RESEARCH ARTICLE Open Access
Understanding the evolutionary relationships and
major traits of Bacillus through comparative
genomics
Luis David Alcaraz1, Gabriel Moreno-Hagelsieb2, Luis E Eguiarte3, Valeria Souza3, Luis Herrera-Estrella1,4,
Gabriela Olmedo1*
Abstract
Background: The presence of Bacillus in very diverse environments reflects the versatile metabolic capabilities of a
widely distributed genus. Traditional phylogenetic analysis based on limited gene sampling is not adequate for
resolving the genus evolutionary relationships. By distinguishing between core and pan-genome, we determined
the evolutionary and functional relationships of known Bacillus.
Results: Our analysis is based upon twenty complete and draft Bacillus genomes, including a newly sequenced
Bacillus isolate from an aquatic environment that we report for the first time here. Using a core genome, we were
able to determine the phylogeny of known Bacilli, including aquatic strains whose position in the phylogenetic
tree could not be unambiguously determined in the past. Using the pan-genome from the sequenced Bacillus, we
identified functional differences, such as carbohydrate utilization and genes involved in signal transduction, which
distinguished the taxonomic groups. We also assessed the genetic architecture of the defining traits of Bacillus,
such as sporulation and competence, and showed that less than one third of the B. subtilis genes are conserved
across other Bacilli. Most variation was shown to occur in genes that are needed to respond to environmental
cues, suggesting that Bacilli have genetically specialized to allow for the occupation of diverse habitats and niches.
Conclusions: The aquatic Bacilli are defined here for the first time as a group through the phylogenetic analysis of
814 genes that comprise the core genome. Our data distinguished between genomic components, especially core
vs. pan-genome to provide insight into phylogeny and function that would otherwise be difficult to achieve.
A phylogeny may mask the diversity of functions, which we tried to uncover in our approach. The diversity of
sporulation and competence genes across the Bacilli was unexpected based on previous studies of the B. subtilis
model alone. The challenge of uncovering the novelties and variations among genes of the non-subtilis groups still
remains. This task will be best accomplished by directing efforts toward understanding phylogenetic groups with
similar ecological niches.
Background
Bacillus is one of the best characterized bacterial genera.
Since the late 19th century, the long history of Bacilli
research has included classical microbiology, biochemis-
try, and more modern genomic and proteomic
approaches. Bacillus is defined as a Gram-positive, rod-
shaped bacterium that can be aerobic or facultative
anaerobic [1] and produces highly resistant dormant
endospores in response to nutritional or environmental
stresses [2].
Bacilli are ubiquitous bacteria that exploit a wide
variety of organic and inorganic substrates as nutrient
sources [1]. However, spore dispersal by air and water
[3] may lead to false conclusions about the ecological
significance of recovered bacillus isolates, since it is
not clear if the robust presence of the bacteria is due
to the resistant nature of the dispersed spores or due
rather to a large adaptive capacity that would allow the
bacteria to be found in an active, vegetative state in
diverse environments [2]. This study of the Bacillus
* Correspondence: golmedo@ira.cinvestav.mx
1Departamento de Ingeniería Genética, Centro de Investigación y de
Estudios Avanzados del I.P.N. Campus Guanajuato, AP 629 Irapuato,
Guanajuato 36500, México
Alcaraz et al. BMC Genomics 2010, 11:332
http://www.biomedcentral.com/1471-2164/11/332
© 2010 Alcaraz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

pan-genome, and in particular the functional categori-
zation of the accessory genomes and their relationship
or lack thereof with the environment, may provide
insight into these important biological questions.
There are several ways to classify this group according
to biochemistry, lifestyles, and/or growth on different
substrates. One classification of the Bacillus splits them
into three major classes [1]: pathogenic, environmental,
and those used for industrial purposes. The pathogenic
class is represented by B. anthracis, B. cereus, and B.
thuringiensis. Environmental Bacilli are quite diverse and
include B. subtilis, B. pumilus, B. halodurans, and B. coa-
huilensis. The strain B. licheniformis is a well known
representative of an industrial strain [1]. This classifica-
tion is useful for introducing the metabolic diversity of
the genus, but it provides no guidance on a phylogenetic
classification of the Bacillus for research purposes. In
addition, this type of classification does not consider any
aquatic Bacillus. There is need for a classification method
that could take advantage of the nearly 130 genome pro-
jects of the genus. With more than 108 complete and
draft genome sequences available to date, Bacillus is one
of the most represented genera in the genomic databases.
Although there are close to 1,000 complete prokaryo-
tic genome sequences to date, the group is highly biased
toward pathogenic isolates [4,5]. Among 85 Bacillus
genomes, 61% are devoted to the cereus-anthracis-thur-
ingiensis group (See Additional file 2: Table S1). Several
researchers have used this overrepresentation as an
advantage to perform comparative genomic studies
aimed at defining the population structure and finding
genetic markers for pathovar identification [6-9]. Despite
the oversampling of pathogens, genomes of Bacillus iso-
lated from a wide range of environments are available,
including hydrothermal vents [10], tidal flats [11], soil
[12], alkaline environments [13,14], shallow marine
water [15], and a shallow water column from an oligo-
trophic environment [16]. The presence of Bacillus in
these different environments reflects the broad meta-
bolic capabilities of a widely distributed genus.
In a report of the intra-species diversity of Streptococ-
cus agalactiae [17], the “pan-genome” concept was
defined as the sum of the core genome (comprising
genes present in all analyzed strains) and the “accessory”
genome (comprising all strain-specific genes) [18,19].
This concept has been expanded for comparisons at
other taxonomic levels, such as family [20,21] and for
defining the universal ancestor hypothetical core [22].
Most traditional markers for species identification, such
as 16S rRNA genes, Comparative Genome Hybridization
(CGH), and the classical measures of phenotypic similar-
ity, mask the real genetic diversity since they rely mainly
on core-genome genes [17,23,24]. Another approach to
unveil the microbial diversity used mostly by population
geneticists and by clinical microbiologists are the Multi
Locus Sequence Typing (MLST) and Multi Locus
Sequence Analysis (MLSA) methods [25] relying on the
analysis of internal fragments of housekeeping genes
(usually 7 genes), which are useful for understanding
populations dynamics, recombination, and pathogen
diagnosis. Phylogenetic relationships can be obtained
through more extensive genomic sampling, such as the
one afforded by the genome sequences. Analyzing the
whole set of conserved genes across a taxonomical level,
such as the core genomes, will shed light about evolu-
tionary and functional relationships among the related
species. Several methods based on pairwise ortholog
comparison and synteny strategies have been developed
to assess the composition of core genomes [17,20,23].
