Expression and recombination of the antigenic variation vlsE gene of the Lyme disease spirochete Borrelia burgdorferi were analyzed in the tick vector. To assess vlsE expression, Ixodes scapularis nymphs infected with the B. burgdorferi strain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression of vlsE in vitro was affected by a rise in pH from 7.0 to 8.0 at 34°C. Hence, vlsE expression appears to be sensitive to environmental cues of the type found in the B. burgdorferi natural history. To assess vlsE recombination, nymphs were capillary fed the B. burgdorferi B31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10 B. burgdorferi clones from the spirochetal isolate of each tick were obtained. The vlsE cassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in the vlsE sequence. In contrast, vlsE cassettes amplified from B. burgdorferi clones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows that vlsE recombination does not occur in the tick vector.

Previously, we had demonstrated the upregulation in the expression of several proteins, including the lipoproteins OspC and P35, of Borrelia burgdorferi in the stationary growth phase. Since the expression of OspC is also known to be affected by culture temperature and pH, we examined the effects of both variables on the expression of the remaining stationary-phase-upregulated proteins. Our study revealed that the expression of each of the remaining stationary-phase-upregulated proteins, P35 included, was also influenced by culture temperature; these proteins were selectively expressed at 34°C but not at 24°C. Significantly, the expression of a majority of these proteins was also affected by culture pH, since they were abundantly expressed at pH 7.0 (resembling the tick midgut pH of 6.8 during feeding) but only sparsely at pH 8.0 (a condition closer to that of the unfed tick midgut pH of 7.4). We propose that this group of B. burgdorferi proteins, which in culture is selectively expressed under conditions of 34°C and pH 7.0, may be induced in the tick midgut during the feeding event. Furthermore, the differential and coordinate expression of these proteins under different environmental conditions suggests that the encoding genes may be coregulated.

bmp gene family 36 of Borrelia burgdorferi, the agent of Lyme disease, comprises four paralogs: bmpA, bmpB, bmpC, and bmpD. The bmpA and bmpB genes constitute an operon. All four genes have been found to be transcribed in cultured spirochetes. Expression from the bmpAB operon results in three distinct transcripts of 1.1, 1.6, and 2.4 kb, and the relative expression of bmpA mRNA is three-to fourfold greater than that of bmpB mRNA. However, thus far only expression of the BmpA protein has been demonstrated. Therefore, in this study we characterized the origins of the three transcripts and compared the relative expression of the BmpA and BmpB proteins. Northern blotting revealed that the three distinct transcripts originated from a single promoter located upstream of bmpA but terminated either 3 to the bmpA (1.1-kb RNA) or bmpB (2.4-kb RNA) gene or, most unusually, within the bmpB gene (1.6-kb RNA). Termination within the bmpB gene was associated with a functional Rho-independent transcription terminator. At the protein level, we also observed a 4.3-fold greater abundance of BmpA compared to that of BmpB. These studies identify a transcription termination mechanism in B. burgdorferi resulting in the disparate expression of the two genes of the bmpAB operon.Paralogous bmp gene family 36 of Borrelia burgdorferi encodes four putative lipoproteins: BmpA (P39), BmpB, BmpC, and BmpD. The corresponding genes are confined to a single locus on the linear chromosome of this organism in the following 5Ј33Ј gene order: bmpD-bmpC-bmpA-bmpB (2,15,33,43). The paralogs share a high degree of homology at both the DNA (56 to 64%) and protein (36 to 46% identity) levels (2,15,33,37,43). Although the function of none of these proteins has been established, the conservation of their genes within the B. burgdorferi sensu lato complex (2,16,33,37,43) and the presence of orthologs in Treponema pallidum (41) and numerous other bacteria (B. burgdorferi genome web site at the Institute for Genomic Research) suggest that these proteins fulfill an essential physiological role.All four paralogs are transcribed in cultured spirochetes (5,11,33,34,35). Whereas the transcription of bmpA, bmpB, and bmpD has been demonstrated by both Northern blotting (33, 34) and reverse transcription (RT-PCR) (11), the very lowlevel expression of bmpC mRNA could be ascertained only by RT-PCR (11). However, thus far, only the expression of BmpA and BmpD proteins in vitro has been demonstrated (33, 42). The expression of these genes at the RNA level was also examined as a function of culture temperature and pH (11,35). In one study, the expression of bmpC and bmpD was found to decrease by 6-and 2.5-fold, respectively, at 37°C and pH 6.8 compared to the expression level at 23°C and pH 7.5 (35). In a different study, there was no difference in the expression of the four genes in spirochetes cultured at 23, 32, and 37°C (11). The expression of these genes in vivo was recently examined by microarray analysis (21). In mice infected with B. burgdorferi, bmpB, bmpC, and bm...