Bacillus coahuilensis sp. nov., a moderately
halophilic species from a desiccation lagoon in the
Cuatro Cie´negas Valley in Coahuila, Mexico
Rene´ Cerritos,1 Pablo Vinuesa,2 Luis E. Eguiarte,1 Luis Herrera-Estrella,3
Luis D. Alcaraz-Peraza,4 Jackeline L. Arvizu-Go´mez,4 Gabriela Olmedo,4
Enrique Ramirez,4 Janet L. Siefert5 and Valeria Souza1
Correspondence
Valeria Souza
souza@servidor.unam.mx
1Departamento de Ecologı´a Evolutiva, Instituto de Ecologı´a, Universidad Nacional Auto´noma de
Me´xico, Apartado Postal 70-275, Me´xico D.F. 04510, Mexico
2Programa de Ingenierı´a Geno´mica, Centro de Ciencias Geno´micas, Universidad Nacional
Auto´noma de Me´xico, Apartado postal 565-A, Cuernavaca, Mor. 62210, Mexico
3Langebio, Cinvestav, Apartado Postal 629, Irapuato, Gto. 36821, Mexico
4Departamento de Ingenierı´a Gene´tica de Plantas, Cinvestav Unidad Irapuato, Apartado Postal 629,
Irapuato, Gto. 36821, Mexico
5Department of Statistics, Rice University, Houston, TX 77251, USA
A moderately halophilic, Gram-positive and rod-shaped bacterium, strain m4-4T, was isolated
from a Chihuahuan desert lagoon in Cuatro Cie´negas, Coahuila, Mexico. Strain m4-4T was found
to grow optimally at 30–37 6C, pH 7.0–8.0 and 5% NaCl and to tolerate from 0.5% to 10%
NaCl. It was shown to be aerobic. The genomic DNA G+C content was about 37 mol%. Strain
m4-4T exhibited minimal or no growth on most sugars tested. Its major cellular fatty acids were
C14 : 0, C16 : 0 and C18 : 1. Based on phylogenetic analysis of 16S rRNA and recA gene
sequences, we observed that the closest relatives of the isolate are moderately halophilic Bacillus
species, with 16S rRNA gene sequence similarity ranging from 96.6 to 97.4% (Bacillus
marisflavi, Bacillus aquimaris and Bacillus vietnamensis). Additionally, using genomic data it was
determined that the type strain contains a total of nine rRNA operons with three slightly different
sequences. On the basis of phenotypic and molecular properties, strain m4-4T represents a novel
species within the genus Bacillus, for which the name Bacillus coahuilensis sp. nov. is
proposed, with the type strain m4-4T (5NRRL B-41737T 5CECT 7197T).
A number of halophilic and moderately halotolerant,
Gram-positive, endospore-forming aquatic isolates in the
genus Bacillus have been described. A large number of
them have been isolated from marine environments
(Siefert et al., 2000; Yoon et al., 2003, 2004; Noguchi
et al., 2004; Yoon & Oh, 2005; Lee et al., 2006). However,
little is known about species inhabiting non-marine, high
salinity aquatic environments (Lim et al., 2006; Souza et al.,
2006). In this study, the Bacillus strain m4-4T was isolated
in August 2003 from a desiccation lagoon in the Churince
system, a hydrological system on the western side of the
Cuatro Cie´negas Valley in Coahuila, Mexico (26u 50.8309N,
102u 09.3359W).
Strain m4-4T was analysed using taxonomic and biochem-
ical methods. Two markers were used for phylogenetic
reconstruction (16S rRNA and recA gene sequences).
Studies have shown that more robust results are obtained
when additional markers such as housekeeping genes are
used, especially in closely related isolates (Stackebrandt
et al., 2002; Zeigler, 2003). We determined the phylogenetic
affiliation of the isolate m4-4T by means of 16S rRNA gene
phylogeny reconstruction and determined its taxonomic
status as a representative of a novel species by using a
polyphasic approach. The study also included genomic
analysis to determine environmental genome size and
diversity of ribosomal operons.
GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences: m4-4T, EF014450, EF014451 and EF014452; and the
recA gene sequences: m4-4T, EF014455; B. marisflavi TF-11,
EF014457; B. vietnamensis NRIC 0530, EF014458; B. vietnamensis
NRIC 0531T, EF014459; B. vietnamensis NRIC 0532, EF014460; B.
vietnamensis NRIC 0533, EF014461.
Photomicrographs of Bacillus coahuilensis m4-4T, a phylogenetic tree of
the recA sequences of m4-4T and other bacilli and a table showing the
fatty acid composition of m4-4T are available with the online version of
this paper.