In this study, we were interested in understanding the
cohesion of the Bacillus genus at the genomic level by
using the core and pan-genomes as the working units
and taking advantage of the large dataset available. We
have recently described the complete genome of Bacillus
coahuilensis [16,26], which possesses one of the smallest
genomes (3.35 Mb; 38% GC) reported for a free-living
bacteria in the group, and have identified genes that
allow this bacterium to survive in an aquatic oligo-
trophic environment. We now report the genome
sequence of another isolate, Bacillus sp. m3-13 with a
genome size of 4.13 Mb from the same environment as
B. coahuilensis. We compared these genomes to ask
whether the common environment has selected for simi-
lar features in the two genomes. These coincidences
would be observed in their gene constitutions as cohe-
sion of similar classes of metabolic genes.
To obtain insight into the group’s biology we describe
the relatedness within the Bacillus using whole genome
information to reconstruct their evolutionary history tak-
ing advantage of the dataset available from the complete
and draft genomes of 20 Bacillus isolated from a wide
range of environments. We compared the use of different
conserved genes as well as pairwise shared genes to
address local phylogenies and measure quantitatively the
relatedness between species using the core genome. Ana-
lysis of the functional categories of the core and pan-gen-
ome revealed a clear separation between different groups
and reflected the niche of the Bacillus strains. Finally,
clustering of conserved/absent genes for competence and
sporulation genes, distinctive processes of the Bacillus
genus, showed that genetic mechanisms for sporulation
are far more diverse across Bacillus than expected from
studies of B. subtilis alone (Figure 1).
Results
Bacillus sp. m3-13 genome summary
Bacillus sp. m3-13 genome was sequenced using a 454
FLX system (454 Life Sciences) with a 20-fold coverage.
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Assembly of the sequences resulted in 50 contigs and a
total of 4,137,575 bp assembled. The entire genome is
4.1 Mb with 40% GC content, 4,417 coding sequences,
and 81 RNAs.
Bacillus sp. m3-13 was isolated from a desiccation
lagoon in Cuatro Cienegas, Coahuila, Mexico within the
same sampling of the previously described B. coahuilen-
sis strain [16,26]. Using a 16S rRNA comparison, we
determined that the B. horikoshii NBSL26 is the closest
strain to Bacillus sp. m3-13 with 99% identity. Both B.
coahuilensis and Bacillus sp. m3-13 live in the same oli-
gotrophic environment, which includes low phosphorous
levels (less than 0.3 μM), but they appear to have differ-
ent strategies for dealing with the poor nutrient environ-
ment. B. coahuilensis produces sulfolipids and replaces
membrane phospholipids [16], In contrast, Bacillus sp.
m3-13 has phn genes, which code for phosphonate ABC
importers, permeases, and a phosphonate-lyase, and we
hypothesize that the strain may use these genes to take
up and assimilate phosphonates. Both strategies seem to
be used by bacteria from the Cuatro Cienegas [27], as
well as by marine bacteria [28,29].
Bacillus core genome phylogenetics
To gain insight into the natural history of Bacillus based
on the complete genome sequence, we performed a phy-
logenetic reconstruction using all of the core genes. We
chose at least one representative from each major divi-
sion within the Bacillaceae family to maintain the meta-
bolic cohesion behind the diverse ecological groups. The
type of strains used for this analysis as well as their gen-
eral genome features, habitats, accession numbers, isola-
tion data, and outstanding phenotypes are summarized
in Table 1.
As noted in the literature, the total number of genes
within a core-genome tends to diminish as more
Figure 1 Overview of this work.
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genomes from related strains are incorporated into the
analysis [5,17,23]. B. coahuilensis has the smallest gen-
ome reported for a Bacillus and thus is a good reference
to address the number of genes shared between all of
the representatives of Bacillus. A full matrix of Recipro-
cal Blast Hits (RBH) was constructed for the identifica-
tion of the 814 orthologous genes shared by all 20
species analyzed and was defined as the core genome.
The average gene content for Bacillus was 4,973 ± 923
genes and thus the core genome reflected only a fifth of
the total content of an average genome. In contrast,
after clustering homologous protein families, an esti-
mated pan-genome size of 155,747 genes was obtained.
All pan-genome genes were grouped into 19,043 families
and reflect the large repertoire of genes within this cos-
mopolitan group.
We reconstructed a Maximum Likelihood (ML) phylo-
geny using concatenated alignments of the 814 trans-
lated core genes across the 20 species (Additional file 1),
resulting in a 308,782 amino-acid length alignment (Fig-
ure 2A). The phylogeny created clusters of the following
major groups: B. clausii-halodurans, B. subtilis-
Table 1 Species, accession numbers, and general features of Bacillus sp. used in this work [30]
Strains Accession CDS GC% Habitat Phenotype Isolation environment Reference
Bacillus sp. m3-13 ACPC00000000
(WGS semi-finished)
4294 40 Fresh
water
N/A Chihuahuan desert lagoon in Cuatro
Cienegas, Coahuila, Mexico in 2005
This work
Bacillusc oahuilensis
m4-4
NZ_ABFU00000000
(WGS semi-finished)
3642 38 Fresh
water
N/A Chihuahuan desert lagoon in Cuatro
Cienegas, Coahuila, Mexico in 2005
[16]
Bacillus subtilis subsp.
subtilis str. 168
NC_000964 4408 43.5 Soil N/A X-ray irradiated strain in Marburg in
1947
[12]
Bacillus halodurans
C-125
NC_002570 4326 43.7 Soil,
Fresh
water
Alkalophile 1977 [14]
Bacillus cereus ATCC
10987
NC_003909 6248 38 Dairy
isolate,
Soil
Pathogen Cheese spoilage in Canada [31]
Bacillus anthracis str.
Ames
NC_003997 5569 35.4 Soil Non-Pathogen N/A [32]
Oceanobacillus
iheyensis HTE831
NC_004193 3736 35.7 Marine Alkalophile Deep sea mud at 1050 m depth
from the Iheya ridge near Okinawa
Japan in 1998
[11]
Bacillus cereus ATCC
14579
NC_004722 5610 35.3 Soil Pathogen N/A [9]
Bacillus anthracis str.
Sterne
NC_005945 5641 35.4 Soil Non-Pathogen N/A Unpublished
Bacillus thuringiensis
serovar konkukian str.
97-27
NC_005957 5590 35.4 Host,
Soil
Pathogen Severe human tissue necrosis [33]
Bacillus licheniformis
ATCC 14580
NC_006270 4371 46.2 Soil Pathogen, Subtilisin
production, Amylase
production
N/A [13]
Bacillus cereus E33L NC_006274 6010 35.4 Soil Pathogen Swab of a zebra carcass in Ethosha
National Park in Namibia in 1996
[33]
Geobacillus
kaustophilus HTA426
NC_006510 3733 52.1 Deep
sea,
Marine
N/A N/A [10]
Bacillus clausii KSM-
K16
NC_006582 4349 44.8 Soil Alkalitolerant, Probiotic,
Protease production
N/A Unpublished
Bacillus anthracis str.
