adapted to very different ecologies.
4.2. B. cereus group
B. cereus, B. anthracis, B. mycoides and B. thuringiensis were
described very early on in the history of the Bacillus genus, on the
H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797
793
Fig. 3. Evolution Canyons diagram simplified from Sikorski and Nevo (2007); the origin of B. simplex samples is coded by geometric shape (circles for Canyon 1, squares for
Canyon 2) and color (blue-green for north-facing slopes, orange for south-facing slopes); generalized phenotypic similarity between the various groups of samples is
represented in the insert box, top right, showing that similarity correlates with type of slope of origin rather than canyon of origin.
basis of purely phenotypic criteria. B. anthracis was described as
mentioned earlier in relation to the pathogenesis of anthrax
disease. B. cereus was described as a microbe commonly associated
with cereals (Frankland and Frankland, 1887). During the same
period, B. mycoides was described on the basis of the striking
rhizoid morphology its colonies exhibit during growth on nutrient
media (Flügge, 1886). Thirty years later, B. thuringiensis was
described based on its production of parasporal protein crystals
and their associated entomocidal properties (Berliner, 1915).
However, the emergence of DNA hybridization techniques, then
16S rDNA and multilocus sequence typing (MLST; http://mlstoslo.uio.no), progressively revealed that these species were much
more closely related than what their taxonomical status implied.
16S rDNA provided insufficient resolution at the species level and
while several MLST schemes proved useful for distinguishing
clades, they could not resolve the traditional species either. The
concept of a B. cereus sensu lato group drawing these species tightly
together was therefore introduced about a decade ago to
acknowledge that proximity, with the qualifier sensu stricto added
to ‘‘regular’’ B. cereus strains to distinguish them from the group
name (Léonard et al., 1997). At roughly the same time, two
additional species were described as members of the group: B.
pseudomycoides as a genetic variant of B. mycoides (Nakamura,
1998) and B. weihenstephanensis as a psychrotrophic variant of B.
cereus s. s. (Lechner et al., 1998). Most recently a cluster of
thermophilic strains of clinical origin was proposed to represent a
new species, to be named Bacillus cytotoxicus (Lapidus et al., 2008).
These strains are still referred to as B. cereus subsp. cytotoxis in the
literature and public databases pending official approval of the
new species designation.
There is in fact a growing consensus in the research community
that the members of the B. cereus group should be considered as
forming one single species from which different ecotypes and
pathotypes emerge in a dynamic fashion, leading in some cases to
the formation of clonal complexes with specific phenotypes
(Helgason et al., 2000; Priest et al., 2004; Rasko et al., 2005;
Tourasse et al., 2006). This view stems from crucial insights
obtained over the last decade from the combination of MLST,
genomics and the study of large plasmids. Indeed, we now know
that the main phenotypical properties that were originally used to
distinguish B. cereus s. s., B. thuringiensis and B. anthracis are directly
related to the presence or absence of large plasmids, which carry
the genetic determinants that are responsible for those phenotypes. In B. anthracis, the virulence plasmids pXO1 (192 kb) and
pXO2 (96 kb) encode the anthrax toxin and capsule genes,
respectively, as well as associated regulatory elements (Koehler,
2009). In B. thuringiensis there can be one or several so-called ‘Cry
plasmids’, most of them conjugative, that encode insecticidal
crystal toxins (Schnepf et al., 1998); B. thuringiensis strains that
have lost their toxin-bearing plasmids are effectively indistinguishable from B. cereus s. s. strains. B. cereus s. s. strains encode a
range of toxins and other extracellular virulence factors on the
chromosome, but it has been shown that the same genes are
present and actively expressed in B. thuringiensis strains (Swiecicka
et al., 2006). Fig. 4 shows a conceptualized view of the B. cereus
sensu lato phylogenomic structure based on MLST data from
multiple studies integrated in the HyperCAT database (Tourasse
et al., 2006, 2010).
Various methods of molecular typing have periodically been
put forward with the claim that they could differentiate B. cereus s.
s. and B. thuringiensis, but the results from genomic studies have
essentially put to rest the idea of them forming two genetically
distinct species. B. cereus s. s. and B. thuringiensis had long been
known to be highly polymorphic, and what the genomics showed
was that the amount of variation within and overlap between each
of these so-called species is such that neither one displays a
coherent individual phenotypic or genomic identity in comparison
to the other beyond plasmid-borne virulence properties (Rasko
et al., 2005).
