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International Journal of Systematic and Evolutionary Microbiology (2008), 58, 919–923 Bacillus coahuilensis sp. nov., a moderatelyhalophilic species from a desiccation lagoon in theCuatro Cie´negas Valley in Coahuila, Mexico Rene´ Cerritos,1 Pablo Vinuesa,2 Luis E. Eguiarte,1 Luis Herrera-Estrella,3Luis D. Alcaraz-Peraza,4 Jackeline L. Arvizu-Go´mez,4 Gabriela Olmedo,4Enrique Ramirez,4 Janet L. Siefert5 and Valeria Souza1 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, 5Department of Statistics, Rice University, Houston, TX 77251, USA A moderately halophilic, Gram-positive and rod-shaped bacterium, strain m4-4T, was isolatedfrom a Chihuahuan desert lagoon in Cuatro Cie´negas, Coahuila, Mexico. Strain m4-4T was foundto 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%. Strainm4-4T exhibited minimal or no growth on most sugars tested. Its major cellular fatty acids wereC14 : 0, C16 : 0 and C18 : 1. Based on phylogenetic analysis of 16S rRNA and recA genesequences, we observed that the closest relatives of the isolate are moderately halophilic Bacillusspecies, with 16S rRNA gene sequence similarity ranging from 96.6 to 97.4 % (Bacillusmarisflavi, Bacillus aquimaris and Bacillus vietnamensis). Additionally, using genomic data it wasdetermined that the type strain contains a total of nine rRNA operons with three slightly differentsequences. On the basis of phenotypic and molecular properties, strain m4-4T represents a novelspecies within the genus Bacillus, for which the name Bacillus coahuilensis sp. nov. isproposed, with the type strain m4-4T (5NRRL B-41737T 5CECT 7197T).
A number of halophilic and moderately halotolerant, 2006). In this study, the Bacillus strain m4-4T was isolated Gram-positive, endospore-forming aquatic isolates in the in August 2003 from a desiccation lagoon in the Churince genus Bacillus have been described. A large number of system, a hydrological system on the western side of the them have been isolated from marine environments Cuatro Cie´negas Valley in Coahuila, Mexico (26u 50.8309N, (Siefert et al., 2000; Yoon et al., 2003, 2004; Noguchi et al., 2004; Yoon & Oh, 2005; Lee et al., 2006). However, Strain m4-4T was analysed using taxonomic and biochem- little is known about species inhabiting non-marine, high ical methods. Two markers were used for phylogenetic salinity aquatic environments (Lim et al., 2006; Souza et al., reconstruction (16S rRNA and recA gene sequences).
Studies have shown that more robust results are obtained GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene when additional markers such as housekeeping genes are sequences: m4-4T, EF014450, EF014451 and EF014452; and the used, especially in closely related isolates (Stackebrandt recA gene sequences: m4-4T, EF014455; B. marisflavi TF-11, et al., 2002; Zeigler, 2003). We determined the phylogenetic EF014457; B. vietnamensis NRIC 0530, EF014458; B. vietnamensis affiliation of the isolate m4-4T by means of 16S rRNA gene NRIC 0531T, EF014459; B. vietnamensis NRIC 0532, EF014460; B.
phylogeny reconstruction and determined its taxonomic status as a representative of a novel species by using a Photomicrographs of Bacillus coahuilensis m4-4T, a phylogenetic tree of polyphasic approach. The study also included genomic the recA sequences of m4-4T and other bacilli and a table showing thefatty acid composition of m4-4T are available with the online version of analysis to determine environmental genome size and Strain m4-4T was isolated from surface water samples that 1500 uC for 3 min, increasing at the rate of 40 uC min21 to were taken and placed in sterile flasks. These were subjected a final temperature of 3000 uC, which was maintained for to a shock temperature of 80 uC for 20 min by means of 20 min. Helium was used as carrier gas with a constant damp heat (Istock et al., 2001). Subsequently, 1 : 100 and flow of 1 ml min21. Fatty acid methyl esters were identified 1 : 1000 dilutions were made. Aliquots (100 ml) from each using the mass spectral library search (NIST MS Data Base) dilution, as well as from the original water samples, were distributed by the National Institute of Standards and placed in Petri dishes with marine agar 2216 medium (MA; Difco) and incubated at 37 uC for 2 days. Cultures were A combination of Sanger (Nunally, 2005) and 454 Life purified by subculturing on the same medium and Sciences sequencing methods (Margulies et al., 2005) were maintained at –80 uC in 5 % MA and 15 % (w/v) glycerol.
used to sequence the m4-4T genome, as described by We studied the cell morphology and sporulation process Alcaraz et al. (2008). The genome sequencing found nine for strain m4-4T using phase-contrast microscopy. Cells ribosomal operons; three of them had slight differences, were negatively stained with 1 % (w/v) malachite green and giving sequences m4-4a, b and c (Fig. 1). The G+C contrasted with 1 % (w/v) safranine. Characterization of content was obtained directly by genomic analysis.