‘Ames Ancestor’
NC_007530 5973 35.4 Soil Pathogen N/A [34]
Bacillus thuringiensis
str. Al Hakam
NC_008600 5090 35.4 Host,
Soil
Pathogen Severe human tissue necrosis [35]
Bacillus pumilus SAFR-
032
NC_009848 3913 41.3 Soil Biomass degrader,
Pathogen, Radiation
resistant
Spacecraft Assembly Facility at NASA
Jet Propulsion Laboratory
[36]
Bacillus
weihenstephanensis
KBAB4
NC_010184 6133 35.4 Soil Non-Pathogen N/A Unpublished
Bacillus sp.
NRRLB14911
NZ_AAOX00000000
(WGS semi-finished)
5869 45.7 Marine N/A 10 meters depth in the Gulf of
Mexico
[15]
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licheniformis-pumilus, B. anthracis-thuringiensis-cereus,
and a novel group, Bacillus sp. NRRLB-14911-coahui-
lensis-m3-13, The strain Geobacillus kaustophilusis
within the major groups in its own leaf but deep into
the Bacillus, whereas Oceanobacillus iheyensis is basally
located outside the major groups. A sister group is
formed by B. halodurans-clausii falling on the edge of
the main Bacillus groups. Another distinctive feature of
the core genome phylogeny, as compared with the tradi-
tional 16S rRNA and the universally conserved COG
phylogenies, is its robustness that is reflected by the
generally high bootstrap replica values. Still, this tree is
not fully resolved, as shown by the position of the G.
kauustophilus leaf.
We further measured the distance between species
with a Genomic Similarity Score (GSS) [37]. This mea-
surement is based on the sum of bit-scores of shared
orthologs, detected as RBH, and normalized against the
sum of bit-scores of the compared genes against them-
selves (self-bit-scores). It has a range from 0 to 1 with a
maximum reached when two compared proteomes are
identical. There is an average of 2,539 ± 561 shared
genes between different Bacillus species and an average
GSS of 0.5637 ± 0.0039. A distance matrix of the GSS
scores of the shared orthologs for the 20 Bacillus groups
was plotted in a Neighbor-Joining tree to evaluate the
resolution of a pairwise, shared orthologs index as an
evolutionary distance tool (Figure 2B). All of the inner
groups shown in the core genome phylogeny appear in
this clustering, although inner sister groups are clustered
showing the ambiguity of the deep nodes. GSS can
therefore be used as a complementary approach, as an
index to clarify relationships among organisms using
whole pairwise shared orthologs. GSS takes into account
the maximum comparable pairwise genome shared, in
contrast to the regular phylogenetic reconstruction,
which can only compare a common dataset of homolo-
gous genes.
A 16S rRNA Maximum Likelihood phylogeny of the
selected strains is shown in Figure 2C. Major groups,
such as the B. cereus and B. subtilis’ groups, are main-
tained with low bootstrap support (< 50), while the
aquatic Bacillus group is paraphyletic within this phylo-
geny. The use of 20 conserved concatenated Cluster of
Orthologous Groups (COGs) as described in [38-40] for
Bacillus (5,299 amino acid alignment length) to perform
an ML phylogeny (Figure 2D) resulted in a tree that
shows also a paraphyletic aquatic Bacillus group and
lower bootstrap support when comparing inner groups
like B. cereus group to the core genome phylogeny. We
noted that the 16S phylogeny places G. kaustophilus as
internal clade within the Bacillus genus, but with con-
siderable substitution rates shown in the large branch
length, while B. clausii and O. iheyensis are placed in
the same clade though with very low bootstrap support;
in contrast, the conserved universal COGs (uCOGs)
phylogeny places O. iheyensis close to a branch formed
by B. halodurans and B. clausii. Neither the 16S rRNA
nor the uCOGs resolve the internal clades with as much
support as the core genome phylogeny.
Functional composition of the core genome of sequenced
Bacillus
To understand the functional roles of the genes that
constitute the core and pan-genome, we took advantage
of the COGs functional classification [41]. This is a clas-
sification system based on orthologous relations among
genes. We used the COGs to map the core genome,
pan-genome, and each of the four groups defined in the
core phylogeny (Figure 3): i) B. anthracis-cereus-thurin-
giensis, ii) B. subtilis-licheniformis-pumilus, iii) B. clau-
sii-halodurans, iv) B. coahuilensis-m313-nrrlb14911, v)
G. kaustophilus-O. iheyensis. These last two do not form
a phylogenetically related group, however, for the func-
tional analysis we chose to group them given the simila-
rities in the environments from which these strains were
recovered. Using this strategy, we grouped Bacillus
representatives that had shared evolutionary and ecolo-
gical features and then searched for over/under repre-
sented gene functions within each group to underscore
relevant gene functions for each evolutionary group. We
observed significant differences in the COGs categories
of core-pan-genome-groups (Chi square = 753.72; d.f. =
126; p-value < 2.2e-16). Figure 3 shows a heat plot map
of the ratio of normalized genes to total gene content
for each COG category. Several expected features arose
from this analysis, such as the predominance of genes in
COG category J (translation and ribosomal structure
genes; core = 0.11/average = 0.06) within the core. This
was expected as most of the conserved universal COGs
are contained within this category [40]. Their conserva-
tion across taxa and functional restrains are precisely
why they are chosen as gold standards for phylogenetics
in addition to 16S rRNA genes. Other over-represented
categories, although only within the core, are C (energy
production and conversion; core = 0.07/average = 0.06)
and L (replication, recombination, and repair; core =
0.07/average = 0.05). Highly represented categories
included COGs E (amino acid transport and metabo-
lism; core = 0.09/average = 0.1), R (function unknown;
core = 0.11/average = 0.12), and S (poorly characterized
genes; core = 0.06/average = 0.09) that were not only in
the core but also in the pan-genome and in all of the
Bacillus groups. Interestingly, several core genes of the
COG R and S are conserved across the entire Bacillus
with some clearly being involved in the sporulation pro-
cess, including spmA, spmB, yaaT, spoIVFB, spoVB,
spoVR, and others (See Additional file 2: Table S2).
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Figure 2 Phylogenetic reconstruction for Bacillus. A. Concatenated 814 translated genes of the core genome maximum likelihood (ML)
phylogeny. Bootstrap values are shown. B. Genome Similarity Score (GSS) distance matrix plotted as a Neighbor-Joining tree. C. ML phylogeny
using 16S rRNA. C. Concatenated 20 Conserved Universal Cogs (uCOGs) ML phylogeny. Note how inner groups are well defined and supported
only in the core genome phylogeny (A) and how the GSS distance (B) resembles the inner groups described in the core’s genome phylogeny.
