TI - Discussion . AB - Although the ArcAB system has been extensively investigated for its role as the global control system of E.coli in anaerobic growth , its role , if any , in aerobic growth is much less understood . We have previously reported that ArcA is necessary for the pathogenic bacterium Salmonella enterica to resist reactive oxygen and nitrogen species under aerobic conditions [38] . In this report , we used E.coli as our model to further explore the role of both ArcA and ArcB in ROS resistance , and to investigate the mechanism of ROS resistance mediated by the ArcAB two-component system . Here we demonstrate that deletion mutants of ArcA and ArcB were more susceptible to H2O2 , suggesting that both ArcA and ArcB were necessary for E.coli to resist the stress caused by H2O2 ( Figure 1 ) , and that their functions were not limited to anaerobic growth of bacteria . Interestingly , we have not detected any growth defects of DeltaarcA or DeltaarcB mutant E.coli under anaerobic conditions ( data not shown ) and to our knowledge no such defect has been reported in the literature . In addition , an DeltaarcA mutant of Salmonella enterica grew normally in anaerobic medium [38] . This further indicates that ArcAB has wider roles in the physiology and metabolism of enteric bacteria besides its well-characterized regulation of anaerobic growth of bacteria . The signaling pathway of the ArcAB system under anaerobic conditions has been extensively characterized [25-28,30-34,42,44] . The membrane-bound sensor kinase ArcB is activated by reduced quinones under anaerobic conditions , and subsequently activates its cognate transcriptional regulator ArcA by PHOSphorylating ArcA at Asp54 [30,42,25] . Matsushika and Mizuno previously reported that ArcB can also PHOSphorylate ArcA directly through His292 under aerobic conditions [45] , however , its physiological relevance to E.coli has not been reported . Our results on the role of ArcAB in ROS resistance suggest that ArcAB can be activated by novel signals other than reduced quinones and anaerobic conditions , and the activation is independent of phosphorylaTION at Asp54 of ArcA as demonstrated under anaerobic conditions [41,42,46] , since PHOSphorylation-defective ArcA expressed from a plasmid fully complemented an DeltaarcA mutant E.coli for its susceptibility to H2O2 ( Figure 3 ) . We would like to point out that our analysis was conducted using a PHOSphorylation-mutant ArcA ( Asp54 - Ala ) expressed from a plasmid . It is yet to be determined if a mutant carrying a corresponding mutation of arcA in the chromosome is susceptible to H2O2. ( Our attempts to generate a mutant arcA encoding an Asp54 - Ala mutation in the chromosome were unsuccessful due to technical difficulties Similar to what we observed for arcB , plasmids carrying arcA were prone to mutations during cloning ) We have also noticed that the wild type ArcA expressed from a plasmid confers a stronger H2O2 resistance phenotype than the PHOSphorylation-defective ArcA . The DeltaarcA mutant E.coli complemented in trans with a wild type arcA allele demonstrated higher H2O2 resistance than the wild type E.coli ( Figure 1 and 3 ) , while the same mutant E.coli complemented with a PHOSphorylation-defective arcA allele has the same H2O2 resistance as the wild type E.coli ( Figure 3 ) . In addition to novel signals and signaling pathways that may mediate the function of the ArcAB system in the ROS resistance , the ArcAB system may also regulate a distinct set of genes under aerobic conditions . Under anaerobic conditions ArcA mostly negatively regulates genes involved in the TCA cycle and electron transport [26-28] . Under aerobic conditions , a microarray study by Oshima et al . demonstrated that expression of a large number of genes in the DeltaarcA or DeltaarcB mutant E.coli was altered [23] . Our results suggest that levels of atleast three proteins ( flagellin , GltI and OppA ) were altered in the DeltaarcA mutant E.coli both constitutively and in response to H2O2 treatment ( Figure 4 and Table 2 ) . Our further analysis on the messenger RNA level of fliC indicates that the RNA levels are higher in the DeltaarcA mutant E.coli and corresponded to the protein levels , suggesting that the regulation is likely on the transcriptional or post-transcriptional level ( Figure 5 ) . Oshima et al . did not detect a significant alteration in the expression of fliC in their microarray analysis , although flagellar synthesis was identified as a system that was affected in the DeltaarcA