strain m4-4T included the study of cultural, physiological The 16S rRNA gene was amplified using the 27F and 1492R and biochemical parameters. Single carbon source assim- primers under conditions described previously (Lane, ilation tests were performed in MA (4 g l21), replacing the 1991) in 100 ml final volume. The recA gene was chosen yeast extract and peptone with the main carbon source.
for sequencing and phylogenetic analysis. Oligonucleotide Nitrate reduction was determined as described by La´nyı´ primers were designed using the recA genes of the complete (1987) in the presence and absence of 3 % (w/v) NaCl.
genomes of Bacillus strains reported in GenBank. These Growth at different temperatures was measured on MA primers extended from position 28 to 48 (59-GATCG- between 30 and 50 uC. Urease activity was determined as TCARGCAGSCYTWGAT-39) and from position 583 to described previously by Cowan & Steel (1965).
602 (59-TTWCCRACCATAACSCCRAC-39), yielding a For quantitative analysis of whole-cell fatty acids, strain 574 bp product. PCR mixtures (25 ml) were prepared with m4-4T was cultivated on MA for 2 days at 37 uC. The 1 U Taq polymerase (Roche), 2.5 mM MgCl2, 1 mM whole-cell fatty acid composition was determined by using dNTPs, 2 mM each recA primer and 1 ml DNA (25– a gas chromatograph (model 5890; Hewlett Packard) 100 ng ml21). The PCR program was one cycle of initial equipped with a capillary column HP-5MS (30 m6 denaturation at 95 uC for 5 min, 30 cycles of denaturation 0.25 mm i.d.; 0.25 mm film thickness) coupled to a mass at 95 uC for 30 s, annealing at 45 uC for 30 s and extension spectrometer detector (model 5972; Hewlett Packard).
at 72 uC for 60 s, and a final extension cycle at 72 uC for Operating conditions were an injection temperature of 5 min. PCR products were purified using a gel extraction Fig. 1. Unrooted phylogenetic tree using theneighbour-joining method and derived from theanalysis of the 16S rRNA gene sequences ofstrain m4-4T and other representative Bacillusstrains. Numbers next to the branches repres-ent bootstrap values expressed as percentagesof 2500 replications; only values greater than70 % are indicated. GenBank accession num-bers of sequences are shown in parentheses.
Bar, 0.01 substitutions per nucleotide position.
International Journal of Systematic and Evolutionary Microbiology 58 DNA kit (Qiagen). For the 16S rRNA gene, a fragment of sequences obtained with the Ribosomal Database Project approximately 1400 bp was sequenced with the primer set and the NCBI databases. Phylogenetic reconstruction for reported previously (Sacchi et al., 2002). For the recA gene, the recA gene was done using seven complete genomes of a 450 bp segment was sequenced from strain m4-4T, B.
Bacillus strains reported in the NCBI database. Sequences marisflavi TF-11T and from four isolates of B. vietnamensis were aligned using the CLUSTAL_W program (Thompson (NRIC 0530, 0531T, 0532 and 0533). The sequencing et al., 1994). Phylogenetic reconstruction for the 16S rRNA reaction had a total volume of 15 ml consisting of 2 ml Big and recA genes was done using the neighbour-joining Dye Terminator sequencing buffer (Applied Biosystems), algorithm with Kimura two-parameter distances, as 1.6 mM primer and 5 ml purified amplified product. The implemented in MEGA3 (Kumar et al., 2004).
amplification conditions were as follows: one cycle of5 min at 95 uC, and 45 cycles of 10 s at 95 uC, 10 s at 50 uC Strain m4-4T was subjected to morphological and physio- and 4 min at 60 uC. Sequencing was done in a capillary logical tests that showed significant differences with respect sequencer (ABI-Avant 100). Sequences (GenBank accession to other closely related Bacillus species (Table 1). Strain numbers EF014450–EF014452, EF014455 and EF014457– m4-4T grew on only three carbon sources (starch, glycerol EF014461) were manually edited with the BioEdit program and trehalose). Cells were rod-shaped, approximately 0.5– (Hall, 1999). In the case of the 16S rRNA gene sequences, 0.7 mm in diameter and 1.5–3 mm in length after 2 days of isolate identities were established by comparing the 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 forutilization of starch, glycerol, L-glutamine, citrate, trehalose and fumarate. All strains were negative for nitrate reduction, H2S and urease.
*C, Central; S, subterminal.
DLY, Light yellow; O, orange; PO, pale orange; PY, pale yellow.
di, iso; ai, anteiso.
IJSEM Online). The G+C content of 37 mol% for strain Description of Bacillus coahuilensis sp. nov.
m4-4T is significantly different from that for B. marisflavi Bacillus coahuilensis (co.a.hui.len9sis. N.L. masc. adj.
(49 mol%) and B. vietnamensis (43–44 mol%), but not coahuilensis in reference to Coahuila, the state in Mexico from that for B. aquimaris (38 mol%).
where the type strain was collected).