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Some categories are at least partially over-represented
in the core when compared to the pan-genome and to
either group and include the following: COG H (coen-
zyme transport and metabolism; core = 0.05/pan-gen-
ome = 0.03), which includes biosynthesis genes for
biotin (birA), riboflavin (ribA), co-enzyme A (ylol), and
a dipicolinate synthase (spoVFB); COG F (nucleotide
transport and metabolism; core = 0.05/pan-genome =
0.03), which includes comEB competence protein; COG
O (protein turnover and chaperones; core = 0.05/pan-
genome = 0.03), which includes genes such as groES;
and COG D (cell division), which includes the DNA
translocase ftsK, the rod shape-determining gene rodA,
and some sporulation-related genes such as spoIID,
spoVE, and soj.
COG categories U (intracellular trafficking and secre-
tion), N (cell motility), and I (lipid transport) are in the
same range and the fractional differences between the
core, pan-genome, and Bacillus groups of these cate-
gories are not noticeable (average = 0.01). Categories K
(transcription; core = 0.06/average = 0.09) and S (gen-
eral functions; core = 0.06/average = 0.09) are underre-
presented in the core. Despite the ubiquity of these
genes within Bacillus, there are only a few transcription-
related proteins that are shared between all of them. It
has been shown, however, that a given transcription fac-
tor ortholog selected solely by RBH may not have a con-
served function, and this aspect depends on the
phylogenetic distance and tempo of the rapidly evolving
regulatory networks [42]. We expected and observed
Figure 3 Heat map comparison of the core genome, pan-genome, and Bacillus groups conservancy of COGs. Normalized Cluster of
Orthologous Groups (COGs) frequencies within each group are shown as a two way clustering; interestingly the clustering supports the groups
formed in the core genome phylogeny (horizontal axis). In vertical axis COGs are grouped according to their abundance showing moderate
conserved COGs in the top, low frequency conserved COGs in the central clusters and highly abundant COGs cluster in the lower clusters. In the
bottom total numbers of proteins families mapped to COG within each row are shown.
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important differences in this category, since the fine
tuning of gene expression reflects the far and wide dis-
tribution of metabolic diversity in Bacillus. We also
expected an underrepresentation of genes in COG T
(signal transduction; core = 0.03/average = 0.04) and
COG V (defense mechanisms; core = 0.01/average =
0.02) in the core, since the different environments
where Bacillus activity takes place vary dramatically
among species. Therefore, the mechanisms for sensing
and responding to stimuli within each niche are not
expected to be conserved but to be a part of the acces-
sory genes. In contrast, the composition of COGs in the
pan-genome shows similarity to that of the B. cereus-
anthracis-thuringiensis group. This simply reflects the
large redundancy of this group within the overall num-
ber of gene families (19,043 families) in the pan-genome.
B. cereus, B. thuringiensis, B. anthracis, and B. weihen-
stephanensis are described as members of a single spe-
cies or taxon, the B. cereus group [43,44], and have the
largest number of sequenced members as well as the
largest genomes among known Bacillus (5,716 ± 354
coding genes). Therefore, it is remarkable that category
G (carbohydrate transport and metabolism; B. cereus =
0.05/average = 0.07) is underrepresented compared to
all other groups, while all other groups have similar
amounts of genes from this category. This finding
reflects a specialization in the metabolism of carbohy-
drates when compared to other groups. It is well docu-
mented that B. cereus has considerably less genes for
the degradation of carbohydrates compared to B. subtilis
[9], and this observation contradicts the hypothesis that
the ancestor of B. cereus was a soil bacterium. The B.
cereus group lacks the metabolic potential for the uptake
and assimilation of plant-derived carbohydrates that
exists in soil bacteria, such as B. subtilis, limiting the
number of polysaccharides that are degraded by this
group to glycogen, starch, chitin and chitosan [9]. The
pathogenic Bacillius are included in the B. cereus group,
and similar to the pan-genome, there is a clear predomi-
nance of genes in COG category V (involved in defense
mechanisms; B. cereus = 0.03/average = 0.02), compared
to a lower basal average for all of the Bacillus groups
and the core genome. As expected, the largest repertoire
of antibiotic resistance genes is present in the B. cereus
group. This group is actively suffering selective pressure
for these traits, a feature that is not observed in any of
the other groups [33,45,46]. Restriction endonucleases,
ABC-type transporters for the detoxification of cells,
cation/multidrug efflux pumps, and enzymes involved in
antibiotic resistance are all found within the COG V
category. However, most pathogenic traits of these
strains are encoded in plasmids and mobile elements.
Examples of this are the Cry toxins of B. thuringiensis,
the anthrax toxin and capsule genes of B. anthracis
located on the pX01 and pX02 plasmids, and the emetic
toxin of B. cereus located on the pX01-like plasmid (this
feature has been used for phenotypic differentiation of
the closely related strains). However, it has recently
been shown that there can be multiple plasmid transfers
among the B. cereus group strains [44], thereby compro-
mising the main genetic and phenotypic differences
within the cereus-thuringiensis-anthracis group.
Two interesting features appear when comparing the
B. coahuilensis-m313-NRRLB14911 group to the other
groups. First, this group contains the largest proportion
of genes in category T (signal transduction; B. coahui-
lensis’ group = 0.05/average = 0.04). These genes are
thought to have been acquired through HGT, most
likely from a Cyanobateria, in a similar manner as the
sensory rhodopsin from B. coahuilensis [16]. The three
members of this group were isolated from shallow
waters exposed to high radiation and oligotrophic condi-
tions. Two of them were isolated from a desiccation
lagoon and the Bacillus sp. NRRLB14911 strain was iso-
lated from the Gulf of Mexico at a depth of 10 m.
These environments may require the strains to be
responsive to sudden changes in conditions [15,16,26],
and thus environmental sensing through signal trans-
duction genes may be of greater importance than for
other groups of Bacillus. This is a particularly noticeable
feature of B. coahuilensis, a strain with a genome that
seems to have undergone extensive size reduction [16].
The second interesting feature is that with exception to
the O. iheyensis group, the B. coahuilensis-m313-
NRRLB14911 group has fewer genes from category K
(transcription factors; B. coahuilensis group = 0.08/aver-
age = 0.09) than all other groups. This group shares
phenotypic traits, such as pigmentation, and are subject
to a similar osmotic pressure due to salinity. This group
also shows an underrepresentation of genes in category
C (energy production and conversion; B. coahuilensis
group = 0.05/average = 0.06), P (transport and metabo-
lism of inorganic ions; B. coahuilensis group = 0.05/
average = 0.06), and E (amino acid metabolism genes; B.
coahuilensis group = 0.09/average = 0.1). All of these
categories are involved in several of the early stages of
amino acid synthesis. Several auxotrophies as well as
specialization within these strains have been shown, par-
ticularly for B. coahuilensis [16]. This group exhibits the
largest genome size variation with a range between 3.3
and 5 Mb. Despite the differences in the number of cod-
ing genes, the largest genome (that of NRRLB14911)
does not bias the result of the COG T nor does it show
an increase in transcription related genes. Finally, a lar-
ger than average gene content in the COG J category
(translation, ribosome structure; B. coahuilensis group =
0.05/average = 0.06) is noticed in the group, which is
congruent with the fact that they have an increase in
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the number of genes from COG L when compared with
the B. subtilis group (replication, recombination and
repair; B. coahuilensis group = 0.06/B. subtilis group =
0.06). We can hypothesize that the latter genes are
needed to repair DNA that is damaged by the high
radiation exposure. An over-representation of transpo-
sons and IS elements may be responsible for acquiring
new genes via HGT or pseudogenization and the reduc-
tion of the genomes [47].
