BMC Genomics 2009, 10:165 http://www.biomedcentral.com/1471-2164/10/165 observed during the exponential growth phase (data not shown), suggesting that GcvB exerts constant AR regula- tory functions throughout the various growth phases. We also individually deleted gcvA and ygdI which are the two genes immediately adjacent to gcvB. Both mutants dis- played comparable AR as the wild type (Fig. 1A). This shows that the observed acid vulnerability in the gcvB mutant is not due to the disruption of the activities of its adjacent genes. Next, we cloned the gene encoding GcvB (together with its native promoter) into plasmid pET32a (+) and overex- pressed the sncRNA in the E. coli MG1655 gcvB mutant. The gcvB mutant carrying an empty vector served as a negative control. We found that overproducing GcvB restored the ability of the gcvB mutant to survive at low pH (Fig 1C). These observations together with the knockout data revealed that GcvB acts as a positive regulator of AR. The known GcvB target OppA plays a minor, if any, role in GcvB-mediated AR GcvB has been demonstrated to repress the translation of oppA and dppA in E. coli by masking the ribosome binding sites of their mRNAs, which encode periplasmic substrate- binding proteins of ABC uptake systems for amino acids and peptides [13-15]. To determine whether these two proteins played a role in GcvB-mediated AR, we individu- ally deleted their encoding genes, and examined the abil- ity of the resultant mutants to survive at low pH. We found that deleting oppA slightly increased AR, as revealed by their acid recovery (OD600) and percent acid survival (cfu) levels, while the absence of dppA had little effect on AR (Fig. 2A, B). Thus DppA is not involved in GcvB-medi- ated AR, and OppA plays a minor, if any, role in this proc- ess. We reasoned that the GcvB sncRNA may be exerting its effects on AR via a previously unidentified molecular target. GcvB mediates AR by upregulating RpoS expression RpoS is the central regulator of glutamate-dependent AR2 genes such as gadA, gadBC [1], gadX [16], gadE [17], and gadY [6,7]. It is also required for the AR1 system [1,18]. We therefore set out to determine whether GcvB regulated AR by modulating RpoS expression levels. We found that deleting gcvB did not lead to a further reduction in acid survival levels in an rpoS mutant background (Fig. 3A). More importantly, the absence of rpoS completely abol- ished AR elicited by the plasmid-encoded GcvB (Fig. 3B), suggesting that GcvB-mediated AR is dependent on RpoS. Next, an rpoS-lacZ translational fusion was constructed to examine whether the presence or absence of the GcvB sncRNA significantly affected rpoS expression levels. Deleting gcvB reduced the expression levels of the rpoS- lacZ fusion by 50% (Fig. 4A), whereas gcvB overexpression increased it 3-fold (Fig. 4B). Furthermore, deleting oppA, aEFnfifgceeuctrisneoE2fscinhdeirvicidhuiaalcloylideleting oppA and dppA on acid resist- Effects of individually deleting oppA and dppA on acid resistance in Escherichia coli. The acid-stress survival abil- ities of two individual deletion mutant strains lacking previ- ously identified targets of GcvB were compared with those of the MG1655 wild type (wt) strain. The abilities of strains to survive a 30 minute acid shock were quantified by two methods: (A) acid recovery measured as optical density at 600 nm (OD600). (B) Percent survival determined by colony forming units (cfu). Error bars represent standard deviation (* P-value < 0.05). whose absence had previously been shown to slightly increase AR levels, had no effect on rpoS-lacZ expression (Fig. 4C). This indicates that the GcvB-mediated regula- tion of RpoS expression does not involve OppA. We then investigated whether GcvB indirectly controls RpoS production via an upstream regulator. In the litera- ture, there are numerous reports outlining mechanisms by which the synthesis of RpoS is regulated at the transcrip- tional [19-22], translational and posttranslational levels [23-25]. Of these regulators of RpoS expression, Crp [20], H-NS [23], GadX [22], and a hybrid sensor kinase called BarA [21] have previously been associated with AR. Although BarA acts as a positive regulator of rpoS tran- scription, its effect is restricted to the exponential phase. It Page 3 of 7 (page number not for citation purposes)