16S rRNA gene copy number in probiotic Lactobacillus strains Lactobacillus salivarius (Claesson et al., 2006) possess seven copies of the 16S rRNA gene per genome, while Lactobacillus delbrueckii has the highest multiplicity, with nine copies of the 16S rRNA gene (Makarova et al., 2006; van de Guchte et al., 2006). Information on other Lactobacillus species is scarce. Thus, in the present study, the 16S rRNA gene copy numbers of 12 Lactobacillus strains from ﬁve differ- ent species (Lactobacillus gallinarum, Lactobacillus panis, L. reuteri, L. brevis and L. salivarius) were determined by Southern hybridization technique. Seven restriction en- zymes that do not cut within the 16S rRNA genes were used to digest the genomic DNA of the strains. The digested genomic DNA was hybridized with a labeled probe which was complementary to the 16S rRNA gene to determine the 16S rRNA gene copy number. Materials and methods Bacterial strains Lactobacillus reuteri C 1, C 10 and C 16; L. brevis I 12, I 23, I 25, I 211 and I 218; L. gallinarum I 16 and I 26; L. salivarius I 24; and L. panis C 17 used in this study were previously isolated from the gastrointestinal tract of chickens by Jin et al. (1996). These Lactobacillus strains were identiﬁed using conventional and molecular techniques, and were used as a probiotic to exert health beneﬁts in poultry (Jin et al., 1998, 2000; Kalavathy et al., 2003; Lee et al., 2008). The Lactobacillus strains were propagated in de Man, Rogosa, Sharpe (MRS) broth (Oxoid, UK) and incubated at 39 1C for 24 h in anaerobic jars (Oxoid) with gas generating kits (Oxoid). Stock cultures were stored in 15% glycerol at 80 1C. Genomic DNA preparation High-molecular-weight DNA was extracted from the 12 Lactobacillus strains according to the method described by Leenhouts et al. (1990) with slight modiﬁcations as follows. The overnight bacterial cultures (24 h) were resuspended in lysis buffer containing 8 mg of lysozyme mL 1 and incu- bated at 37 1C for 2 h. After 1-h incubation with proteinase K and sodium dodecyl sulphate (SDS), the lysate was extracted twice with an equal volume of phenol– chloroform–isoamyl alcohol (25 : 24 : 1) and once with an equal volume of chloroform–isoamyl alcohol (24 : 1). Genomic DNA was dissolved in 50 mL of TE buffer after precipitation with 3 M sodium acetate and ethanol. Restriction enzyme digestion of DNA and agarose gel electrophoresis The 16S rRNA gene sequences of the 12 Lactobacillus strains were analyzed using NEB cutter (Vincze et al., 2003) to FEMS Microbiol Lett 287 (2008) 136–141 137 determine the enzymes that do not cut within the 16S rRNA gene of the strains (GenBank accession numbers EF412975–EF412986). Genomic DNA (10 mg) was digested with a range of noncutting restriction enzymes (BamHI, BclI, EcoRI, EcoRV, HindIII, NheI and SalI; New England BioLabs). Independent digestions were carried out with 20 units of restriction enzyme and incubated overnight at their optimum conditions as recommended by the manu- facturer. Digested DNA was subjected to electrophoresis on a 0.8% (w/v) agarose gel in Tris-acetate buffer at 60 V for 4 h. DNA fragments were transferred to Amersham Hybond N1(GE Healthcare) by capillary blotting (Sambrook et al., 1989) with 20 SSC as the transfer solution. After the transfer, the nylon membrane was dried and cross-linked by UV irradiation. Preparation of biotinylated 16S rRNA gene probe The probe was prepared by PCR ampliﬁcation of a fragment of the 16S rRNA gene from L. reuteri C 10 which corre- sponded to the conserved sequence complementary to the positions 8-519 region of the rRNA gene (Escherichia coli numbering; Brosius et al., 1978). PCR mixture contained 50–100 ng of bacterial DNA, 50 pmol of each primer, 10 mmol of dNTPs (Finnzyme, Finland), 1 U of DyNAzyme II DNA polymerase (Finnzyme), and 1 PCR buffer, and the volume was adjusted to 50 mL with deionized water. PCR ampliﬁcation with universal primers 8f (50-AGAGTTTGAT CCTGGCTCAG-30) and 519r (50-GTATTACCGCGGCTGC TGG-30) was carried out by an initial denaturation at 95 1C for 5 min, 30 cycles of 95 1C for 1 min, 55 1C for 1 min, 72 1C for 2 min and ﬁnal extension at 72 1C for 7 min. The PCR products were electrophoresed and puriﬁed using the Qia- quick PCR puriﬁcation kit (Qiagen, Germany). The puriﬁed products were biotinylated by random labeling with the NEBlot Phototope Kit (New England BioLabs) according to the manufacturer’s instructions. Southern hybridization analysis Before hybridization, prehybridization was carried out for 1 h at 60 1C in 5 Denhardt’s solution, 6 SSC, 0.5% (w/v) SDS, and 10 mg mL 1 denatured salmon sperm DNA. Hy- bridization was accomplished by the addition of 20 ng mL 1 of denatured biotinylated probe to the same solution and incubated at 60 1C overnight. After hybridization, the blots were washed twice with 2 SSC containing 0.1% (w/v) SDS at room temperature for 5 min each and twice with 0.5 SSC containing 0.1% (w/v) SDS for 15 min each at 60 1C. Detection of hybridized probe was performed using a chemiluminescent detection system (Phototope-Star Detec- tion Kit, New England BioLabs). Hybridized DNA was visualized by exposure of the membrane to X-ray ﬁlm (Kodak). c 2008 The Authors Journal compilation c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd.