Vol.2, No.10, 1113-1118 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.210138
Assessment of a short phylogenetic marker based on
comparisons of 3’ end 16S rDNA and 5’ end 16S-23S ITS
nucleotide sequences of the Bacillus cereus group
Sabarimatou Yakoubou1,2, Jean-Charles Côté1*
1Agriculture and Agri-Food Canada, Research Centre, Gouin Blvd, St-Jean-sur-Richelieu, Canada; *Corresponding Author:
2Département des Sciences Biologiques, Université du Québec à Montréal, Succ.“Centre-Ville” Montréal, Canada.
Received 28 May 2010; revised 30 June 2010; accepted 5 July 2010.
ABSTRACT
A short phylogenetic marker previously used in
the reconstruction of the Order Bacillales and
the genus Bacillus was assessed here at a lower
taxa level: species in the Bacillus cereus group:
B. anthracis, B. cereus, B. thuringiensis and B.
weihenstephanensis. This maker is 220 bp in
length. It is a combination of 150 bp at the 3’ end
of the 16S rDNA and 70 bp at the 5’ end of the
16S-23S ITS sequence. Three additional Bacillus
species, B. halodurans, B. licheniformis and B.
subtilis, and Clostridium tetani were included
for comparison purposes. A total of eight bacte-
rial species and 12 strains were analyzed. A boot-
strapped neighbor-joining tree was inferred from
comparative analyses of all allelic sequences of
the bacterial species and strains under study.
Based on its topology, four major Groups were
revealed at the 90% nucleotide sequence identi-
ties, Group I to IV. Group I contains all alleles of
the Bacillus cereus group. Group II contains all
alleles of B. halodurans. Group III contains all
alleles of B. licheniformis and B. subtilis. Group
IV contains all alleles of Clostridium tetani. The
220 bp phylogenetic marker used here could
resolve different species from different genera.
At the genus level, distant species could be dis-
tinguished. Very closely-related species, how-
ever, were undistinguishable. Species in the B.
cereus group, most notably B. cereus, B. anth-
racis and B. thuringiensis, could not be distin-
guished. After successfully inferring the phylo-
genies of the Order Bacillales and the genus
Bacillus, we have met the resolving limit of this
short phylogenetic marker: B. cereus, B. an-
thracis and B. thuringiensis.
Keywords: Bacillus cereus; 16S rDNA; 16S-23S
ITS; Phylogeny
1. INTRODUCTION
The Bacillus cereus group comprises six genetically
highly related species: B. cereus sensu stricto, B. an-
thracis, B. thuringiensis, B. weihenstephanensis, B. my-
coides [1] and B. pseudomycoides [2]. They are Gram-
positive, rod-shaped, endospore-forming, either obligate
or facultative aerobic bacteria [1].
Bacillus cereus is a ubiquitous soil bacterium. It can
be a contaminant of a variety of foods: meats, vegetables
and dairy products [3,4]. It can cause diarrheal, and em-
etic food poisoning syndromes [5]. It can also be the eti-
ologic agent of some opportunistic infections [6,7]. Bac-
illus anthracis is the etiologic agent of anthrax, an acute
disease in herbivorous mammals, transmissible to other
animals, including humans [8]. This species has been st-
udied and developed as a biological weapon [9]. Virul-
ent strains of B. anthracis carry two plasmids, pXO1
(181 kb) and pXO2 (96 kb) which may be transmitted to
others members of Bacillus cereus group [10]. Bacillus
thuringiensis is an insect pathogen. It is characterized by
the synthesis upon sporulation of a parasporal inclusion
body. This inclusion body is made of proteins, the δ-en-
dotoxins, which are toxic to several insect larvae [11,12]
and other invertebrates [13]. B. thuringiensis formula-
tions have been developed for the control of insect pests
in agriculture and forestry [14-16] and for the control of
insect vectors of human diseases such as malaria, yel-
low fever, onchocerciasis, etc [17]. Bacillus weihenstep-
hanensis is a psychotolerant species characterized by the
ability to grow at 7°C and the absence of growth at 43°C.
