burgdorferi genome shows 36% identity at the amino acid level (E-value is 7.9e-08) to bb0259, which has a GEWL domain. We did not attempt to knockout this gene, but it may be a target to consider in future studies. Since chitobiose transport is important for chitin utilization in other organisms [24, 31], we evaluated the S63845 solubility dmso role of chbC during chitin utilization in B. burgdorferi. As expected from previous studies [14, 17], RR34 (chbC mutant) was unable to grow on chitobiose in the absence of free GlcNAc (Fig. 5A). Similarly, no growth was observed when RR34 cells were
cultured in the absence of GlcNAc and supplemented with chitotriose or chitohexose, demonstrating that chbC is also required for the utilization of GlcNAc oligomers longer than chitobiose. Complementation of the chbC mutant by introduction of the wild-type chbC gene on a shuttle vector (Fig. 5B) restores the wild-type phenotype. Together, these results demonstrate that chitobiose transport is necessary for the utilization of chitobiose and longer GlcNAc oligomers, and suggest that an unidentified enzyme(s) involved in the degradation of chitin is secreted, either extracellularly or into the periplasm. In addition, these results show that chitobiose transport is necessary for utilization of sequestered GlcNAc in the second exponential phase, and support our hypothesis
that GlcNAc oligomers are not the source of sequestered LY2606368 clinical trial GlcNAc in the second exponential phase. Previous work conducted in our laboratory suggested that RpoS, one of two alternative sigma factors present in B. burgdorferi, regulates chitobiose utilization in the B31-A background
by partially regulating expression of chbC during GlcNAc starvation . Here we cultured an rpoS mutant in BSK-II lacking GlcNAc and supplemented with chitobiose or chitohexose and 7% unboiled (Fig. 6A) or boiled (Fig. 6B) rabbit serum. Biphasic growth of the rpoS mutant in the presence of chitobiose was nearly identical in unboiled and boiled rabbit serum. This is important because it further demonstrates that unboiled serum does not possess a β-N-acetylglucosaminidase activity that cleaves chitobiose to monomeric GlcNAc. In contrast, growth of the rpoS mutant supplemented with chitohexose was delayed in boiled serum compared Tacrolimus (FK506) to that in unboiled rabbit serum. This delay supports the data presented in Table 1 showing an inherent chitinase activity in unboiled rabbit serum as rpoS mutant growth on chitohexose in unboiled serum (Fig. 6A) mirrors that on chitobiose, suggesting the chitinase activity in the rabbit serum degraded the chitohexose to chitobiose. In addition, the delay in chitohexose utilization in boiled serum strongly suggests that RpoS regulates chitin utilization not only through the regulation of chbC , but also through the regulation of other gene(s) important for degradation of chitin.