The ‘‘B. cereus vs. B. thuringiensis’’ problem (i.e., that B.
thuringiensis exists as a separate species on paper only) raises
questions of policy that extend beyond the realm of scientific
accuracy. Until now, the separate species status of B. thuringiensis
has been a key point in the safety evaluation of strains used in
commercial biopesticide formulations, which represent a worldwide for-profit industry. In the USA, the regulatory process for the
registration of biopesticidal strains is currently being updated by
the Environmental Protection Agency to address concerns regard-
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H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797
Fig. 4. Phylogenomic structure of the B. cereus group stylized as an unrooted tree diagram based on HyperCAT (Tourasse et al., 2010); top-level clusters identified in HyperCAT
are represented as leaves and identified by roman numerals (I–VII); classical species designations indicate the dominant contents of each cluster according to the established
pre-genomic taxonomy; topology of relationships between clusters is respected but branch length and leaf size are not necessarily to scale, and branching within leaves is
shown for aesthetic purposes only. Note that the B. anthracis complex is shown as a budding sub-group colored in red to emphasize its close relationship to B. cereus group
strains in Cluster III. Source data may be accessed at: http://mlstoslo.uio.no.
ing potential toxin content, but to date the ‘‘old’’ taxonomy is still
used as a basis for strain classification. What this means for the
reliability of the process and safety of registered products is still
unclear, but certainly warrants further investigation.
At first glance B. anthracis appears to pose less of a taxonomical
problem, because in its canonical form it is highly monomorphic
and possesses clearly identifiable phenotypic and genetic chromosomal features (Keim et al., 1999; Kolstø et al., 2009; Van Ert et al.,
2007). Specifically, the key chromosomal feature is a nonsense
mutation in the gene encoding PlcR (Easterday et al., 2005; Gohar
et al., 2008), a master regulator that is responsible for numerous
phenotypic traits in all B. cereus s. l. group organisms. In B. anthracis,
the plcR gene is inactivated by a single point mutation and the
master regulator is AtxA, which is encoded by the atxA gene on
pXO1 (Fouet and Mock, 2006; Mignot et al., 2001). Combined with
prophage content (Sozhamannan et al., 2006), sensitivity to
gamma phage (Abshire et al., 2005; Schuch et al., 2002) and the
presence of the pXO1 and pXO2 virulence plasmids, this was for a
time thought to be sufficient to characterize B. anthracis
unambiguously (Klee et al., 2006).
However, the phage-based criteria have since been shown to be
non-exclusive (Marston et al., 2006), and recent studies have
shown that plasmids closely related to pXO1 and pXO2 can be
found widely in environmental isolates of B. cereus sensu lato (Bahl
and Rosenberg, 2010; Hu et al., 2009). In addition, a number of
strains of B. cereus s. s. and B. thuringiensis, many of them
pathology-associated clinical isolates, have been found displaying
phenotypic characteristics of B. anthracis (including production of
anthrax toxins) and, significantly, containing plasmids very closely
related to the anthrax virulence plasmids pXO1 and pXO2
(Hoffmaster et al., 2004; Klee et al., 2010, 2006; Okinaka et al.,
2006; Pannucci et al., 2002a,b). Genomic analyses revealed that
these strains cluster closer to the canonical B. anthracis clonal
complex than to the bulk of B. cereus s. s./B. thuringiensis strains,
although they do not possess the point mutation that inactivates
PlcR. However, not all B. cereus strains that are genomically very
close to B. anthracis necessarily contain pXO1-like and/or pXO2like plasmids. The existence of all these so-called ‘‘close neighbors’’
of B. anthracis demonstrates a degree of phylogenomic continuity
that can be argued to invalidate the maintenance of B. anthracis as a
separate species in purely genetic terms.
How to deal with these issues is the subject of much debate.
One proposal under consideration is for strains possessing the
canonical PlcR mutation and select virulence factors to be classified
as B. anthracis sensu stricto, while borderline isolates lacking those
characteristics would be classified as B. anthracis sensu lato
(Okinaka et al., 2006). Another is for those borderline isolates to
be classified as B. cereus var. anthracis (Kolstø et al., 2009), which
H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797
could be subjected to specific regulatory controls without leading
to an overhaul of safety grading for the rest of the B. cereus s. l.
group.
