Growth of an rpoS mutant on chitin Previous work in our laboratory demonstrated that the alternative sigma factor RpoS partially regulates #Selleck BVD-523 randurls[1|1|,|CHEM1|]# chitobiose utilization, by regulating the expression of chbC during GlcNAc starvation [17]. Since chbC is necessary for chitin utilization, we hypothesized that RpoS may also be involved in the regulation of other genes in this pathway. To test this, we cultured an rpoS mutant (A74) in BSK-II without free GlcNAc, supplemented with 75 μM chitobiose or 25 μM chitohexose and containing either 7% unboiled (Fig. 6A) or boiled (Fig. 6B) rabbit serum. As in our previous report [17], culturing

the rpoS mutant with chitobiose in the absence of free GlcNAc resulted in biphasic growth. This was observed in the presence of both unboiled (Fig. 6A) and boiled (Fig. 6B) rabbit serum with the second exponential phase starting at 142 hours in either

medium. Comparison of chitohexose utilization by the rpoS mutant in unboiled (Fig. 6A) or boiled (Fig. 6B) serum revealed biphasic growth under both conditions, but with a delay in the initiation of the second Staurosporine order exponential growth phase only in a medium supplemented with boiled serum. The delay in second exponential phase growth ranged from 72 to 120 h in the three replicate experiments conducted. These data suggest a role for RpoS in the regulation of chitin utilization separate from its role in regulating chbC expression. Figure 6 RpoS regulates Urease chitobiose and chitin utilization. Growth of A74 (rpoS mutant) in BSK-II without GlcNAc and supplemented with 7% unboiled (A) or boiled serum (B). Late-log phase cells were diluted to 1.0 × 105 cells ml-1 and cultures were supplemented with the following substrates: 1.5 mM GlcNAc (closed circle), No addition (open circle), 75 μM chitobiose (closed triangle) or 25 μM chitohexose (open triangle). Cells were enumerated daily by darkfield microscopy. This is a representative experiment that was repeated three times.

Discussion Chitin is one of the most abundant polymers in the environment [32] and is a major structural component of arthropods, including Ixodid ticks, the vector hosts for B. burgdorferi. B. burgdorferi must obtain GlcNAc from its tick and vertebrate hosts and does so by transporting either free GlcNAc or chitobiose into the cell [14–17]. Recently, Tilly et al [14, 15] reported that B. burgdorferi cells exhibit biphasic growth in the absence of free GlcNAc in vitro. It was proposed that the second growth phase observed during GlcNAc starvation was due to the up regulation of chbC and the utilization of chito-oligomers present in the yeastolate component of BSK-II [14]. While we were able to confirm that the induction of chbC expression during GlcNAc starvation is responsible for chitobiose utilization, our observations suggested that yeastolate is not the source of sequestered GlcNAc for second exponential phase growth [17].