In contrast, expression of chbC in the rpoS mutant did not change from baseline levels for the first 340 h. However, expression did increase by 6-fold at 381 h, which may correspond to this PF-01367338 solubility dmso strain beginning to enter a second exponential phase after 400 h (Fig. 4B). When expression of chbC was evaluated in the rpoS complemented mutant (WC12), levels increased as cells entered the second exponential phase similar to that observed in the wild type. A 27-fold increase was observed at 216 h as cells started to grow in the second exponential phase, and expression peaked with a 40-fold increase at 239 h before declining as cells entered stationary phase. Statistical analysis was performed to determine

the significance of chbC expression between B31-A and A74 and between WC12 and A74, and fold differences were determined to be statistically significant between 216 and 340 h (p < 0.001). Figure 4 Mutation of rpoS delays ARS-1620 chitobiose utilization. Growth of B. burgdorferi strains (A) B31-A (WT), (B) A74 (rpoS mutant) and (C) WC12 (rpoS

complemented mutant) in BSK-II lacking GlcNAc (open circle) and supplemented with 1.5 mM GlcNAc (closed circle), or high (closed triangle, 75 μM or 150 μM) or low (open triangle, 5 μM or 15 μM) concentrations of chitobiose. Late-log click here phase cells were diluted to 1.0 × 105 cells ml-1 in the appropriate medium, incubated at 33°C, and enumerated daily as described in the Methods. This is a representative experiment that was repeated three times. Effect of RpoS on chitobiose utilization In order to evaluate the importance of RpoS in chitobiose utilization, we compared growth of B31-A, A74 and WC12 in BSK-II lacking GlcNAc and supplemented with low (5 μM or 15 μM) or high (75 μM or 150 μM) concentrations of chitobiose (Fig. 4A–C). As expected from the previous study

[10], B31-A exhibited a single exponential phase when cultured with a high concentration (150 μM) of chitobiose, reaching a peak cell density of 7.8 × 107 cells ml-1 by 166 h (Fig. 4A). In contrast, when B31-A was cultured with ten-fold less chitobiose (15 μM) biphasic growth was observed. Biphasic growth in the presence of 15 μM chitobiose differed from that observed in cells cultured without both free P-type ATPase GlcNAc and chitobiose, as cells in the first exponential phase grew to a density that was 6.3-fold higher in the presence of low levels of chitobiose (1.6 × 107 cells ml-1) compared to no added chitobiose or GlcNAc (2.5 × 106 cells ml-1). To determine if RpoS is required for chitobiose utilization, we cultured A74 in BSK-II without GlcNAc and supplemented with low (15 μM) or high (150 μM) concentrations of chitobiose (Fig. 4B). In contrast to the wild type, the rpoS mutant was initially unable to utilize chitobiose at either concentration, as cells only grew to 2.0 × 106 cells ml-1 before blebbing and entering a death phase.