Current trends in the published record as well as discussion in
the B. cereus research community suggest it is unlikely that the
classification of B. cereus s. l. group organisms will be changed to
single-species status in the foreseeable future. It seems that, for
now, we will keep the notion of a B. cereus s. l. group as a ‘‘wrapper’’
and attempt to work around the problems within by defining
genomic criteria that allow rational management of ‘‘science vs.
policy’’ issues.
795
identification of closely related strains/species, which could then
be followed by the more in-depth genomic characterization of
interesting groups of strains. Such a study has recently been done
in B. subtilis using microarray and sequencing technologies and has
uncovered a great deal of genomic diversity within this group of
closely related B. subtilis strains (Earl et al., 2007, 2008). Although
this may currently seem unreasonable to do with all bacterial
species of interest, advances in sequencing technologies coupled
with decreasing costs, in addition to advances in single-cell
genomics technologies and metagenomics methods, should
eventually enable a thorough description and classification of
the amazing molecular and ecological diversity of Bacillus.
5. Reconciling molecules and ecology
In the previous examples it is important to note that 16S-rDNA
based taxonomy does not disagree with methods offering more
resolution, but is simply unable to accurately estimate the
ecological diversity present within identical sequences. Let us
now conduct a brief ‘‘reductio ad absurdum’’ experiment to
estimate the number of Bacillus species based on ecology. As
discussed above, it is essentially meaningless to treat B. cereus and
B. thuringiensis as distinct species, at least in genetic terms.
However, for this exercise we will purposefully ignore that fact,
and proceed to calculate the average pairwise nucleotide difference between 16S rDNA sequences identified in the RDP database
as belonging to B. cereus (n = 513), B. thuringiensis (n = 277), or B.
anthracis (n = 136). We then use the resulting value of 0.7%
difference as a cutoff to predict the number of ecologically distinct
Bacillus species that could be said to be currently represented in the
sequence databases if the B. cereus group species distinctions were
considered valid and an appropriate yardstick for the rest of the
genus.
The first striking observation we make is that the divergence
between these three species is statistically insignificant. For
example, many B. cereus isolates are more closely related to B.
anthracis isolates than they are to other B. cereus strains, basically
confirming that these are not different species. But if we ignore this
issue for now and apply the 0.7% divergence to all 7510 Bacillus 16S
sequences we predict the existence of 1034 species of Bacillus, an
almost tenfold increase from our earlier prediction of 116 using the
conventional cutoff of 97% nucleotide identity.
There are two ways of interpreting this order of magnitude
increase in number of species predicted. On the one hand, this may
mean we are vastly underestimating the number of ecologically
distinct species of Bacillus species in nature by using cutoffs that
are too conservative. On the other, this brings us back to the
fundamental flaw of defining species on the basis of ecological
traits that may not be congruent with phylogenomic structure. In
fact, these interpretations are not mutually exclusive, and it is
likely that the Bacillus classification, in its current state, is
misleading in both directions. Because the ecology of most species
is virtually unknown and diversity in those areas of the tree is
estimated exclusively on the basis of 16S rDNA sequence, we are
probably missing out on a lot of ecological richness. At the same
time, we are blinded to the extreme genomic proximity of
historically defined species such as those in the B. cereus group
where phenotypes were mistakenly assumed to follow phyletic
divergences.
What is the solution? Clearly molecular based taxonomy is the
way forward, but significant improvements can be made by the
addition of ecological data as in the case of B. simplex and the B.
cereus group. It is unlikely that truly relevant ecological data will be
obtained by present culturing methods, leaving us to predict
ecology based on site of isolation and further genomic and
metagenomic sequencing efforts. For example, sequencing of 16S
rDNA or other conserved loci can be used for initial clustering and
Acknowledgements
We wish to thank Dr. Ashlee M. Earl for the 16S rDNA analyses
and conceptual discussions of the content in this review, and Dr.
William Schneider at the EPA Office of Pesticide Programs for
providing information about the registration of B. thuringiensis
biopesticides. GVdA is a postdoctoral research fellow funded by the
Fonds National de la Recherche Scientifique (FNRS-FRS, Belgium).
HM is funded by a Canadian Cystic Fibrosis Foundation postdoctoral fellowship.
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