The B. subtilis-licheniformis-pumilus group has a
slightly higher than average number of genes related to
carbohydrate transport and metabolism (COG G; B.
subtilis group = 0.08/average = 0.07) as expected for a
group isolated from the soil and in close contact with
plants and their products [2,9]. This group has a
reduced number of genes involved in replication,
recombination and repair (COG L) that correlates with
the scarce repetitive elements such as IS, transposons,
and transposases present in B. subtilis, B. pumilus, and
other sister species [10]. Therefore, it seems that chro-
mosome remodeling and genome reduction [48-50] is
not a prevalent feature of B. subtilis. Interestingly, B.
pumilus has a reduced number of genes involved in
DNA repair and oxidative stress as well as small acid
soluble proteins (SASP) that mitigate DNA damage
and are involved in the desiccation and UV resistance
of spores as compared to B. subtilis and close relatives
[36,51]. In contrast, transcription-related genes (COG
K; B. subtilis group = 0.1/average = 0.09) are slightly
over-represented in the subtilis group. This suggests
that the genetic response within this group is finely
tuned as previously observed when the large gene
families of B. subtilis and B. coahuilensis were com-
pared [16].
B. clausii-halodurans form the group of alkalophiles
and halotolerant strains. Interestingly, this group has
fewer genes involved in the cell wall/membrane/envel-
ope category (COG M; B. clausii group = 0.05/average
= 0.06) than the average. This could be explained by
the loss of 13 genes involved in the synthesis of tei-
choic acid and the loss of 6 genes involved in teichuro-
nic acid biosynthesis. In addition, there are several
known differences in the cell wall composition of B.
clausii-halodurans compared to B. subtilis, such as the
presence of the major cell wall component teichurono-
peptide [14]. The number of genes in B. clausii-halo-
durans is similar to other Bacillus for the following
categories: coenzyme transport and metabolism (COG
H; B. clausii group = 0.03/average = 0.03), nucleotide
transport and metabolism (COG F; B. clausii group =
0.03/average = 0.03), and protein turnover and chaper-
ones (COG O; B. clausii group = 0.03/average = 0.03).
The high number of genes from COG category G (car-
bohydrate metabolism; B. clausii group = 0.09/average
= 0.07) stand out in this group. There are two possible
explanations for this observation. First, Bacillus soil
strains are expected to have a vast repertoire of genes
for sugar assimilation, as is the case of B. subtilis [9],
and B. clausii was isolated from soil [52]. Second,
sugars, such as trehalose, function as osmoprotectants
and therefore play an important role in halophilic bac-
teria [53]. The B. clausii genome has also an unusually
high number of ABC transporter permeases (N = 36)
that increase the number of genes within the COG G
category. A noticeably high number of replication,
recombination, and repair genes (COG L; B. clausii
group = 0.07/average = 0.06) are present within this
group. This may be due to the high number of repeti-
tive elements in B. halodurans, which posseses 112
genes similar to transposases or recombinases [14] and
numerous IS sequences [10]. Therefore, these repeti-
tive elements may be considered as factors important
for environment specialization [54-56].
O. iheyensis and G. kaustophilus were isolated from
deep sea environments (1,050 and 3,000 m depth,
respectively) but have different niche specializations. O.
iheyensis, isolated from sediment and adapted to
extreme salinity, is a facultative alkaliphilic. In contrast,
G. kaustophilus, though also isolated from sediment, is
associated with a marine trench and has an optimal
growth temperature of 60°C. An important shared func-
tional feature of this group is the resistance to osmotic
pressure of up to 30 MPa, a unique characteristic when
compared to normal atmospheric pressures of 0.1 MPa
[10,14]. These two genomes are small in size (~3.6 Mb;
see Table 1) and have less genes for several COG
including nucleotide metabolism (COG F; O. iheyensis
group = 0.03/average = 0.03). The essential genes of B.
subtilis, such as ymaA and ydiO, are absent in G. kaus-
tophilus[10]. There are only 12 genes in category Q for
each of these genomes (secondary metabolism; O.
iheyensis group = 0.01/average = 0.02), which is low
when compared to an average of 14-21 genes per gen-
ome seen in category Q from other groups such as B.
cereus. The number of genes categorized for transcrip-
tion (COG K; O. iheyensis group = 0.08/average = 0.09)
is also lower within this group and resembles the history
of the B. coahuilensis group. The small genomes of this
group and B. coahuilensis seem to be a result of genome
reduction and adaptation to specific niches, such as oli-
gotrophic environment, high salinity, high osmotic pres-
sure, and thermal environments [47,54,56].
Conservation of genes for competence and sporulation
among members of Bacillus
Two post-exponential key processes have been sub-
jected to extensive study for the genus Bacillus, and in
particular the model system B. subtilis: genetic
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competence and sporulation. Genetic competence,
defined as a state that permits the uptake of exogenous
DNA, is widespread among both gram-positive and
gram-negative bacteria. It is a genetically programmed
state during which a small percentage of cells in a
population can uptake DNA from the environment
and integrate it into their chromosome [57]. Particu-
larly, in the case of B. subtilis 168, it is thought that
the greater DNA uptake efficiency of the strain is a
consequence of a laboratory selection process [2].
Most proteins that form the complex responsible for
mediating the binding and uptake of DNA are part of
the core (ComEC, ComFA, ComGA) or are highly con-
served (ComEA). The interaction of several of these
competence-specific proteins in the complex has been
demonstrated, as well as interactions with the highly
conserved proteins RecA, SsbB, and Smf [58]. The
master regulator of the process, the ComK transcrip-
tion factor, binds to competence promoters to activate
transcription of many genes. A feature of competence
development is the stabilization of ComK by protein
ComS [59]. While the gene coding for the ComK tran-
scription factor is conserved in most Bacillus (absent
in B. halodurans, B. clausii, and B. coahuilensis),
ComS seems to be a specialization of the B. subtilis
group. ComK is itself synthesized in response to the
signal-transduction network, but most genes coding for
the regulatory proteins that constitute this network are
not conserved. Given the conservation of the transfor-
mation machinery, it is of considerable interest to
understand to what extent natural genetic competence
can explain genetic variability by gene acquisition, at
what frequency it occurs, and which signals trigger the
competence state under specific environmental
conditions.