It is also characterized by the presence of specific signa-
ture sequences on the 16S rRNA gene (small subunit
ribosomal RNA gene) and the cspA gene (gene encoding
the major cold shock protein) [18]. B. mycoides is char-

S. Yakoubou et al. / Natural Science 2 (2010) 1113-1118
1114
acterized by the formation of rhizoid colonies and the
absence of motility [19]. B. pseudomycoides is pheno-
typically similar to B. mycoides and is distinguished by
DNA relatedness and fatty acid composition [2].
The 16S rDNA is the macromolecule of choice in the
reconstruction of bacterial phylogenies [20-24]. The 16S
rDNA, however, cannot distinguish among species in the
Bacillus cereus group [25,26]. Genomic approaches have
been used in an attempt to elucidate the genetic diversity
of three highly closely related species in the B. cereus
group: B. cereus, B. anthracis and B. thuringiensis. They
appear as a single species on the basis of genetic eviden-
ce [27].
In a previous study, a 220 bp marker was developed
and used to infer the phylogeny of species in the genus
Bacillus and closely-related genera [28]. This marker
was a combination of the last 150 bp at the 3’ end of the
16S rDNA and the first 70 bp at the 5’ end of the 16S-
23S rDNA internal transcribed spacer (ITS). More recent-
ly, we assessed the usefulness of the 220 bp marker at a
higher taxonomic level, the Order Bacillales [29]. This
marker showed several advantages over the use of 16S
rDNA sequences or the generation of extensive phenoty-
pic and genotypic data in phylogenetic analyses. First,
the 150 bp at the 3’ end of the 16S rDNA allowed dis-
crimination among distantly related species. Owing to its
higher rate of nucleotide substitutions, the 70 bp at the 5’
end of the 16S-23S rDNA (ITS) added discriminating
power among closely related species from same genus
and closely related genera from same family. Because of
its higher percentage of nucleotide sequence divergence
than the 16S rDNA, the 220 bp marker could better dis-
criminate among closely related Bacillus [28] and Ba -
cillales [29] species. Second, the method was simple, ra-
pid, suited to large screening programs and easily acces-
sible to most laboratories. Third, the marker also revea-
led species which appeared misassigned and for which
additional characterization appeared warranted.
In the current study, we further analyze the resolving
power of this short marker in inferring phylogenies at a
much lower taxa level: the Bacillus cereus group.
2. MATERIALS AND METHODS
2.1. Bacterial Species and Strains
Four species in the Bacillus cereus group: B. anthracis,
B. cereus, B. thuringiensis and B. weihenstephanensis
were analyzed. Three additional Ba cillus species, B. ha-
lodurans, B. licheniformis and B. subtilis, and Clostrid-
ium tetani were included for comparison purposes. A to-
tal of eight bacterial species and 12 strains were analy-
zed (Table 1). They were selected on the basis that their
complete genome sequences were freely available in Ge-
nBank at the National Center for Biotechnology Infor-
mation (NCBI) completed microbial genomes database
(http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi,Augus
t 2009). Bacillus mycoides and B. pseudomycoides were
not included because their complete genome sequences
have not been determined.
Table 1. Bacterial species used in this study.
Genera Species Strain GenBank accession no.
Bacillus anthracis
cereus
thuringiensis serovar konkukian
weihenstephanensis
halodurans
licheniformis
subtilis subsp. subtilis
Ames
Ames Ancestor
Sterne
ATCC 14579
ATCC 10987
E33L
97-27
KBAB4
C-125
ATCC 14580
168
AE016879
AE017334.2
AE017225.1
AE016877.1
AE017194.1
CP000001.1
AE017355.1
NC_010184.1
BA000004.3
AE017333.1
AL009126.3
Clostridium tetani E88 AE015927.1

S. Yakoubou et al. / Natural Science 2 (2010) 1113-1118
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2.2. Sequences
The 16S rDNA and 16S-23S ITS for the 12 bacterial
species and strains were retrieved from GenBank, for a
total of 129 allelic sequences. The last 150 bp at the 3’
end of 16S rDNA and the first 70 bp at the 5’ end of
16S-23S ITS were merged into a single 220 bp sequence
for each of the 129 alleles under study as described be-
fore [28]. This 220 bp sequence will be used as a phy-
logenetic marker for the 12 bacterial species and strains
under study.