International Journal of Systematic and Evolutionary Microbiology (2008), 58, 919–923 DOI 10.1099/ijs.0.64959-0
64959 G 2008 IUMS Printed in Great Britain 919

Strain m4-4T was isolated from surface water samples that
were taken and placed in sterile flasks. These were subjected
to a shock temperature of 80 uC for 20 min by means of
damp heat (Istock et al., 2001). Subsequently, 1 : 100 and
1 : 1000 dilutions were made. Aliquots (100 ml) from each
dilution, as well as from the original water samples, were
placed in Petri dishes with marine agar 2216 medium (MA;
Difco) and incubated at 37 uC for 2 days. Cultures were
purified by subculturing on the same medium and
maintained at –80 uC in 5 % MA and 15 % (w/v) glycerol.
We studied the cell morphology and sporulation process
for strain m4-4T using phase-contrast microscopy. Cells
were negatively stained with 1 % (w/v) malachite green and
contrasted with 1 % (w/v) safranine. Characterization of
strain m4-4T included the study of cultural, physiological
and biochemical parameters. Single carbon source assim-
ilation tests were performed in MA (4 g l21), replacing the
yeast extract and peptone with the main carbon source.
Nitrate reduction was determined as described by La´nyı´
(1987) in the presence and absence of 3 % (w/v) NaCl.
Growth at different temperatures was measured on MA
between 30 and 50 uC. Urease activity was determined as
described previously by Cowan & Steel (1965).
For quantitative analysis of whole-cell fatty acids, strain
m4-4T was cultivated on MA for 2 days at 37 uC. The
whole-cell fatty acid composition was determined by using
a gas chromatograph (model 5890; Hewlett Packard)
equipped with a capillary column HP-5MS (30 m6
0.25 mm i.d.; 0.25 mm film thickness) coupled to a mass
spectrometer detector (model 5972; Hewlett Packard).
Operating conditions were an injection temperature of
1500 uC for 3 min, increasing at the rate of 40 uC min21 to
a final temperature of 3000 uC, which was maintained for
20 min. Helium was used as carrier gas with a constant
flow of 1 ml min21. Fatty acid methyl esters were identified
using the mass spectral library search (NIST MS Data Base)
distributed by the National Institute of Standards and
Technology (NIST).
A combination of Sanger (Nunally, 2005) and 454 Life
Sciences sequencing methods (Margulies et al., 2005) were
used to sequence the m4-4T genome, as described by
Alcaraz et al. (2008). The genome sequencing found nine
ribosomal operons; three of them had slight differences,
giving sequences m4-4a, b and c (Fig. 1). The G+C
content was obtained directly by genomic analysis.
The 16S rRNA gene was amplified using the 27F and 1492R
primers under conditions described previously (Lane,
1991) in 100 ml final volume. The recA gene was chosen
for sequencing and phylogenetic analysis. Oligonucleotide
primers were designed using the recA genes of the complete
genomes of Bacillus strains reported in GenBank. These
primers extended from position 28 to 48 (59-GATCG-
TCARGCAGSCYTWGAT-39) and from position 583 to
602 (59-TTWCCRACCATAACSCCRAC-39), yielding a
574 bp product. PCR mixtures (25 ml) were prepared with
1 U Taq polymerase (Roche), 2.5 mM MgCl2, 1 mM
dNTPs, 2 mM each recA primer and 1 ml DNA (25–
100 ng ml21). The PCR program was one cycle of initial
denaturation at 95 uC for 5 min, 30 cycles of denaturation
at 95 uC for 30 s, annealing at 45 uC for 30 s and extension
at 72 uC for 60 s, and a final extension cycle at 72 uC for
5 min. PCR products were purified using a gel extraction
Fig. 1. Unrooted phylogenetic tree using the
neighbour-joining method and derived from the
analysis of the 16S rRNA gene sequences of
strain m4-4T and other representative Bacillus
strains. Numbers next to the branches repres-
ent bootstrap values expressed as percentages
of 2500 replications; only values greater than
70% are indicated. GenBank accession num-
bers of sequences are shown in parentheses.
Bar, 0.01 substitutions per nucleotide position.