A defining feature of the Bacilli is the formation of a
highly resistant non-reproductive structure called the
spore. Within the Firmicutes, the genus Bacillus and
Clostridium produce endospores. The primary function
of most spores is to ensure the survival of a bacterium
through periods of environmental stress. The resistance
of a spore could be considered as a crucial survival fea-
ture, and therefore sporulation genes would be expected
to be part of the core genome. However, previous stu-
dies have found, both experimentally and in silico, that
there is great intra-specific [2,60] and inter-specific var-
iation in sporulation genes. The variability of sporulation
genes has been only superficially reported in these stu-
dies and they have mainly included non-subtilis models
such as B. cereus and B. anthracis.
The sporulation process has been subjected to exten-
sive review [51,61-64]. This morphogenetic process is
triggered by conditions of starvation that result in a dis-
tinct asymmetrically positioned septum that delimits the
fore-spore and is surrounded by two membrane layers.
Peptidoglycan is deposited in the space between the
membranes to form the cortex and additional proteins
are deposited around the cortex to form the so-called
spore coat of the forming endospore. Some Bacilli also
have an outer membrane composed of lipid and protein
called an exosporium. The spore can quickly outgrow
into a vegetative bacterium upon stimulation by an
environmental cue.
Each of these stages has been documented and more
than 200 regulatory and structural genes are known to
be expressed in B. subtilis in a temporally regulated
manner. Figure 4 depicts the conservation pattern of
Kyoto Encyclopedia of Genes and Genomes’ (KEGG)
BRITE hierarchy [65] that comprises 185 sporulation
related genes. These sporulation and germination genes
are arguably the best studied transition regulators. Clus-
tering of the conserved/absent sporulation and compe-
tence genes resulted in 4 clear groups that were
arbitrarily denominated A through D. Group A, with 52
genes, contains all of the sporulation/competence core
genes; group B, with 52 genes, shows great variability
with an absence bias of genes in the extremophyle/aqua-
tic Bacillus; Group C, with 43 genes, is conserved
mainly among strains close to B. subtilis; and group D,
with 35 genes, seems to represent the specialized genes
of B. subtilis.
Within the sporulation core (Group A), we found
genes required for the temporal and spatial regulation of
sporulation gene expression that depends on four sporu-
lation-specific sigma factors (SigE, F, G, and K), all of
which are part of the core (the apparent lack of conser-
vation of sigK is due to the fact that in B. subtilis it is
encoded by two separate genes which are merged upon
entrance into sporulation). As shown for B. subtilis, the
initiation of sporulation is dependent on the phosphory-
lation of the two-component protein Spo0A, a transcrip-
tion factor that controls a large number of genes. Spo0A
is regulated through a phosphorylation cascade known
as the phosphorelay. Sense input signals to Spo0A are
given by histidine kinases, such as KinB, which are
highly conserved (missing only in O. iheyensis). KbaA,
the activator of KinB, is also present in the sporulation
core. The number of sensor kinases that participate in
this phosphorelay has been shown to differ between B.
subtilis and B. cereus (5 and 9, respectively) [66]. The
highly variable nature of the amino-terminal domains of
the sporulation sensor kinases of the different Bacillus
species has been suggested to represent differences in
the signals used to initiate the developmental program.
Recently, the B. anthracis kinase BA2291 was shown to
be remarkably different from other sensor kinases by
having a unique specificity for GTP [67]. Response regu-
lator aspartate phosphatases as well as their cognate
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Figure 4 Bacillus selected sporulation and competence genes conservancy. In this heat map shows the presence/absence of the set of 185
sporulation and competence related genes according to KEGG’s BRITE hierarchies http://www.genome.jp/kegg/brite.html. Each column stands for
a Bacillus strain and each row represents a gene and here are shown present (in gray) or absent genes (in black). Is possible to distinguish
between 4 major clusters according to the conservancy level of the genes, (A) the first upper cluster stands for the sporulation core genes; (B)
second is a group of sporulation genes diminished mostly in the extremophile and aquatic species of Bacillus; (C) cluster is defined as genes
present in almost all the strains of B. subtilis’ related group but depleted in the B. cereus’ group; and (D) are B. subtilis’ specialized sporulation
genes. A comprensive list of each cluster of genes is available in Additional file 2: Table S3. Figure Abbreviations: B. subtilis (bsu), B. pumilus(bpu),
B. licheniformis ATCC 14580 (bli), B. licheniformis DSM13 (bld), B. halodurans (bha), O. iheyensis (oih), B. coahuilensis (bcoa), B. clausii (bcl), Bacillus
sp. m3-13 (M3.13), G. kaustophilus (gka), Bacillus sp.NRRLB-14911 (b14911), B. cereusATCC 14579 (bce), B. weihenstephanensis (bwe), B. thuringiensis
Al Hakam (btl), B. thuringiensis97-27 (btk), B. cereus ZK (bcz), B. cereus ATCC 10987 (bca), B. anthracis Sterne (bat), B. anthracis Ames (ban),
B. anthracis Ames 0581 (bar).
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aspartate phosphatases are either not conserved or exhi-
bit changes that make them difficult to recognize by
sequence similarity. The only coat gene present in the
core is cotE, which is involved in the outer layer of the
spore coat [68]. Germination genes are also found
within the core. GerD is involved in the early germina-
tion response to amino acids such as L-alanine and
L-asparagine [69], whereas GerM has been hypothe-
sized to bind to peptidoglycan [70]. The spore matura-
tion proteins SpmA and SpmB are also part of the
sporulation core and are involved in spore dehydration.
These proteins provide spore resistance to moist heat,
as was shown in Clostridium perfringens [71,72]. Dipi-
colinic acid has been recognized as a core molecule that
gives the spore radiation resistance [51]. The two units
of dipicolinate synthase, spoVFA and spoVFB, are also
present in the sporulation core. SspI appears to be the
only universal SASP.
An interesting observation of group B is the seemingly
generalized gene loss in the aquatic/halophile Bacillus.
This finding is not seen in the B. subtilis and B. anthra-
cis-cereus-thuringiensis groups. Diverse sporulation
genes, germination genes, coat and small acid proteins
are the predominant categories absent from this group.
Variation of conservancy decreases with several SASPs,
such as SspB, SspD, SspH, and SspF. This suggests that
despite these proteins being very abundant in the spore,
they are diverse and exhibit redundancy in places where
they are bound to DNA (3-6% of the total spore protein)
[51]. Therefore, the absence of some genes may be com-
pensated by the presence of others.
The phosphatases RapF and RapI are involved in the
phosphorelay and are poorly conserved across all Bacil-
lus. The low conservation of phosphatase-related pro-
teins is more prominent in groups C and D (RapA, B,
C, D, E, G, and K;and PhrA, C, E, F, and G), suggesting
that phosphorelay cascades may almost be strain-speci-
fic. A similar situation is observed for several coat pro-
teins that first appear in group B (CotB, D, F, H, JA, JB,
JC, SA, and Z) and for germination proteins (GerE, PA,
PC, PD, and PE).
