Alternative pathways for Escherichia coli biofilm formation revealed by sRNA overproduction

Summary 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 yeast extract casamino acids (YESCA) media, biofilm was dependent on 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. Depending on the growth medium, Escherichia coli make biofilms that use different genetic pathways and show responses to different regulatory RNAs. These regulatory RNAs act post‐transcriptionally to modify major regulatory pathways, with DsrA inhibiting translation of the transcriptional regulatory H‐NS and GadY inhibiting the activity of the translational regulator CsrA. And MicF stimulates the pathway dependent on σS (RpoS) and CsgD. The biofilm pathways depicted appear to be mutually exclusive.