mutant but not the DeltaarcB mutant E.coli [23] . The discrepancy is possibly due to the differences in experimental conditions ( shaking bacterial cultures at 120 rpm versus 225 rpm ) and detection methods ( microarray versus Real-Time Reverse Transcriptase PCR and 2-D gel electrophoresis ) . Since we detected an elevation of both mRNA and protein levels of flagellin in the DeltaarcA mutant E.coli ( Figures 4 and 5 ) , we believe that our observation is valid . The regulation of ArcA on flagellin is likely to be indirect , as we did not detect specific binding of recombinant ArcA protein to the upstream sequence of fliC ( data not shown ) . Given that the ArcAB system regulates a large number of genes in E.coli , its role in the ROS resistance is likely to be complex . We have demonstrated that mutation of ArcA or ArcB did not alter the H2O2 scavenging ability of E.coli ( Figure 2 ) , however , the precise molecular mechanism on how ArcA regulates ROS resistance in E.coli is yet to be elucidated . ArcA was reported to be necessary for the ROS resistance of Haemophilus influenzae due to its regulation of Dps , a ferritin-like small protein that was previously reported to be involved in ROS resistance of Salmonella [39,47] . The mechanism of the ROS resistance mediated by ArcA is likely to be different in E.coli , since dps is expressed close to the wild type level in the DeltaarcA or DeltaarcB mutant ( 84% and 99% respectively ) , and our preliminary microarray analysis with Salmonella DeltaarcA mutant indicated that dps responded normally to H2O2 in the DeltaarcA mutant ( unpublished results ) . One possible clue on the mechanism of how ArcAB contributes to the ROS resistance of E.coli came from our proteomic analysis that showed altered expression of flagellin , GltI and OppA between the wild type and DeltaarcA mutant E.coli ( Table 2 ) . The constitutive GltI and OppA levels are higher in the DeltaarcA mutant than in the wild type E.coli , suggesting that the mutant may have a higher need for amino acid transport . In contrast to the GltI and OppA levels in the wild type E.coli that increased 6 - and 24-fold respectively in response to H2O2 exposure ( possibly due to a higher need for amino acid transport under ROS stress ) , the level of neither protein in the DeltaarcA mutant increased under the same condition ( Table 2 ) . A higher level of flagellin in the DeltaarcA mutant likely put further constraint on the protein synthesis , and as a result the DeltaarcA mutant E.coli might have become less fit under H2O2 stress . Our genetic study demonstrating that deletion of fliC "rescued" the survival defect of the DeltaarcA mutant E.coli under H2O2 stress ( Figure 6 ) supports the hypothesis . ROS stress conditions induce growth arrest in E.coli . Chang et al . has reported that in growth arrest induced by either glucose-lactose diauxie , entry into stationary phase or H2O2 treatment , genes involved in amino acid biosynthesis pathways are down-regulated except those of histidine and arginine biosynthesis [24] . Recently , Jang and Imlay have shown that H2O2 damages enzymes with iron-sulfur and impairs bacterial metabolism , especially the biosynthesis of leucine [48] . This down regulation of amino acid synthesis may cause a strain on the protein synthesis of bacteria . Our results indicate that protein synthesis is important for E.coli to survive H2O2 treatment . Chloramphenicol , an antibiotic inhibiting protein synthesis , reduced the survival of both the wild type and DeltaarcA mutant E.coli after H2O2 treatment , while ampicillin did not ( Figure 8 ) . Consistently , amino acid supplementation enhanced the survival of E.coli after H2O2 treatment ( Figure 7 ) . This is in agreement with the report by Calioz and Touati that amino acid supplementation facilitates the survival of superoxide dismutase-deficient E.coli under aerobic conditions [49] . Although our results and results from other investigators suggest that protein synthesis and amino acid availability are important for E.coli to survive ROS stress and the global regulatory system ArcAB plays a role this aspect of ROS stress resistance , protein synthesis and amino acid availability may be only one aspect of the pleiotropic effect of ArcAB system on E.coli , since chloramphenicol-treated DeltaarcA mutant was still more susceptible than the similarly treated wild type E.coli . Further studies are necessary to elucidate more molecular mechanisms that control the ROS resistance mediated by the ArcAB global regulatory system .