1). Growth of B31-A and A74 were similar in complete medium, although the wild-type strain reached a slightly higher cell density of 8.6 × 107 cells ml-1 compared to 3.2 × 107 cells ml-1 for the rpoS mutant. When cells were cultured in the absence of free GlcNAc there was a considerable difference in the ability of the two strains to initiate a second exponential phase. Initially, both strains grew from a starting cell density of 1.0 × 105 cells selleck ml-1 to ~2.5 × 106 cells ml-1 by 72 h before entering a death phase characterized by a loss of motility and the formation
of blebs near the cell midpoint (Fig. 2B and 2D). As expected, the wild-type strain exhibited biphasic growth, initiating a second exponential phase by 200 h and reaching a peak cell density of 3.65 × 107 cells ml-1 by 290 h. During the second exponential phase cells exhibited normal morphology characteristic of cells cultured in the presence of GlcNAc (Fig. 2A and
2C). In contrast, the rpoS mutant strain did not Z-IETD-FMK supplier initiate a second exponential phase by 381 h. Figure 1 Mutation of rpoS delays biphasic growth during GlcNAc starvation. Growth of B. burgdorferi strains B31-A (WT), A74 (rpoS mutant) and WC12 (rpoS complemented mutant) in BSK-II with GlcNAc (closed circle, B31-A; closed triangle, A74; closed square, WC12) and without GlcNAc (open circle B31-A; open unless triangle, A74; open square, WC12). Late-log phase cells from each strain 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. Figure 2 Morphology of B. burgdorferi during GlcNAc
starvation. Phase contrast microscopy of B. burgdorferi strain B31-A at 400× (A and B) and 1000× (C and D). Spirochetes were cultured for 72 h in BSK-II with GlcNAc (A and C) and without GlcNAc (B and D). Similar growth experiments were AZD1480 conducted with the rpoS complemented mutant, WC12, in an attempt to recover the second exponential phase in A74 (Fig. 1). In complete BSK-II, WC12 showed a growth rate similar to the wild-type and rpoS mutant strains, and reached a peak cell density of 8.2 × 107 cells ml-1. When cultured in the absence of free GlcNAc, WC12 exhibited a growth pattern similar to the wild-type B31-A strain. The cells grew to 1.5 × 106 cells ml-1 by 72 h before entering the characteristic death phase, and then initiated a second exponential phase by 200 h. Taken together, these results suggest that RpoS plays a role in the initiation of the second exponential phase when cells are cultured in the absence of free GlcNAc, possibly due to the regulation of genes important to the process.