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 targets 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.
Upon rpoS deletion, GcvB had no influence on AR. Furthermore, 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 singularly 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-inhibitory stem loop structure that probably occludes the Shine-Dalgarno ribosome binding site and prevents translation [24
]. DsrA [24
] and RprA [28
] 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
]. 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 by an unknown mechanism. Computational sequence analyses using the RNAhybrid server [31
] have indicated that GcvB does not contain any extensive regions of sequence complementary to the 5' leader of rpoS
mRNA (data not shown). As such, the mechanism by which GcvB regulates rpoS
expression remains a subject of further investigation.