1 0 0 1 0 Page 4 of 16 Amplified fragment size (bp) Number of alleles in L. taiwanensis strains 1552 740 908 1176 908 Sarmiento-Rubiano et al. BMC Genomics 2010, 11:504 http://www.biomedcentral.com/1471-2164/11/504 Table 2 Multilocus sequence analysis Gene 16S rRNA recA pheS tufF pyrG Primer 16S-27 for 16S-1552rev RecAfor RecArev PheSfor PheSrev Tuf-for Tuf-rev PyrGfor PyrGrev Nucleotide sequence 5′-AGAGTTTGATCCTGGCTCAG 5′-AAGGAGGTGWTCARCCGCA 5′-GAAAARRAYTTYGGWAARGGYKCDRTBATGCG 5′-TACATRATRTCDACTTCWSMNMSYTTRAATGG 5′-KGGDCGYAAGGGTGAATTAAC 5′-ACATCRTTWGTRTAGAARTCACGAATATC 5′-ATGGCAGAAAAAGAACATTACG 5′-AGTAACYTGACCRGCACCAAC 5′-TTATGTTACYGAYGATGGTAC 5′-ACCACGWGTACCAAAACCAC DNA-DNA hybridization assays DNA macro-arrays on nylon membranes (Hybond N, Amersham Biosciences) were performed by using a dot blot assay according to Hänninen et al. [37] with some modifications. Three aliquots (160, 80 and 40 ng) of denatured chromosomal DNA samples of 26 Lactobacil- lus strains were spotted on duplicated membranes. Six digoxigenin-labelled probes were prepared by random priming using the DIG DNA Labelling kit (Roche) and genomic DNA isolated from the type strains of L. intes- tinalis, L. johnsonii, L. crispatus, L. gasseri, L. acidophi- lus or L. taiwanensis. Those probes were hybridized against six identical macroarrays with 26 Lactobacillus strains. Hybridization, washing and staining was carried out as recommended by the manufacturer using the CDP-Star chemiluminescent substrate (Roche). Chemi- luminescence was detected in a Fujifilm LAS 1000 ima- ging system (Fuji Photo Film Co. Ltd.) and it was measured using the Image Gauge Version 4.0 pro- gramme. The DNA-DNA hybridization experiments were done in triplicate. Comparative genomic hybridization on microarrays Based on the genome sequence of L. johnsonii NCC533 [13], Agilent 60-mer oligo microarrays were designed with 5 to 6 probes spreading the coding sequence of each gene (Agilent technologies Inc., USA). Genomic DNA was prepared and labelled as previously described [26]. Hybridization reactions were performed in a volume of 210 μl with 10 μl of labelled DNA, 70 μl of nuclease free water, 25 μl of control target Agi- lent and 105 μl of Agilent hybridization buffer, follow- ing the recommendations of the manufacturer. The slides were washed 10 min at RT in 6× SSC, 0.005% Triton x-100 and 10 min on ice in 0.1× SSC, 0.005% Triton x-100. The slides were immediately dried by centrifugation and scanned at 10 μm with a Scanarray 4000 (Packard Biochip Technologies, Billerica, MA, USA). Data were extracted with Imagene 5.6 (Biodis- covery, El Segundo, CA, USA) and treated with home- made scripts in Python language http://www.python. org. Probes were eliminated from the analysis if their signal strength was lower than twice the standard deviation of the local background when hybridized with L. johnsonii NCC533 genomic DNA. The signal ratio (Cy3-labeled unknown strain DNA versus Cy5- labeled L. johnsonii NCC533 DNA) of each spot was calculated without background subtraction and nor- malized based on our previous experience of CGH [26]. Having 5 to 6 probes per gene, each gene signal ratio was given by the median of the corresponding probes values. Considering the low level of strain variability for the tested molecule (DNA), the large number of probes per gene and the robustness of the median, biological replicates of hybridizations focused only on the most critical analysis: self-NCC533 (valida- tion of the arrays), ATCC33200T/NCC533 (intra-spe- cies comparison), and BL263/NCC533 (inter-species comparison with the novel Lactobacillus). Accession numbers The 16S rRNA gene, recA, pheS, tuf and pyrG sequences generated in this study were deposited in the GenBank under accession numbers FJ556999 to FJ557013, FJ557014 to FJ557023, GU121623 to GU121628 and HM777006, GU121629 to GU121634 and HM777008, and GU121635 to 121640 and HM777007, respectively. The microarrays data have been deposited in NCBI’s Gene Expression Omnibus [38] and are accessible through GEO Series accession number GSE21627.