2.3. Phylogenetic Analyses
All 129 allelic sequences were aligned using ClustalW
[30] (data not shown). A neighbor-joining tree was cons-
tructed [31], based on the alignment of the 129 alleles of
the 220 bp sequence. The tree was bootstrapped using
1,000 random samples. The neighbor-joining tree was dr-
awn and printed with Tree Explorer, all components of
the Molecular Evolutionary Genetics Analysis (MEGA,
version 3.1) software package [32].
3. RESULTS AND DISCUSSIONS
In a previous study, a 220 bp sequence was developed
as a DNA marker and used to infer the phylogeny of
species in the Gram-positive genus Bacillus and closely-
related genera [28]. This marker was a combination of
the last 150 bp at the 3’ end of the 16S rDNA and the
first 70 bp at the 5’ end of the 16S-23S rDNA internal
transcribed spacer (ITS). More recently, we assessed the
usefulness of the 220 bp marker by extending its analy-
ses at a higher taxonomic level, the Gram-positive Order
Bacillales [29]. In parallel, a similar marker was used to
infer the phylogeny of the Gram-negative Class γ-pro-
teobacteria [33]. In the current study, we further analyze
the resolving power of this marker in inferring the phy-
logeny at a much lower taxa level: the Bacillus cereus
group.
A bootstrapped neighbor-joining phylogenetic tree
was inferred from comparative analyses of the 220 bp
marker from the 129 alleles from the bacterial species
and strains under study (Figure 1). Four major Groups
were revealed based on the topology of the neighbor-
joining tree at the 90% nucleotide sequence identities,
Group I to IV. Group I contains all alleles of the species
in the Bacillu s cereus group. Group II contains all alleles
of B. halodurans. Group III contains all alleles of B.
licheniformis and B. subtilis. Group IV contains all al-
leles of Clostridium tetani. Based on nucleotide se-
quence identities, sub-groups and branches can be re-
vealed. Group I can be sub-divided into three sub-groups
at the 95% nucleotide sequence identities. Sub-group I-1
encompasses 27 alleles from the B. anthracis strains, 36
alleles from the B. cereus strains, 11 alleles from B.
thuringiensis and one allele from B. weihenstephanensis.
Sub-group I-2 encompasses six alleles from B. anthracis,
two alleles from B. cereus and one allele from B. thur-
ingiensis. Sub-group I-3 encompasses 12 alleles from B.
weihenstephanensis. A branch corresponding to an allele
from B. weihenstephanensis is present between sub-gro-
ups I-2 and I-3. Group II contains all eight alleles from B.
halodurans. They show at least 20% nucleotide sequen-
ce divergences with alleles from the other Bacillus spe-
cies. Group III contains all alleles from B. licheniformis
and B. subtilis. It can be sub-divided into two sub-groups
at the 95% nucleotide sequence identities. Sub-group
III-1 encompasses all seven alleles from B. licheniformis.
Sub-group III-2 encompasses all ten alleles from B. sub-
tilis. Group IV contains all six alleles from Clostridium
tetani. These alleles show at least 26% nucleotide se-
quence divergence with alleles from species and strains
in the genus Bacillus.
In accordance with our previous work on the Order
Bacillales, the 220 bp sequence used as a phylogenetic
marker was able to group alleles from same species for B.
halodurans, B. licheniformis, B. subtilis, and Clostrid-
ium tetani, respectively. However, this 220 bp sequence
could not group most alleles from same species, exclu-
sive of alleles from others, for the B. cereus group. Sub-
group I-1 is heterogeneous. It contains alleles from all
four species from the B. cereus group. The close prox-
imity of B. cereus, B. anthracis and B. thuringiensis is in
agreement with previous works based on whole-genome
DNA hybridization [34], pulsed-field gel electrophoresis
(PFGE) [35], multilocus enzyme electrophoresis (MEE)
[36], amplified fragment length polymorphism (AFLP)
fingerprinting [37] and multilocus sequence typing (ML-
ST) [38,39], which showed that all three species are ge-
netically highly related. They appear as a single species
on the basis of genetic evidence [27]. Sub-groups I-2 is
more homogeneous. It mostly contains alleles from B. an-
thracis. Sub-groups I-3 is homogeneous. It only contains
alleles from B. weihenstephanensis. As shown earlier, on
the genus Bacillus [28] and the Order Bacillales [29],
this 220 bp sequence contains 150 bp at the 3’ end of
16S rDNA which allowed discrimination among dis-
tantly related species and 70 bp at the 5’ end of 16S-23S
ITS which, owing to its higher percentage of nucleotide
sequence divergence, added resolving power among clo-
sely related species.