The cereus and subtilis group are similar in the A and
B gene categories, but lack almost all genes described in
categories C and D. Our results are consistent with pre-
vious studies [6-9,66] on comparative genomics within
the B. cereus group that show that the main differences
among the groups reside in HGT mobilized elements
[31,73] and not in the core genome.
Our findings suggest that the sporulation sensor
kinases, coat proteins, and SASPs of the various Bacillus
species have evolved to be responsive to signals specific
for particular environments. Even two very close strains,
such as B. licheniformis DSM13 and ATCC 14580, differ
in the preservation of some genes, including sspJ and
sspL.
Discussion
The increasing number of sequenced microbial genomes
provides an ideal opportunity to re-evaluate approaches
in understanding phylogenetic and functional differences
among bacteria. Much of the understanding of microbial
biodiversity has been studied by comparison of rRNA
sequences. However, this approach has clear limitations,
such as arbitrary cut-off values for sequence identity
and the inability to resolve relationships between closely
related groups [74]. For very close relatives, MLST and
similar approaches can be used successfully to describe
intra-diversity and resolve discrete clusters [75]. Both
rRNA and MLST approaches use genes from the core
genome [17], and in our analysis, we expanded the gene
set for use in phylogenetic reconstruction in order to
greatly increase our ability to resolve clusters. Testing
inter-species phylogenetic cohesion of a group, such as
the Bacillus, and taking advantage of 814 concatenated
core genes, allowed us to obtain a robust phylogenetic
reconstruction of the inner clusters that failed when
comparing the same species with rRNA or universally
conserved genes. Data obtained using metrics of taxa
distance, such as GSS, for whole genome pairwise com-
parisons that made use of the entire shared genetic
information agreed with the cluster resolution. Of note,
we described the aquatic Bacillus as a new group. We
predict that this group will quickly gain importance
given the numerous examples of aquatic representatives
that have been identified through 16S rRNA gene
sequencing in multiple environmental samplings.
Today, given the constant improvement in cost/benefit
of massive sequencing technologies, it is possible to
think in whole genome shotgun (WGS) approaches to
try to answer global internal group diversity. Although it
is not yet the cheapest/feasible option for the regular
laboratory, we suggest that the rapidly growing micro-
bial genome database can be used to regularly and auto-
matically build core genomes at intra-species, genus,
family, order, and other taxonomic levels. This approach
will aid in defining the functions of the genes behind
different taxonomic ranks and provide the whole
research community with specific genetic markers to
perform detailed ecological and evolutionary analysis.
Similar to the RBH approach, researchers can benefit
from WGS projects in progress to define core genomes,
since the number of unfinished genomes (1,777) almost
doubles the amount of complete genomes (892). In this
study, we used data from the sequenced genome of
Bacillus sp. m3-13 with 22-fold coverage, the previously
sequenced genome of B. coahuilensis [16], and the WGS
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assembly of Bacillus sp. NRRL-B14911 [15]. A core gen-
ome is a dynamic entity, since the incorporation of new
genomes into the database will reduce the total number
of genes within core genomes. How does core data com-
pare with experimental data, such as the essential genes
of B. subtilis [76]?. In this study, we found that the core
genome for the sequenced Bacillus includes 61 of the
79 essential genes of B. subtilis. Another approach,
using synteny [20] rather than RBH, defined 761 genes
of the core genome. That particular study used several
definitions of a core-genome and only considered com-
plete genomes in the analysis. Our approach defined
814 genes and therefore obtained 54 more genes than
the synteny approach. The use of a synteny strategy to
identify a core genome would clearly miss rearranged
orthologs and would also be limited if it were applied to
WGS assemblies. Thus, working with orthologs as RBH
may be a better approach.
Pan-genomes at genus level are only beginning to be
described, and the number of gene families in these is
expected to grow as more genomes are included. This
approach however is highly valuable to reveal niche spe-
cific genes, to guide future studies linking genes to ecol-
ogy, and even for selecting new genomes to sequence
that could balance and enrich our knowledge of the
Bacillus genus. The genetic features that explain meta-
bolic diversity and that are part of the pan-genome and
the non-core genes (known also as dispensable or
“accessory” genes) are missing from the usual rRNA and
MLST analysis. Genes involved in niche adaptation are
amongst the dispensable genes and the whole pan-gen-
ome. Several of these genes are mobilized by means of
HGT, such as genomic islands or individual elements
involved in pathogenicity, as is the case for the B. cereus
group [77], responses to environmental stresses like
phosphorous deprivation found for B. coahuilensis [16].
In addition to core genome phylogenetics and the deter-
mination of ecological and geographical features, we
suggest taking into account gene functions, such as
COG comparative distributions, in order to describe
species clusters and compare the phylogenetic profile
with the functional profile. These data will aid in the
analysis of the concordance or incongruence between
them.
The existence of model organisms, such as B. subtilis
str. 168, has been crucial for inferring homologous gene
functions through all bacteria using physiological,
genetic, and molecular biology approaches. The power
and unequivocal value of the model organism in tackling
biological mysteries is clear, but recent work on the
intra-specific diversity of this model organism [78] has
led us to recognize the great genomic variation that
exists. These findings suggest that strain-specific genes
are at the base of broad adaptations of B. subtilis to the
terrestrial and aquatic environments from where it has
been isolated. The B. subtilis str. 168 genome was
sequenced in early 1997, but concerns regarding the
effects of domestication of the strain in the laboratory
and the subsequent genomic changes, as well as reeva-
luation of the quality of the original sequence, led to the
re-sequencing of str. 168 [79]. Interestingly, other strains
within the subtilis species show differences in the con-
servancy of genes. This is true even within the so-called
essential genes, sporulation and competence genes,
which are a central part of Bacillus biology. Therefore,
the variability of gene content in the sporulation and
essential genes amongst other representatives of the
genus comes as no surprise. Although spore formation
is central to the definition of Bacilli, it is clear that
variability in the sensing of stress conditions, spore
resistance, and germination is the result of specific
niche constrains.
Use of comparative genomics, ecological and evolu-
tionary facts, and the lowering costs of genomic sequen-
cing are together aiding in the understanding of
microbial diversity. Our next steps must focus on ana-
lyzing the temporal and spatial patterns of genes present
or absent using high throughput genetic expression in
order to understand the roles of microbes in their envir-
onments. Science is moving into a paradigm shift in the
study of bacteria from single individuals to populations
with the boost from metagenomic approaches. The chal-
lenges of unveiling comprehensive relationships between
the environment, genes, and evolution of the bacterial
species remain ahead.