The major cellular fatty acids of strain m4-4T were C14 : 0 Vegetative cells are rod-shaped, occurring in large chains (29.4 %), C16 : 0 (22.3 %), C18 : 1 (15.2 %) and C17 : 0 (7.9 %).
(Supplementary Fig. S1a), approximately 0.5–0.7 mm in Fatty acids occurring in minor amounts were C12 : 0 diameter by 1.5–3 mm in length. Central ellipsoidal (1.3 %), anteiso-C17 : 0 (4.7 %) and anteiso-C15 : 0 (4.8 %) endospores are observed in swollen sporangia and are (Supplementary Table S1). Fatty acids profile comparisons 1.0 mm wide and 1.5–1.7 mm long (Supplementary Fig. S1b, between strain m4-4T and other species of the genus c). Colonies on MA are light yellow and 2–5 mm in Bacillus reveal significant differences (Table 1).
diameter after 2 days growth at 37 uC; they are low, convex, 16S rRNA gene sequence similarity between strain m4-4T circular and slightly irregular. Optimal growth temperature and type strains of other phylogenetically closely related is 30–37 uC and the maximum growth temperature is 45 uC.
Bacillus species (B. marisflavi, B. aquimaris and B.
Minimum pH for growth lies between 5.0 and 5.5, the vietnamensis) ranged from 96.6 to 97.4 %. Values obtained optimum pH for growth is between 7 and 8 and the in this study meet widely accepted criteria for delineating maximum pH for growth is 9. Acid is produced from species in current bacteriology (Stackebrandt & Goebel, glycerol, but not from D-glucose or lactose. Citrate and 1994). A 16S rRNA gene-sequence-based neighbour-join- fumarate can be utilized. Nitrate reduction was not present.
ing phylogeny analysis revealed that the three different H2S and urease are not produced. Does not utilize sucrose, ribosomal operons of strain m4-4T formed a tight and lactose, arabinose, dulcitol, fructose, adonitol, D-sorbitol, highly supported clade (100 % bootstrap support) nested salicin, D-mannitol, D-xylose, L-rhamnose and L-glutamine within a deeper cluster that comprises B. aquimaris, B.
as sole carbon and energy sources. DNA G+C content of marisflavi, B. vietnamensis and Bacillus seohaeanensis at a the type strain is 37 mol%. Halotolerant, growing in NaCl bootstrap confidence level of 87 % (Fig. 1). In addition, a salt concentration from 0.5 to 10 %. The major fatty acids recA-based neighbour-joining tree also grouped strain are C14 : 0, C16 : 0 and C18 : 1. Additionally, based on genome m4-4T as a strongly supported monophyletic lineage analysis, strain m4-4T showed nine ribosomal operons with (Supplementary Fig. S2), which is distinct from the clade comprising B. marisflavi and B. vietnamensis.
The type strain, m4-4T (5NRRL B-41737T 5CECT 7197T), Our results show that strain m4-4T can grow in medium was isolated from a desiccation lagoon in the Cuatro containing NaCl in the range 0.5 to 10 % (w/v). From these Cie´negas Valley in Coahuila, Mexico.
data we concluded that this Bacillus strain is moderatelyhalophilic (Ventosa et al., 1998).
In this study we described a Bacillus isolate using This research was supported by a CONACyT scholarship to C. R. The biochemical and genomic data as well as phylogenetic project was funded by SEMARNAT/CONACyT and SEP CONACyT reconstructions involving 16S rRNA and recA gene (C01-0237/A1 and 44673 Q) to V. S. and L. E. F. We thank Antonio sequences. This approach showed that m4-4T is a member Cruz, Laura Espinosa and Jose´ Luis Herna´ndez for specialized of a distinct group within the genus Bacillus. The strain technical assistance and Morena Avitia and Miguel Contreras for displayed characteristics typical of Bacillus species, like laboratory work. Special thanks to Alejandro Rooney for incorporat- spore production and low DNA G+C content (37 mol%).
ing the strains to the NRRL collection and Luisa Falcon and Ana M.
Noguez for thoughtful comments and Mark Schneegurt for providing However, the fatty acid composition for strain m4-4T is completely different from those of other closely relatedBacillus species (Supplementary Table S1). Chains C14, C16,and C18 are characteristic for this novel isolate.
Phylogenetic analysis using 16S rRNA gene sequencesshowed that the novel isolate formed a distinct clade Alcaraz, L. D., Olmedo, G., Bonilla, G., Cerritos, R., Herna´ndez, G., compared with the closely related type strains of B.
Cruz, A., Ramı´rez, E., Putonti, C., Jime´nez, B. & other authors (2008).
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survival in the relic of an ancient marine environment. Proc Natl Acad vietnamensis (NRIC 0531T) and B. seohaeanensis (DSM 16464T). Phylogenetic reconstruction using recA gene Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of sequences also showed that the novel isolate formed a Medical Bacteria. Cambridge: Cambridge University Press.
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