16S rRNA gene copy number in probiotic Lactobacillus strains 139 Fig. 3. Southern hybridization with genomic DNA of Lactobacillus gallinarum I 16 (a) and I 26 (b). The genomic DNA was cleaved with several restriction enzymes that do not cut within the 16S rRNA gene. Lane 1, BamHI; lane 2, BclI; lane 3, EcoRI; lane 4, EcoRV; lane 5, HindIII; lane 6, NheI; lane 7, SalI. Fig. 4. Southern hybridization of Lactobacillus brevis I 211 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. rRNA genes located on the same DNA fragment. Recently, Makarova et al. (2006) reported that based on the complete genome sequences of L. brevis ATCC 367 in the GeneBank, the strain possessed ﬁve copies of the 16S rRNA genes. Digestion of chromosomal DNA of L. reuteri C 1, C 10 and C 16 with BamHI, BclI, EcoRI and EcoRV yielded four hybridizing bands of varying intensities, indicating the existence of at least four copies of the 16S rRNA gene (Fig. 5). It was also observed that the hybridizing patterns of these digests and those of HindIII, NheI and SalI for L. reuteri C 1 and C 16 were identical (Fig. 5a and b). The full genome sequencing of L. reuteri DSM 20016T (GenBank accession number CP000705) was completed recently and it was found that the 16S rRNA gene was present in six copies. The discrepancy between this result and that of the present FEMS Microbiol Lett 287 (2008) 136–141 study suggests that the hybridizing bands with strong signals in the present study could contain more than one 16S rRNA gene. The 16S rRNA gene copy numbers showed relatively little variation among strains within a species. As pointed out by Johansen et al. (1996), variations in 16S rRNA gene copy numbers between different bacterial species are well docu- mented, but strain-dependent differences are reported at a lower rate. In contrast to the 16S rRNA gene copy number, the restriction fragment patterns on Southern blots can be diverse among the strains. The variations of the hybridizing patterns may be due to genomic heterogeneity caused by point mutations, genetic rearrangements and additive ge- netic changes within and between the gene (Liu & Sander- son, 1998; Acinas et al., 2004; Gonza´ lez-Escalona et al., 2005). These events may change the cutting site frequency of restriction enzyme and the size of restricted fragment which subsequently generate substantial differences in the hybridization patterns. Thus, 16S rRNA gene restriction patterns seem to represent a promising tool in typing bacterial strains. Hybridization patterns of the 16S rRNA gene have been successfully used to discriminate strains of some lactic acid bacteria (Bjo¨rkroth & Korkeala, 1997) and Lactobacillus strains (Rodtong & Tannock, 1993; Bjo¨rkroth & Korkeala, 1996; Simpson et al., 2001). Similarly, in the present study, L. reuteri C 10 could be differentiated from L. reuteri C 1 and C 16, and L. gallinarum I 16 from L. gallinarum I 26 based on the hybridization patterns of these strains. Although many studies have been carried out on the multiplicity of the 16S rRNA gene in prokaryotic organisms, little information is available for Lactobacillus species. To our knowledge, no data on the 16S rRNA gene copy numbers of L. gallinarum and L. panis have been reported and our study is the ﬁrst record. Information on 16S rRNA c 2008 The Authors Journal compilation c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd.