BMC Genomics 2009, 10:165 http://www.biomedcentral.com/1471-2164/10/165 FhEnfifgse,ucctrrsepoo5frdgealedtXing gcvB on acid resistance in mutants lacking Effects of deleting gcvB on acid resistance in mutants lacking hns, crp or gadX. Cell cultures of E. coli MG1655 mutant strains containing single deletions of the hns, crp or gadX genes were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (gadX mutant) or 30 min (hns and crp mutants). After neutralization by adding alkalinized LB (pH 9.3), cells were cultured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent standard deviation (* P-value < 0.05). tional level, we deleted the region spanning the rpoSp, nlpDp1 and 2 promoter sites (Fig. 4). As removing these promoters will eliminate rpoS expression, a constitutive promoter encoded in the knockout cassette (described in Materials and Methods) was used to drive rpoS transcrip- tion in the absence of rpoSp, nlpDp1 and 2. The resulting mutant was referred to as d567–1342. As shown in Fig. 6, replacing the native promoters of rpoS with this heterolo- gous promoter had little effect on GcvB-induced AR. These results indicate that GcvB does not regulate rpoS expression at the transcriptional level. Discussion In this study, we constructed a library comprising single- gene knockout mutants of seventy-nine sncRNA genes in E. coli. We used this mutant collection to identify sncRNAs involved in acid resistance (AR) within this bacterium. Consequently, we were able to identify the sncRNA GcvB as a novel regulator of AR. DppA and OppA, which were previously identified as tar- gets of GcvB, were shown not to be responsible for GcvB- mediated AR. While the dppA deletion did not affect AR, the absence of OppA slightly improved the ability of E. coli to survive under extreme acid conditions. Since GcvB- mediated inhibition of oppA and dppA only weakly increased AR, we reasoned that the GcvB sncRNA may be regulating AR via some other mechanistic pathway. lEFaficfgekucintrsgeoh6fnGs, cvrpB, ogavdeXrexoprrneastsiivoenpornomacoidterressiosftarnpcoeS in mutants Effects of GcvB overexpression on acid resistance in mutants lacking hns, crp, gadX or native promoters of rpoS. The mutant lacking the native promoters of rpoS was named d567–1342. Cell cultures of E. coli MG1655 strains con- taining single deletions were grown in LB broth (pH 7.0) at 37°C for 5 hrs and then acid challenged (pH 2.0) for 15 min (gadX mutant) or 30 min (hns and crp mutants and d567–1342). After neutralization by alkalinized LB (pH 9.3), cells were cul- tured for an additional 3 hrs before measurement of optical density at 600 nm (OD600 nm). Error bars represent stand- ard deviation (* P-value < 0.05). Upon rpoS deletion, GcvB had no influence on AR. Fur- thermore, we found that GcvB positively regulated rpoS expression. Deleting oppA had no effect on the levels of RpoS, providing evidence that the GcvB-mediated control of RpoS does not involve the OppA protein. Also, GcvB does not regulate RpoS via H-NS, Crp, or GadX; as GcvB- mediated AR was unaffected when these genes were singu- larly deleted. The region spanning from -567 to -1342 nt harbors all three of the of the rpoS promoters (rpoSp, nlpDp1 and 2). Replacing these native promoters with the constitutive promoter from a Cm knockout cassette had no affect on GcvB-mediated AR. This suggests that GcvB does not regulate rpoS expression at the transcriptional level. Like GcvB, the sncRNAs DsrA and RprA also act as positive regulators of AR in E. coli. DsrA and RprA bind to the same region of the 5' leader of rpoS mRNA. A sequence within this region was previously predicted to form a self-inhibi- tory stem loop structure that probably occludes the Shine- Dalgarno ribosome binding site and prevents translation [24,27]. DsrA [24,27] and RprA [28,29] bind to this stem- loop structure, free the ribosome binding site, and thereby mediate rpoS translation. This region also contains RNase III cleavage sites that result in rapid decay of the rpoS tran- script [30]. The base-pairing of DsrA within this region stabilizes rpoS mRNA by creating an alternative RNase III cleavage site [30]. As for GcvB, it regulates rpoS expression Page 5 of 7 (page number not for citation purposes)