Conclusions
We have determined and defined a set of 814 genes that
make up the core genome of Bacillus. From the core
genome, we have reconstructed a robust phylogeny of
the group using GSS index data, which use the total
number of pairwise shared genes to resolve phylogenetic
relationships within the group. Both the core genome
and GSS phylogenies describe a new group of aquatic
Bacilli that have similar habitats. To understand the
biology of each group of Bacillus as defined by the
respective phylogenies, we have described functional
roles of their genes as well as differences between the
core and pan-genomes. Our results show that a total of
53 genes comprise the sporulation and competence core
genome. In addition, we have highlighted the differences
in gene set conservancy across all Bacillus species that
have been previously defined for B. subtilis. Our work
will be a valuable resource for understanding the evolu-
tionary and functional relationships within the Bacillus
genus. The core genome defined here may also be used
as a list of genetic markers for future population genet-
ics studies. The lack of conservancy in non-subtilis
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groups of genes for processes such as sporulation and
competence underlines the natural variability of the
genus and emphasizes the need for further exploration
of these differences.
Methods
Reciprocal Best Hits (RBH)
We undertook an RBH approach as previously described
[80,81]in order to identify all of the orthologous pairs
among 20 complete genomes of the Bacillus. Predicted,
translated gene models for each genome were used and
required coverage of 70% of both genes with an e-value
of 10-5 at an effective database size of 107.
Core genome
All pairwise RBH shared genes were compared and the
common dataset of shared genes amongst all strains was
defined as the core genome. We use the COG classifica-
tion schema [41] to classify gene functions. The acro-
nyms utilized in this study for the B. subtilis genes are
the most widely used and we therefore used them for
the entire core genome.
Pan-genome
Here, we define the pan-genome as the total set of genes
within the 20 Bacillus genomes, including plasmid and
extra-chromosomal elements (when available). A total set
of 19,043 homologous families were comprised of a total
of 155,747 genes, as identified by RBH, with a cut-off e-
value of 10-5[80]. COG classification was conducted with
each representative from the homologous families.
Evolutionary Analysis
To compute similarity between genomes using RBH pair-
wise information, we took advantage of our RBH bit-score
results using a Genomic Similarity Score (GSS) [37]that
had a range from 0 to 1.A maximum score was obtained
when two compared proteomes were identical and a GSS
distance matrix was used to build a Neighbor-Joining tree.
Alignments for 16S rRNA were done using MUSCLE [82]
and the phylogenetic tree was created using PhyML [83].
The 20 universally conserved gene COG phylogeny was
constructed as previously described[16,40]. Each translated
gene of the core genome was aligned and concatenated
using ClustalW-MPI [84]. The phylogenetic tree was con-
structed using PhyML [83] with the JTT substitution
model as has been done before with translated and conca-
tenated sequences [40], given the alignment length
(308,782 aa). The gamma distributed rates and 1,000 boot-
straps were estimated from the dataset.
Statistical analyses
All statistical analyses were conducted on R (2.6.2) [85].
Heat maps were generated with the gplots library of R [86].
Sporulation genes
The genes were defined by the Kyoto Encyclopedia of
Genes and Genomes KEGG BRITE Hierarchies [65].
RBH analysis, as previously described, was conducted
with each KO gene annotated for B. subtilis in the
BRITE hierarchy. RBH results were then parsed into
presence/absence to map the conservancy of sporulation
genes across the Bacillus.
Bacillus sp. m3-13 isolation
The strain was isolated from a desiccation lagoon in the
Churince system located in Cuatro Cienegas in Coa-
huila, Mexico (26°50.830’N, 102°09.335’W) by Rene Cer-
ritos as described for B. coahuilensis [26].
Genome sequencing, assembly, and annotation
Bacillus sp. m3-13 was sequenced using the 454 FLX
system (454 Life Sciences) with a 20-fold coverage.
Assembly was done with Newbler, Celera Assembler
[87], and Phrap [88] resulting in 50 contigs and a total
of 4,137,575 bp assembled. Gene prediction was done
using Glimmer v3.0 [89] and GeneMark.hmm [90].
Automated annotation was performed with BASys [91]
checked and proofed manually.
The Whole Genome Shotgun (WGS) Bacillus sp. m3-13
project has been deposited at DDBJ/EMBL/GenBank
under the project accession [ACPC00000000]. The ver-
sion described in this paper (ACPC01000000) is the first
version.
Additional file 1: Core genome concatenate alignment in FASTA
format.
Additional file 2: Supplementary tables. Table S1. Current Bacillus sp.
genome projects. Table S2. GI numbers, gene acronyms, and annotation
for the 814 core genome genes. Table S3. Sporulation core genes.
Abbreviations
CGH: Comparative Genome Hybridization; COG: Cluster of Orthologous
Groups; GSS: Genome Similarity Score; HGT: Horizontal Gene Transfer; KEGG:
Kyoto Encyclopedia of Genes and Genomes; ML: Maximum likelihood; MLSA:
Multi Locus Sequence Analysis; MLST: Multi Locus Sequence Typing; RBH:
Reciprocal Blast Hits; SASP: Small Acid Soluble Protein; WGS: Whole Genome
Shotgun.
Acknowledgements
This work was supported by a CONACyT-SEP grant 57507to VS, VS and LEE
worked on this manuscript during their sabbatical at UCI with a DGAPA and
an UCMExus grant respectively, Howard Hughes Medical Institute Grant
55005946 (to LHE), and a research grant from Cinvestav to GO. LDA was a
recipient of a fellowship from CONACyT. We acknowledge Beatriz Jiménez
and Gustavo Hernandez for sequencing assistance at Langebio-Cinvestav.
We are grateful for the work of Rene Cerritos in the isolation of Bacillus sp.
m3-13, Michael Travisano (U. Minnesota), and Alex Mira (CSISP, Spain) for
their comments on the manuscript.
Author details
1Departamento de Ingeniería Genética, Centro de Investigación y de
Estudios Avanzados del I.P.N. Campus Guanajuato, AP 629 Irapuato,
Alcaraz et al. BMC Genomics 2010, 11:332
http://www.biomedcentral.com/1471-2164/11/332
Page 14 of 17

Guanajuato 36500, México. 2Department of Biology, Wilfrid Laurier University,
75 University Ave. W. Waterloo, ON, N2L 3C5, Canada. 3Departamento de
Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de
México, CU, AP 70-275 Coyoacán 04510 México DF. 4Laboratorio Nacional de
Genómica para la Biodiversidad (Langebio), Centro de Investigación y de
Estudios Avanzados del I.P.N. Campus Guanajuato, AP 629 Irapuato,
Guanajuato 36500, México.
Authors’ contributions
LDA, GO, VS, and GM-H conceived and designed the study, LDA, GO, VS and
LEE analyzed data, and LHE and GM-H contributed with reagents/materials/
analysis tools. LDA, GO, and GM-H wrote the paper. All authors read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 29 December 2009 Accepted: 26 May 2010
Published: 26 May 2010
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doi:10.1186/1471-2164-11-332
Cite this article as: Alcaraz et al.: Understanding the evolutionary
relationships and major traits of Bacillus through comparative
genomics. BMC Genomics 2010 11:332.
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