Double bands were selected only when two distinct bands could be seen on the gel image and in the bionumerics densitometric curve window. Phylogenetic analyses were performed using the Dice similarity coefficient (Dice, 1945) and the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis based on numbers and positions of bands by bionumerics (Sneath & Sokal, 1973). Gel-purified LpF1 was cloned into the pCR-Blunt II-TOPO vector (Invitrogen) and sequenced using the M13 forward (−20) (5′-GTAAAACGACGGCCAG-3′), M13 reverse

(5′-CAGGAAACAGCTATGAC-3′), P1-FBA1 (5′-CAGATGGTCAATCAACGATC-3′), KU-60019 in vitro and P2-FBA1 (5′-CCGGGTGGTGGATTTAAACC-3′) primers using a BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems) in a 3730 Genetic Analyzer (Applied Biosystems). LpF1 was subsequently characterized by sequence similarity searches against the GenBank database using the blast

algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1997). The FBA1-specific fragment (LpF2) was amplified using 35 ng of template DNA, P3-FBA1 (5′-TCTATAATTTGTGATACAGGGGTTGCC-3′), and P4-FBA1 (5′-CTCGTAATCACACAGAAATTATGCTGC-3′) under the following cycling conditions: an initial 94 °C for 3 min; 35 cycles at 94 °C for 15 s, 59 °C for 35 s, and Z-VAD-FMK supplier 68 °C for 2 min; and a final 68 °C for 7 min. Genomic DNA (1 μg) from L. paraplantarum strains digested by Dra I were separated by a 1% agarose gel and transferred to nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany). The LpF2 fragment (946 bp) was purified using a PCR purification kit (Qiagen) and labeled using a Digoxigenin (DIG) High Prime Kit (Roche Diagnostics GmbH) according to the manufacturers’ instructions. Hybridization was carried out at 42 °C. Membrane was washed under conditions of high stringency at 68 °C. Detection was

Metalloexopeptidase performed using an anti-DIG antibody alkaline phosphatase conjugate and CSPD. Membrane was activated at 37 °C for 10 min and developed to an X-ray film (Roche Diagnostics GmbH). Strains were preliminarily classified by sequence analyses of pheS, rpoA (Naser et al., 2005), and 16S rRNA genes (Table 1) and further confirmed using PCR-based methods (Berthier & Ehrlich, 1999; Torriani et al., 2001a, b). To discriminate these strains, we evaluated repetitive element sequence-based (REP-) (Jersek et al., 1999), triplicate arbitrarily primed (TAP-) (Cusick & O’Sullivan, 2000), RAPD-, and ERIC-PCRs, but those except ERIC did not yield a band that was specific to L. paraplantarum strains (data not shown). In ERIC-PCR, the L. paraplantarum strains tested had similar band profiles (Fig. 1a, lanes 7–13); the shared bands agreed with the type strain of L. paraplantarum (JCM 12533T, lane 7). The DNA bands of approximately 2.8, 1.1, 0.9, and 0.55 kb generated with the primer set ERIC-1R and ERIC-2 were common to strains of the species L. paraplantarum (Fig. 1a, horizontal arrows).