They also demonstrated that mutation of the chbC gene resulted in a failure of the cells to initiate a second exponential phase by 200 h . From these data they concluded that chbC expression is critical for initiation and growth of B. burgdorferi cells in the second exponential phase when cultured in the absence of free GlcNAc . Since we have shown the rpoS mutant failed to initiate a second exponential phase in the absence Fludarabine datasheet of free GlcNAc by 381 h (Fig. 1), we hypothesized that the rpoS mutant may not exhibit a second exponential phase because RpoS is involved, directly or indirectly,
in the regulation of chbC transcription. To test this hypothesis, RNA was collected from B31-A, A74 and WC12 at various times during growth in media lacking free GlcNAc, and the expression of chbC was evaluated by real time quantitative reverse selleckchem transcription PCR (qRT-PCR) (Fig. 3). Figure 3 Mutation of rpoS delays the up regulation of chbC expression during GlcNAc starvation. Growth of B. burgdorferi strains B31-A (WT), A74 (rpoS mutant), and WC12 (rpoS complemented mutant)
in BSK-II without GlcNAc (closed circle, B31-A; closed triangle, A74; closed square, WC12) and expression of chbC transcript in each strain (open circle, B31-A; open triangle, A74; open square, WC12). Late-log phase cells from each strain were diluted to 1.0 × 105 cells ml-1in BSK-II lacking GlcNAc, and RNA was extracted from each strain at various times during selleck screening library growth. Expression of chbC was determined by qRT-PCR and the fold change from the initial time point (44 h) was calculated. For expression analyses, duplicate measurements were performed for two biological replicates. Error bars represent the standard error of the mean. Cells were collected for RNA extraction at 44 h after initiation of the growth experiment and at various time points thereafter. Fold differences in chbC expression
were calculated by comparing expression at the various time points to the expression at 44 h (Fig. 3). This time point was chosen as the baseline as cells are still in the first exponential phase and in the presence of residual free GlcNAc Etofibrate or chitobiose from yeastolate or rabbit serum (see below). Prior expression studies conducted by Tilly et al  demonstrated that chbC levels remain low in the presence of free GlcNAc. In addition, we evaluated the expression of chbC in cells cultured in the absence of GlcNAc and supplemented with high or low concentrations of chitobiose (data not shown). As in complete a medium, chbC expression levels remained low until chitobiose was exhausted and cells became starved for GlcNAc (data not shown). In wild-type cells, chbC levels increased by 22-fold at 195 h just as cells entered the second exponential phase.