Sarmiento-Rubiano et al. BMC Genomics 2010, 11:504 http://www.biomedcentral.com/1471-2164/11/504 Table 1 List of strains used in this study Species Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus acidophilus Lactobacillus crispatus Lactobacillus crispatus Lactobacillus gasseri Lactobacillus gasseri Lactobacillus gasseri Lactobacillus gasseri Lactobacillus gasseri Lactobacillus intestinalis Lactobacillus intestinalis Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus johnsonii Lactobacillus murinus Lactobacillus reuteri Lactobacillus taiwanensis Lactobacillus taiwanensis Lactobacillus taiwanensis Lactobacillus taiwanensis Lactobacillus taiwanensis Lactobacillus taiwanensis Straina Culture collectionb BL17 CECT 903T BL279 CECT 4529 BL280 CECT 4179 BL221 BL278 BL223 BL277 BL292 BL294 BL296 BL260 BL288 BL261 BL281 BL287 BL295 BL297 BL298 BL299 BL262 BL259 BL263 BL301 BL302 BL303 BL304 BL340 DSMZ 20584T ATCC 33323T NCC 2858 NCC 2856 NCC 2857 DSMZ 6629T CECT 289 ATCC 33200T NCC 2822 NCC 1741 NCC 533 NCC 2767 CECT 7394 DSMZ 21401T Host human chicken intestine rat feces human feces human eye human feces human unknown unknown unknown rat intestine rat intestine rat intestine unknown human blood dog feces unknown human feces dog feces rat intestine rat intestine rat intestine rat intestine rat intestine rat intestine rat intestine silage aBL, Culture Collection from our laboratory. Strains BL263, BL260, BL261, BL262 and BL259 were previously named as AD102, AD38, AD99, AD100 and AD23, respectively [29]. bCECT, The Spanish Type Culture Collection; DSMZ, German Collection of Microorganisms and Cell Cultures; NCC, Nestlé Culture Collection; ATCC, American Type Culture Collection. A (recA), phenylalanyl-tRNA synthase (pheS), CTP synthetase (pyrG) and translational elongation factor Tu (tuf). PCR reactions were performed with the Expand High Fidelity PCR System (Roche), using chromosomal DNA and the primers listed in Table 2. DNA sequen- cing was performed by the Central Service of Research Support of the University of Valencia (Spain) by using the Dideoxynucleotide DNA chain termination method. Both strands of the PCR fragments were sequenced. In the case of the recA gene, the PCR fragments were cloned in Escherichia coli using the pMOS Blue vector Page 3 of 16 contained in the Blunt- ended cloning kit (GE Health- care) following the manufacturer instructions. Plasmid DNA was isolated from each clone containing a portion of the recA gene from the Lactobacillus strains studied here. The partial recA genes were sequenced using the M13 universal and reverse primers. Phylogenetic analysis The sequences were either obtained in this work, retrieved from the GenBank database or from the Ribo- somal Database Project II (RDP) [32]. Multiple align- ments were obtained using ClustalX [33]. Positions of doubtful homology and gaps were removed by using Gblocks [34]. The phylogenetic reconstruction was per- formed by maximum likelihood as implemented in PHYML [35] with the GTR, substitution model in com- bination with estimation of the transtition/transversion ratio by maximizing the likelihood of the phylogeny, and estimations from the data set of the proportion of invar- iants and the shape parameter (alpha) of the gamma dis- tribution to account for substitution rate heterogeneity among sites. Bootstrap support values were obtained through the analysis of 500 pseudoreplicates. Physiological characterization Carbohydrate fermentation patterns were determined by using the API 50 CH (BioMérieux, S.A.) according to the supplier instructions. Results were recorded after 48 h at 37°C. Growth at 15°C, 45°C, pH 4.5 or in the pre- sence of NaCl (4.5% and 7.0%) was determined in MRS broth (Difco). All the growth analyses were done at least in duplicate. Data were analysed using Pearson’s correla- tion coefficient (XLSTAT programme). A tree was cre- ated by cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA). PAGE analysis of whole-cell protein Cultures were grown in 10 ml MRS at 37°C to an OD550 of 1.0. Cells were collected by centrifugation, washed with Tris-HCl 100 mM, pH 7.5, resuspended in 1 ml of the same buffer plus sucrose 0.5 M and lyso- zyme 2.5 mg/ml. After incubation for 1 h at 37°C, cells were washed, collected by centrifugation and resus- pended in 50 μl of SDS-PAGE loading buffer. Cells were lysed at 100°C 5 min and 20 μl of total extract were separated on a 12% SDS-PAGE using standard protocols [36]. The gels were stained with Coomassie brilliant blue. For each strain among two and four inde- pendent extracts were performed and analysed by SDS- PAGE. A PAGE analysis representative of each strain is shown in the results. Data were analysed using Pear- son’s correlation coefficient (BioNumerics 4.6 Applied Maths Kortrijk). A tree was created by cluster analysis using UPGMA.