Here, species in the B. cereus group, most notably B.
cereus, B. anthracis and B. thuringiensis, are too closely
related to be discriminated with the 220 bp sequence
previously used as a phylogenetic marker. Our work,
however, has shown that the alleles in sub-group I-3
could distinguish B. weihenstephanensis from all other
species.

S. Yakoubou et al. / Natural Science 2 (2010) 1113-1118
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87
96
93
100
88
61
100
55
95
74
99
55
99
64
81
96
65
Bacillus anthracis Ames rRNA E5
Bacillus anthracis Ames Ancestor rRNA F
Bacillus cereus 14579 rRNA C
Bacillus anthracis sterne rRNA 9
Bacillus cereus 14579 rRNA F5
Bacillus cereus 10987 rRNA A5
Bacillus cereus E33L rRNA 9
Bacillus anthracis Ames rRNA C
Bacillus cereus 14579 rRNA G
Bacillus cereus E33L rRNA 10
Bacillus anthracis sterne RNA1
Bacillus anthracis Ames rRNA D
Bacillus cereus E33L rRNA 8
Bacillus cereus 10987 rRNA D
Bacillus cereus E33L rRNA 7
Bacillus anthracis Ames Ancestor rRNA E
Bacillus anthracis sterne RNA2
Bacillus cereus 14579 rRNA L
Bacillus cereus E33L rRNA 2
Bacillus thuringiensis konkukian str. 97-27 rRNA 5
Bacillus cereus E33L rRNA 12
Bacillus thuringiensis konkukian str. 97-27 rRNA 3
Bacillus cereus 14579 rRNA J
Bacillus cereus E33L rRNA 3
Bacillus cereus 14579 rRNA I
Bacillus thuringiensis konkukian str. 97-27 rRNA 8
Bacillus thuringiensis konkukian str. 97-27 rRNA 13
Bacillus anthracis Ames Ancestor rRNA B
Bacillus anthracis Ames Ancestor rRNA D
Bacillus cereus 10987 rRNA J
Bacillus cereus 14579 rRNA H
Bacillus anthracis sterne RNA5
Bacillus anthracis Ames Ancestor rRNA H
Bacillus anthracis Ames Ancestor rRNA C
Bacillus cereus 14579 rRNA D
Bacillus cereus 10987 rRNA C
Bacillus cereus 10987 rRNA E4
Bacillus cereus 14579 rRNA K
Bacillus cereus E33L rRNA 4
Bacillus thuringiensis konkukian str. 97-27 rRNA 10
Bacillus thuringiensis konkukian str. 97-27 rRNA 9
Bacillus cereus 10987 rRNA K
Bacillus thuringiensiskonkukian str. 97-27 rRNA 11
Bacillus cereus 10987 rRNA I
Bacillus anthracis sterne RNA6
Bacillus cereus 10987 rRNA I
Bacillus thuringiensiskonkukian str. 97-27 rRNA 7
Bacillus anthracis Ames Ancestor rRNA G
Bacillus anthracis sterne rRNA 9
Bacillus anthracis Ames Ancestor rRNA I
Bacillus anthracis Ames Ancestor rRNA A
Bacillus cereus E33L rRNA 6
Bacillus anthracis Ames rRNA I
Bacillus anthracis sterne RNA7
Bacillus thuringiensis konkukian str. 97-27 rRNA 2
Bacillus cereus E33L rRNA 1
Bacillus anthracis Ames rRNA F
Bacillus thuringiensis konkukian str. 97-27 rRNA 4
Bacillus anthracis Ames rRNA A
Bacillus cereus 10987 rRNA F
Bacillus cereus E33L rRNA 11
Bacillus cereus 14579 rRNA M
Bacillus anthracis sterne RNA4
Bacillus cereus E33L rRNA 5
Bacillus anthracis Ames rRNA G
Bacillus anthracis Ames rRNA H
Bacillus anthracis sterne RNA3
Bacillus anthracis Ames rRNA B
Bacillus cereus 10987 rRNA B
Bacillus cereus 10987 rRNA H5
Bacillus cereus E33L rRNA 13
Bacillus weihenstephanensis rRNA R0001
Bacillus cereus 14579 rRNA E
Bacillus cereus 10987 rRNA G
Bacillus thuringiensis konkukian str. 97-27 rRNA12
Bacillus thuringiensis konkukian str. 97-27 rRNA1
