A vector that expresses the full-length ospA gene from B. burgdogferi in L. plantarum was previously described [12] . To generate a vector expressing the OspA mutant, we designed primers in which we replaced the codon corresponding to Cys 17 with Asp 17 . Expression vectors were then transformed into L. plantarum strain 256 to obtain the clones LpA and LpA D17 , respectively. Protein expression was checked by immunoblot as follows. Recombinant L. plantarum cells were disrupted with a FrenchH press (Thermo Electron Corporation, Milford, MA), supernatants were analyzed on a 12% denaturing polyacrilamide gels and electrotransferred to a polyvinylidene difluoride membrane (PVDF, Millipore, Billerica, MA) for analysis with an OspAspecific monoclonal antibody (mAb 184.1). To evaluate export and lipidation of OspA and its mutant expressed by recombinant Lactobacillus, we performed Triton X-114 phase partitioning [24] . L. plantarum cultures were grown overnight at 30uC, harvested and resuspended to an OD 600 of 1.0 in PBS. Bacteria were disrupted with a FrenchH press and the insoluble material (membrane and cell wall) was separated from the cytosol fraction by centrifugation. This cell envelope fraction was suspended in 1 ml of ice-cold 2% Triton-X114 (v/v) in PBS. The fractions were rotated end over end at 4uC for 1 h and were phase-separated by warming the solution for 30 min in a water bath at 37uC followed by centrifugation for 15 min at 25uC. The separated detergent and aqueous phases were each washed three times. The solutions were then rewarmed and recentrifuged as described and the detergent and aqueous phases were collected. Ten (10) ml of each phase was analyzed on 15% denaturing polyacrylamide gels, electrotransferred to PVDF filters, and used for immunoblot analysis. OspAspecific monoclonal antibody LA2.2 (1:100) was used as primary antibody, goat anti-mouse IgG (H+L) conjugated to alkaline phosphatase (1:1000; Pierce Rockford, IL) was used as secondary antibody and the immunoblot was developed by BCIP/NBT TM (KPL, Washington, DC). The protein bands corresponding to each OspA antigen were quantified by densitometry using a Multi Image TM Light Cabinet and the AlphaEase TM software (Alpha Innotech Corporation, San Leandro, CA). The results were plotted as a percentage of the total OspA content for each recombinant Lactobacillus.