R. Cerritos and others
920 International Journal of Systematic and Evolutionary Microbiology 58

DNA kit (Qiagen). For the 16S rRNA gene, a fragment of
approximately 1400 bp was sequenced with the primer set
reported previously (Sacchi et al., 2002). For the recA gene,
a 450 bp segment was sequenced from strain m4-4T, B.
marisflavi TF-11T and from four isolates of B. vietnamensis
(NRIC 0530, 0531T, 0532 and 0533). The sequencing
reaction had a total volume of 15 ml consisting of 2 ml Big
Dye Terminator sequencing buffer (Applied Biosystems),
1.6 mM primer and 5 ml purified amplified product. The
amplification conditions were as follows: one cycle of
5 min at 95 uC, and 45 cycles of 10 s at 95 uC, 10 s at 50 uC
and 4 min at 60 uC. Sequencing was done in a capillary
sequencer (ABI-Avant 100). Sequences (GenBank accession
numbers EF014450–EF014452, EF014455 and EF014457–
EF014461) were manually edited with the BioEdit program
(Hall, 1999). In the case of the 16S rRNA gene sequences,
isolate identities were established by comparing the
sequences obtained with the Ribosomal Database Project
and the NCBI databases. Phylogenetic reconstruction for
the recA gene was done using seven complete genomes of
Bacillus strains reported in the NCBI database. Sequences
were aligned using the CLUSTAL_W program (Thompson
et al., 1994). Phylogenetic reconstruction for the 16S rRNA
and recA genes was done using the neighbour-joining
algorithm with Kimura two-parameter distances, as
implemented in MEGA3 (Kumar et al., 2004).
Strain m4-4T was subjected to morphological and physio-
logical tests that showed significant differences with respect
to other closely related Bacillus species (Table 1). Strain
m4-4T grew on only three carbon sources (starch, glycerol
and trehalose). Cells were rod-shaped, approximately 0.5–
0.7 mm in diameter and 1.5–3 mm in length after 2 days of
cultivation at 37 uC (Supplementary Fig. S1, available in
Table 1. Differential characteristics of strain m4-4T and closely related strains
Strains: 1, m4-4T; 2, B. marisflavi JCM 11544T; 3, B. aquimaris JCM 11545T; 4, B. vietnamensis NRIC 0531T. The four strains were positive for
utilization of starch, glycerol, L-glutamine, citrate, trehalose and fumarate. All strains were negative for nitrate reduction, H2S and urease.
Characteristic 1 2 3 4
Spore position* C S or C C C
Colony colourD LY PY PO O
Utilization of:
D-Mannose 2 + 2 2
Raffinose 2 + 2 2
Sucrose 2 + + +
D-Glucose 2 + + +
Lactose 2 + + +
Arabinose 2 + + +
Dulcitol 2 + + +
Fructose 2 + + +
Adonitol 2 + + +
D-Sorbitol 2 + + +
Salicin 2 + + +
D-Mannitol 2 + + +
D-Xylose 2 + + +
L-Rhamnose 2 + + +
Optimal temperature for growth (uC) 30–37 30–37 30–37 30–40
Growth at 45 uC 2 + 2 +
Optimal pH for growth 7.0–80 6.0–8.0 6.0–7.0 6.0–8.0
Growth at:
pH 4.5 2 + 2 2
pH 9.0 + + 2 +
NaCl for growth (% w/v):
Optimum 5 5 9 5
Maximum 10 10 15 15
Major cellular fatty acids (% of total)d C14 : 0 (29.4 %),
C16 : 0 (22.3 %),
C18 : 1 (15.2 %)
ai-C15 : 0 (27.7 %),
i-C15 : 0 (22.9 %),
i-C14 : 0 (9.1 %)
i-C15 : 0 (46.9 %),
ai-C15 : 0 (22.2 %),
i-C14 : 0 (6.5 %)
ai-C15 : 0 (48.3 %),
i-C15 : 0 (16.2 %),
ai-C17 : 0 (13.6 %)
DNA G+C content (mol%) 37 49 38 44
*C, Central; S, subterminal.
DLY, Light yellow; O, orange; PO, pale orange; PY, pale yellow.
di, iso; ai, anteiso.
Bacillus coahuilensis sp. nov.
http://ijs.sgmjournals.org 921

IJSEM Online). The G+C content of 37 mol% for strain
m4-4T is significantly different from that for B. marisflavi
(49 mol%) and B. vietnamensis (43–44 mol%), but not
from that for B. aquimaris (38 mol%).
The major cellular fatty acids of strain m4-4T were C14 : 0
(29.4 %), C16 : 0 (22.3 %), C18 : 1 (15.2 %) and C17 : 0 (7.9 %).
Fatty acids occurring in minor amounts were C12 : 0
(1.3 %), anteiso-C17 : 0 (4.7 %) and anteiso-C15 : 0 (4.8 %)
(Supplementary Table S1). Fatty acids profile comparisons
between strain m4-4T and other species of the genus
Bacillus reveal significant differences (Table 1).
