The primary structures of the 16s rRNA genes of the type strains of Lactobacillus casei and related taxa were determined by PCR DNA-sequencing methods. The sequences of Lactobacillus casei, Lactobacillus zeae, Lactobacillus paracasei, and Lactobacillus rhumnosus were different. The K,,, values ranged from 0.0040 to 0.0126. On the basis of the K,,, values and the levels of DNA-DNA relatedness among the strains of these species, the L. casei-related taxa should be classified in the following three species: L. zeae, which includes the type strains of L. zeae and L. casei; a species that includes the strains of L. paracasei and L. casei ATCC 334; and L. rhamnosus.Members of Lactobacillus casei are gram-positive, facultatively anaerobic, catalase-negative, facultatively heterofermentative, non-spore-forming rods and are isolated from many habitats (e.g., meats, milk, dairy products, sour dough, silage, and sewage). The study of 16s rRNA sequences is now considered to be a powerful technique for determining the phylogenetic relationships of microorganisms (1, 4-6, 13, 19, 22, 23 (10). Collins also determined the 16s rRNA sequences of the type strains of L. casei, L. paracasei, and L. rhamnosus (6).However, the 16s rRNA sequences of all of the type strains of the L. casei subspecies and the phylogenetic relationships among these strains have not been analyzed, so the classification of the L. casei group is not yet stable. Therefore, we analyzed ca. 1,520 nucleotides of the genes coding for the 16s rRNA (16s rDNA) in all of the type strains of the L. casei subspecies and closely related strains and then determined the phylogenetic relationships of these organisms.
MATERIALS AND METHODSBacterial strains and cultivation. The strains used in this study are shown in Table 1. These strains were grown aerobically on Briggs agar (Nissui Seiyaku, Inc., Tokyo, Japan) slants for 2 to 3 days at 30°C.Extraction and determination of the 16s rDNA sequence. Total DNA was extracted and purified from a loopful of cells of each strain with an InstaGene purification matrix (Bio-Rad Laboratories, Richmond, Calif.) according to the protocol for bacteria (2). The 16s rDNA fragments in the total DNA were amplified by the modified PCR methods of Wittwer and Garling (21) by using a model 1605 air thermocycler (Idaho Technology, Inc.) and 5O-pl glass capillary tubes. Eleven fragments of the 16s rDNA were amplified to facilitate amplification and sequencing. The 11 pairs of primers used for amplification are shown in Table 2. The sequence of primer -21M13 was connected to the 5' end region of one primer from each pair, and so a strand of the PCR product contained a complementary sequence of -21M13 in the 3' end region to which the -21M13 universal fluorescent primer annealed for sequencing. The nucleotide sequences of the strands which contained the complementary sequence of -21M13 were determined with a model 373A DNA sequencer (Applied Biosystems, Inc., Foster City, Calif.) by the dideoxynucleotide chain termination method modified for use with...
