At 170 h the complemented
mutant entered a second exponential phase, which peaked at a cell density of 1.5 × 107 cells ml-1. These results lend further support to the hypothesis that RpoS plays a role in the utilization of chitobiose. Effect of RpoN on chitobiose utilization Several reports have demonstrated Selleckchem Proteasome inhibitor that under certain conditions rpoS expression is regulated directly by RpoN [19, 20]. To determine if RpoN plays a role in chitobiose utilization, we generated an rpoN mutant in the B31-A background (RR22) and evaluated its growth in BSK-II lacking GlcNAc and supplemented with a high concentration of chitobiose (Fig. 5). In the complete medium, RR22 exhibited growth similar to the wild type, reaching a peak cell density of 7.7 × 107 cells ml-1 by 172 hours. In BSK-II lacking GlcNAc RR22 exhibited biphasic growth similar to the wild type, as initiation of the second exponential phase occurred at 235 hours. When cultured in a medium lacking GlcNAc and supplemented with 75 μM chitobiose RR22 exhibited only one exponential phase, and reached a peak cell density of 8.6 × 107 cells ml-1 by 172 h. These results suggest RpoN is not necessary for chitobiose
utilization. It is important to note that growth curves of the rpoN mutant were conducted in parallel with the wild type, rpoS mutant and rpoS complemented mutant growth experiments (Fig. 4). Figure 5 RpoN is not required for chitobiose utilization.
Growth of B. burgdorferi strain RR22 selleck chemical in BSK-II lacking GlcNAc and supplemented with 75 μM chitobiose. Late-log phase cells were diluted to 1.0 × 105 cells ml-1 in the appropriate medium (closed circle, 1.5 mM GlcNAc; open circle, No addition, i.e. without GlcNAc; closed triangle, 75 μM chitobiose), incubated at 33°C and enumerated daily as described in the Methods. This Adenosine triphosphate is a representative experiment that was repeated three times. Identification of the chbC transcriptional start site and promoter analysis The results above demonstrate that RpoS regulates the expression of chbC, at least partially, and is important in chitobiose utilization in vitro. To determine if the chbC gene has a promoter similar to other RpoS-dependent genes, we performed 5′ RACE to identify the transcriptional start site of chbC and compared the promoter region with previously described RpoD, RpoS and RpoN-dependent promoter sequences in B. burgdorferi. Total RNA was extracted from B31-A and used to generate chbC-specific cDNA in a reverse transcription reaction. The cDNA was purified and a homopolymeric dA-tail was added. mTOR inhibitor Subsequent PCR with the oligo dT-anchor primer and a nested chbC-specific primer (BBB04 5′ RACE R2) resulted in an approximate 410 bp product (Fig. 6A; lane 2). The PCR product was sequenced, and the transcriptional start site was determined to be between 42 and 44 base pairs upstream of the translational start site (Fig. 6B).