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(ii) PFGE and Southern blotting. Bacteria grown in MRS broth were har- vested during the exponential growth phase, washed twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8]), resuspended in T100E buffer (10 mM Tris-HCl, 100 mM EDTA [pH 7.5]), and embedded in 1% agarose slices. DNA was extracted by incubating the gel slices for 8 h at 37°C in T100Ebuffer containing 10 mg of lysozyme per ml, followed by 16 h at 37°C in TE buffer supplemented with 1.5% N-lauryl sarcosine and 2 mg of pronase per ml. The gel slices were subsequently transferred into T100E buffer and stored at 4°C until use. To obtain NotI digests, the gel slices were washed four times with TE buffer, rinsed with water, and incubated for 16 h at 25°C in 120- lreaction mixtures containing 150 U of NotI (New England Biolabs) according to the manufactur- er’s instructions. Pulsed-field gel electrophoresis (PFGE) was performed in a 1% agarose gel using the CHEF-DRIII system (Bio-Rad) with pulse times of 1 to 25 s for 20 h at 6 V/cm and 15°C in 0.5 TEB buffer (45 mM Tris-OH [pH 8], 45 mM boric acid, 1 mM EDTA). DNA was transferred to aHybond-N membrane (Amersham Biosciences) and hybridized by the method of Maniatis et al. (37). The DNA probe corresponded to a 950-bp internal region of the gtf gene amplified by PCR from total DNA from O. oeni IOEB 0205 cells using primers PF1 and PF8 (see Table 2). The probe was labeled with digoxigenin-11- dUTP by using the digoxigenin DNA labeling kit (Roche), and detection wasdone by chemiluminescence with an antidigoxigenin antibody and CDP-Star (Roche).

(iii) Sequencing of the gtf locus. The sequence of the gtf locus of O. oeni IOEB 0205 was determined by the linker-mediated PCR strategy with the Topo-Walker kit (Invitrogen). The sequences of the primers used are shown in Table 2. Purified DNA from O. oeni IOEB 0205 was used in order toidentify restriction enzyme cleavage sites located between 1 and 5 kb from the 950-bp internal fragment of the gtf gene amplified with primers PF1 and PF8. Two primers located inside this fragment and directed toward the upstream (GspC) or down- stream (GspD) regions were used to elongate DNA fragments containing an EcoRI site and a BamHI site, respectively, at theirextremities (Fig. 1). The Topo linker was added to the extremities of the elongated DNA by topoisomerase- mediated ligation, providing two DNA templates that were amplified by PCR with the primers for upstream GspB and downstream GspE and a primer located inside the Topo linker. The two PCR products were gel purified and sequenced (Milligen, France). Subsequent PCR experimentsusing primers designed from orf1 orthologs in lactic acid bacteria and primer GspE enabled the complete region shown in Fig. 1 to be amplified.

(iv) PCR analysis. PCR mixtures (25 l) contained 0.5 l of each primer (50 pmol), 0.5 l of genomic DNA, and 2 l of custom-made PCR master mix 12.5X (Qbiogene, France). The reaction mixture was preheated for 5 min at 95°C, and 35 cycles(denaturing step of 30 s at 95°C, annealing step of 30 s at 55°C, and extension step of 1 min at 72°C) were carried out. Using primers GspB and C2 enabled the amplification of a 1,277-bp fragment, while primers GspE and C1 generated a 997-bp fragment from the DNA from gtf strains. The primer sets E/D, F/D, and G/H were used to search for the genes surrounding gtf in DNA from various O.oeni gtf mutant strains. The relative positions of the primers are indicated in Fig. 1, and their sequences are shown in Table 2.

(v) Reverse transcription-PCR (RT-PCR) analysis. The 50- l reaction mix- ture contained 25 l of the 2 Sybr green PCR supermix (Bio-Rad), 5 pmol each of primers FQ1 and FQ2 for gtf amplification and LDH1 and LDH2 for D-lactate dehydrogenase (ldhD)...
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