Our results indicate that after exposure to both toxic compounds, arcB transcript levels remain unchanged while those of
ompD and ompC are lowered as compared to untreated cells (Figure 3). Therefore, all the evidence indicates that OM permeability is tightly regulated in response to ROS and could represent a novel mechanism of resistance when bacteria are exposed to these toxic compounds. Figure 3 Effect of H 2 O 2 and HOCl on ompW expression. Wild type (14028s) exponentially growing cells were treated with H2O2 (1.5 mM) or NaOCl (530 μM) for 20 min and ompW, ompD, ompC and arcB mRNA levels were measured by qRT-PCR. Control cells received no treatment. 16S rRNA levels were used for normalization. learn more Values represent the average of four independent experiments ± SD. ArcA binds the ompW promoter region In addition to the soxRS and oxyR systems, several studies have provided evidence that the ArcAB JMJD inhibitor two component system plays an important role in the resistance to ROS induced damage. For example, ArcA is essential for S. Enteritidis and Typhimurium resistance to ROS [24, 27] and E. coli mutant strains of the sensor ArcB and the regulator ArcA, show an increased susceptibility to H2O2. However, neither of these studies identified genes directly regulated by the system under oxidative stress. We recently
demonstrated that ArcA negatively regulates the expression of S. Typhimurium ompD after H2O2 exposure selleck screening library by direct interaction with its promoter region . To determine if ArcA mediates ompW down-regulation in response to H2O2 and HOCl, a search for putative ArcA binding sites at the ompW promoter region was performed using Virtual Footprint many 3.0 . The analysis
predicted the presence of three ArcA binding sites (ABS) located at positions −61 to −70 (ABS-1, forward orientation), -230 to −239 (ABS-2) and −286 to −295 (ABS-3, both in reverse orientation) relative to the experimentally determined transcription start site . Comparison with the extended core region 5′-GTTAATTAAATGTTA-3′ described by Evans et al. (2011) further revealed that only ABS-1 presented a high degree of identity (14 out of 15 nucleotides) with the consensus sequence. To confirm or rule out a direct interaction between ArcA and the predicted binding sites, deletions of the promoter region were generated by PCR (schematized in Figure 4B) and used to perform non-radioactive EMSAs with ArcA and phosphorylated ArcA (ArcA-P). The purity of the protein was assessed by PAGE and ArcA was the dominant product. Electrophoretic mobility shift with ArcA-P was only observed when incubated with fragments that included ABS-1 (Figure 4C and D, W1 and W4). No shifts were observed in fragments that include both ABS-2 and ABS-3 (W3, even at three-fold excess) or control fragments that did not include any ABS (W2 and W5).