138 Results and discussion The estimation of 16S rRNA gene copy number is very much dependent on the restriction digestion of genomic DNA by appropriate restriction endonucleases. Over or underesti- mation of copy numbers may occur. Although overestima- tion of the copy numbers of 16S rRNA genes may be relatively rare, it could happen if the target sequence of the 16S rRNA gene probe is cleaved by the restriction enzyme (Maslunka et al., 2006). Thus, in the present study, sequence analysis of the 16S rRNA gene was performed using NEB cutter (Vincze et al., 2003) to select restriction enzymes that do not cleave within the 16S rRNA gene, for the estimation of the gene copy number. A poor electrophoretic separation of DNA fragments is one of the factors that results in the underestimation of 16S rRNA gene copy numbers. Large DNA fragment or two DNA fragments of very similar size are always difﬁcult to separate and this will result in a single hybridization fragment containing two or more 16S rRNA genes. Besides, large DNA fragments which contain more than one gene with no cutting site between them will also contribute to the underestimation of 16S rRNA gene copy numbers. Performing several independent restriction digests is, therefore, essential in order to obtain an accurate estima- tion of 16S rRNA gene copy number of a bacterium. In the present study, different digestions of genomic DNA that produced similar number of hybridizing bands were used to determine the copy numbers of the 16S rRNA gene and, for each digest, a distinct hybridizing band detected on the blot corresponded to at least one 16S rRNA gene copy per genome. The results of Southern hybridization of L. salivarius I 24 genomic DNA with the probe complementary to the 16S rRNA gene revealed that the hybridization of EcoRI and HindIII fragments yielded seven bands (Fig. 1), which indicated that L. salivarius I 24 probably possessed seven copies of the 16S rRNA gene in the genome. This result is in agreement with the recent ﬁnding of Claesson et al. (2006) in which the complete genome sequence analysis of L. salivarius strain UCC 118 shows that the strain has seven copies of the 16S rRNA gene. There has been no information on the 16S rRNA gene copy numbers of L. gallinarum and L. panis. In the present study, the 16S rRNA gene probe hybridized to ﬁve bands in the BamHI, BclI, EcoRV and HindIII digests for L. panis C 17 (Fig. 2). Thus, the 16S rRNA gene copy number was estimated to be ﬁve. As for L. gallinarum I 16 and I 26, four 16S rRNA gene copy numbers were estimated from EcoRV, HindIII, and NheI digests (Fig. 3). However, unlike the gene copy number, the hybridization patterns of these digests were not similar between the two L. gallinarum strains. When Southern blots of L. brevis strains I 12, I 23, I 25, I 211 and I 218 were hybridized with the 16S rRNA gene c 2008 The Authors Journal compilation c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. C.M. Lee et al. Fig. 1. 16S rRNA gene copy number of Lactobacillus salivarius I 24 as determined by Southern hybridization with biotinylated 16S rRNA gene probe. Genomic DNA of L. salivarius I 24 was cleaved with several noncutting restriction enzymes. Lane 1, BamHI; lane 2, BclI; lane 3, EcoRI; lane 4, EcoRV; lane 5, HindIII; lane 6, NheI; lane 7, SalI. Fig. 2. Southern hybridization of Lactobacillus panis C 17 genomic DNA digested with different restriction enzymes. Lane 1, BamHI; lane 2, BclI; lane 3, EcoRI; lane 4, EcoRV; lane 5, HindIII; lane 6, NheI; lane 7, SalI. probe, uniform hybridization patterns were obtained for the ﬁve Lactobacillus strains (representative gel in Fig. 4). Similar hybridization patterns and number of hybridizing bands were produced for all ﬁve strains when their chromo- somal DNA was digested with BamHI, BclI, EcoRI, HindIII and NheI. The results of the Southern blots revealed the presence of four hybridizing bands of varying intensities, which suggests the existence of at least four copies of 16S rRNA gene in the L. brevis strains. As some of the bands have much stronger signals, it may suggest the presence of two or more comigrating DNA fragments or two or more 16S FEMS Microbiol Lett 287 (2008) 136–141