16S rRNA gene sequence similarity between strain m4-4T
and type strains of other phylogenetically closely related
Bacillus species (B. marisflavi, B. aquimaris and B.
vietnamensis) ranged from 96.6 to 97.4 %. Values obtained
in this study meet widely accepted criteria for delineating
species in current bacteriology (Stackebrandt & Goebel,
1994). A 16S rRNA gene-sequence-based neighbour-join-
ing phylogeny analysis revealed that the three different
ribosomal operons of strain m4-4T formed a tight and
highly supported clade (100 % bootstrap support) nested
within a deeper cluster that comprises B. aquimaris, B.
marisflavi, B. vietnamensis and Bacillus seohaeanensis at a
bootstrap confidence level of 87 % (Fig. 1). In addition, a
recA-based neighbour-joining tree also grouped strain
m4-4T as a strongly supported monophyletic lineage
(Supplementary Fig. S2), which is distinct from the clade
comprising B. marisflavi and B. vietnamensis.
Our results show that strain m4-4T can grow in medium
containing NaCl in the range 0.5 to 10 % (w/v). From these
data we concluded that this Bacillus strain is moderately
halophilic (Ventosa et al., 1998).
In this study we described a Bacillus isolate using
biochemical and genomic data as well as phylogenetic
reconstructions involving 16S rRNA and recA gene
sequences. This approach showed that m4-4T is a member
of a distinct group within the genus Bacillus. The strain
displayed characteristics typical of Bacillus species, like
spore production and low DNA G+C content (37 mol%).
However, the fatty acid composition for strain m4-4T is
completely different from those of other closely related
Bacillus species (Supplementary Table S1). Chains C14, C16,
and C18 are characteristic for this novel isolate.
Phylogenetic analysis using 16S rRNA gene sequences
showed that the novel isolate formed a distinct clade
compared with the closely related type strains of B.
marisflavi (JCM 11544T), B. aquimaris (JCM 11545T), B.
vietnamensis (NRIC 0531T) and B. seohaeanensis (DSM
16464T). Phylogenetic reconstruction using recA gene
sequences also showed that the novel isolate formed a
distinct group compared with B. marisflavi JCM 11544T
and B. vietnamensis strains NRIC 0531T, 0530, 0532, and
0533. We suggest, on the basis of the data described above,
that strain m4-4T should be placed within the genus
Bacillus as a representative of a novel species, for which the
name Bacillus coahuilensis sp. nov. is proposed.
Description of Bacillus coahuilensis sp. nov.
Bacillus coahuilensis (co.a.hui.len9sis. N.L. masc. adj.
coahuilensis in reference to Coahuila, the state in Mexico
where the type strain was collected).
Vegetative cells are rod-shaped, occurring in large chains
(Supplementary Fig. S1a), approximately 0.5–0.7 mm in
diameter by 1.5–3 mm in length. Central ellipsoidal
endospores are observed in swollen sporangia and are
1.0 mm wide and 1.5–1.7 mm long (Supplementary Fig. S1b,
c). Colonies on MA are light yellow and 2–5 mm in
diameter after 2 days growth at 37 uC; they are low, convex,
circular and slightly irregular. Optimal growth temperature
is 30–37 uC and the maximum growth temperature is 45 uC.
Minimum pH for growth lies between 5.0 and 5.5, the
optimum pH for growth is between 7 and 8 and the
maximum pH for growth is 9. Acid is produced from
glycerol, but not from D-glucose or lactose. Citrate and
fumarate can be utilized. Nitrate reduction was not present.
H2S and urease are not produced. Does not utilize sucrose,
lactose, arabinose, dulcitol, fructose, adonitol, D-sorbitol,
salicin, D-mannitol, D-xylose, L-rhamnose and L-glutamine
as sole carbon and energy sources. DNA G+C content of
the type strain is 37 mol%. Halotolerant, growing in NaCl
salt concentration from 0.5 to 10 %. The major fatty acids
are C14 : 0, C16 : 0 and C18 : 1. Additionally, based on genome
analysis, strain m4-4T showed nine ribosomal operons with
three different sequences (Fig. 1).
The type strain, m4-4T (5NRRL B-41737T 5CECT 7197T),
was isolated from a desiccation lagoon in the Cuatro
Cie´negas Valley in Coahuila, Mexico.
Acknowledgements
This research was supported by a CONACyT scholarship to C. R. The
project was funded by SEMARNAT/CONACyT and SEP CONACyT
(C01-0237/A1 and 44673 Q) to V. S. and L. E. F. We thank Antonio
Cruz, Laura Espinosa and Jose´ Luis Herna´ndez for specialized
technical assistance and Morena Avitia and Miguel Contreras for
laboratory work. Special thanks to Alejandro Rooney for incorporat-
ing the strains to the NRRL collection and Luisa Falcon and Ana M.
Noguez for thoughtful comments and Mark Schneegurt for providing
useful bacillus strains.
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Bacillus coahuilensis sp. nov.
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