Bacillus anthracis Ames Ancestor rRNA J
Bacillus anthracis sterne RNA10
Bacillus cereus14579 rRNA A
Bacillus anthracis Ames rRNA J
Bacillus anthracis Ames Ancestor rRNA K
Bacillus cereus 14579 rRNA B
Bacillus anthracis Ames rRNA K
Bacillus anthracis sterne RNA11
Bacillus thuringiensis konkukian str. 97-27 rRNA 6
Bacillus weihenstephanensis rRNA R0008
Bacillus weihenstephanensis rRNA R0027
Bacillus weihenstephanensis rRNA R0146
Bacillus weihenstephanensis rRNA R0030
Bacillus weihenstephanensis rRNA R0071
Bacillus weihenstephanensis rRNA R0059
Bacillus weihenstephanensis rRNA R0068
Bacillus weihenstephanensis rRNA R0054
Bacillus weihenstephanensis rRNA R0094
Bacillus weihenstephanensis rRNA R0065
Bacillus weihenstephanensis rRNA R0015
Bacillus weihenstephanensis rRNA R0062
Bacillus weihenstephanensis rRNA R0074
Bacillus halodurans C-125 rRNAB
Bacillus halodurans C-125 rRNAC
Bacillus halodurans C-125 rRNAD
Bacillus halodurans C-125 rRNAH
Bacillus halodurans C-125 rRNA A
Bacillus halodurans C-125 rRNA F
Bacillus halodurans C-125 rRNA C
Bacillus halodurans C-125 rRNA G
Bacillus licheniformis 14580rrna 0010
Bacillus licheniformis 14580rrna 0021
Bacillus licheniformis 14580rrna 0016
Bacillus licheniformis 14580rrna 0001
Bacillus licheniformis 14580rrna 0013
Bacillus licheniformis 14580rrna 0004
Bacillus licheniformis 14580rrna 0008
Bacillus subtilisrrn O
Bacillus subtilisrrn A
Bacillus subtilisrrn W
Bacillus subtilisrrn J
Bacillus subtilisrrn B
Bacillus subtilisrrn I
Bacillus subtilisrrn E
Bacillus subtilisrrn D
Bacillus subtilisrrn H
Bacillus subtilisrrn G
Clostridium tetaniE888RNA 5
Clostridium tetaniE888RNA 5
Clostridium tetaniE888RNA 4
Clostridium tetaniE888RNA 3
Clostridium tetaniE888RNA 1
Clostridium tetaniE888RNA 2
Sub- GroupsGroups
I
II
III
IV
I-1
I-2
I-3
III-1
III-2
0.01
Figure 1. Bootstrapped neighbor-joining tree inferred from comparative alignment of the 220 bp marker from the 129 alleles from
the 12 bacterial species and strains under study. Major groups are indicated in capital roman numerals. Sub-groups are indicated in
arabic numerals. Bootstrap values higher than 50% are indicated (expressed as percentage of 1000 replication). The horizontal bar
represents 1% nt difference. Bacillus anthracis, B. cereus, B thuringiensis and B. weihenstephanensis’s alleles are written in red, blue,
green and brown, respectively. B. halodurans, B. licheniformis, B. subtilis and Clostridium tetani’s alleles are written in black ink.

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4. CONCLUSIONS
Previous genetic analyses have shown that B. cereus,
B. anthracis and B. thuringiensis should be regarded as a
single species. We have shown here that a 220 bp marker,
used to reconstruct the phylogeny of the Order Bacillales
and the family Bacillacea e, was unable to discriminate
between these three highly-related species. We have rea-
ched the limit of the resolving power of the 220 bp se-
quence as a phylogenetic marker: B. cereus, B. anthracis
and B. thuringiensis.
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