The blue light using FAD (BLUF)-EAL protein YcgF is a known blue-light sensor of Escherichia coli, but its direct regulatory output and physiological function have remained unknown. Here, we demonstrate that unlike other EAL domain proteins, YcgF does not degrade the signaling molecule c-di-GMP, but directly binds to and releases the MerR-like repressor YcgE from its operator DNA upon blue-light irradiation. As a consequence, a distinct regulon of eight small proteins (of 71-126 amino acids) is strongly induced. These include YmgA and YmgB, which, via the RcsC/RcsD/RcsB two-component phosphorelay system, activate production of the biofilm matrix substance colanic acid as well as acid resistance genes and the biofilm-associated bdm gene and down-regulate adhesive curli fimbriae. Thus, small proteins under YcgF/YcgE control seem to act as ''connectors'' that provide additional signal input into a two-component signaling pathway. Moreover, we found ycgF and ycgE expression to be strongly activated at low temperature, and we elucidate how blue light, cold, and starvation signals are integrated in the expression and activity of the YcgF/YcgE/small protein signaling pathway. In conclusion, this pathway may modulate biofilm formation via the two-component network when E. coli has to survive in an extrahost aquatic environment.[Keywords: Biofilm; connectors; cyclic-di-GMP; GGDEF; RcsB; RpoS] Supplemental material is available at http://www.genesdev.org.

RprA is a small regulatory RNA known to weakly affect the translation of σS (RpoS) in Escherichia coli. Here we demonstrate that csgD, which encodes a stationary phase-induced biofilm regulator, as well as ydaM, which encodes a diguanylate cyclase involved in activating csgD transcription, are novel negatively controlled RprA targets. As shown by extensive mutational analysis, direct binding of RprA to the 5′-untranslated and translational initiation regions of csgD mRNA inhibits translation and reduces csgD mRNA levels. In the case of ydaM mRNA, RprA base-pairs directly downstream of the translational start codon. In a feedforward loop, RprA can thus downregulate > 30 YdaM/CsgD-activated genes including those for adhesive curli fimbriae. However, during early stationary phase, when csgD transcription is strongly activated, the synthesis of csgD mRNA exceeds that of RprA, which allows the accumulation of CsgD protein. This situation is reversed when csgD transcription is shut off – for instance, later in stationary phase or during biofilm formation – or by conditions that further activate RprA expression via the Rcs two-component system. Thus, antagonistic regulation of csgD and RprA at the mRNA level integrates cell envelope stress signals with global gene expression during stationary phase and biofilm formation.