Small regulatory RNAs have major roles in many regulatory circuits in Escherichia coli and other bacteria, including the transition from planktonic to biofilm growth. We tested Hfq-dependent sRNAs in E. coli for their ability, when overproduced, to inhibit or stimulate biofilm formation, in two different growth media. We identify two mutually exclusive pathways for biofilm formation. In LB, PgaA, encoding an adhesion export protein, played a critical role; biofilm was independent of the general stress factor RpoS or CsgD, regulator of curli and other biofilm genes. The PgaA-dependent pathway was stimulated upon overproduction of DsrA, via negative regulation of H-NS, or of GadY, likely by titration of CsrA. In YESCA (Yeast Extract Casamino acids) media, biofilm was dependent upon RpoS and CsgD, but independent of PgaA; RpoS appears to indirectly negatively regulate the PgaA-dependent pathway in YESCA medium. Deletions of most sRNAs had very little effect on biofilm, although deletion of hfq, encoding an RNA chaperone, was defective in both LB and YESCA. Deletion of ArcZ, a small RNA activator of RpoS, decreased biofilm in YESCA; only a portion of this defect could be bypassed by overproduction of RpoS. Overall, sRNAs highlight different pathways to biofilm formation.

Bacteria use multidrug efflux pumps to export drugs and toxic compounds out of the cell. One of the most important efflux pumps in Escherichia coli is the AcrAB-TolC system. Small regulatory RNAs (sRNAs) are known to be major posttranscriptional regulators that can enhance or repress translation by binding to the 5= untranslated region (UTR) of mRNA targets with the help of a chaperone protein, Hfq. In this study, we investigated the expression of acrA, acrB, and tolC translational fusions using 27 Hfq-dependent sRNAs overexpressed from plasmids. No significant sRNA regulation of acrA or acrB was detected. SdsR (also known as RyeB), an abundant and well-conserved stationary-phase sRNA, was found to repress the expression of tolC, the gene encoding the outer membrane protein of many multidrug resistance efflux pumps. This repression was shown to be by direct base pairing occurring upstream from the ribosomal binding site. SdsR overexpression and its regulation of tolC were found to reduce resistance to novobiocin and crystal violet. Our results suggest that additional targets for SdsR exist that contribute to increased antibiotic sensitivity and reduced biofilm formation. In an effort to identify phenotypes associated with single-copy SdsR and its regulation of tolC, the effect of a deletion of sdsR or mutations in tolC that should block SdsR pairing were investigated using a Biolog phenotypic microarray. However, no significant phenotypes were identified. Therefore, SdsR appears to modulate rather than act as a major regulator of its targets. IMPORTANCEAcrAB-TolC is a major efflux pump present in E. coli and Gram-negative bacteria used to export toxic compounds; the pump confers resistance to many antibiotics of unrelated classes. In this study, we found that SdsR, a small RNA expressed in stationary phase, repressed the expression of tolC, resulting in increased sensitivity to some antibiotics. This extends the findings of previous studies showing that sRNAs contribute to the regulation of many outer membrane proteins; manipulating or enhancing their action might help in sensitizing bacteria to antibiotics.

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