RcsB and H-NS Both Contribute to the Repression the Expression of the csgDEFG Operon

Curli fimbriae are a major component of biofilm formation in <i>Escherichia coli</i>, and their expression is regulated by numerous transcription factors and small regulatory RNAs (sRNAs). The RcsD-RcsC-RcsB phosphorelay system, which is involved in the envelope stress response, plays a role in this regulation. In this study, we report that DNase-I footprinting analysis revealed that the response regulator RcsB interacts with the -31 to +53 region of the promoter region of <i>csgD</i>, which encodes a major regulator of biofilm formation, and thus contributes to its transcriptional repression. Additionally, overexpression of RcsB or RcsB D56A that could not be phosphorylated by the histidine kinases RcsC and D both significantly reduced <i>csgD</i> expression and suppressed Curli formation. This indicates that the phosphorylation of RcsB has an insignificant impact on its affinity for its operator sites. Furthermore, we confirm that RcsB binds cooperatively to the <i>csgD</i> promoter region in the presence of the nucleoid-associated protein H-NS. Our study also confirms that RcsB positively regulates the expression of an sRNA, RprA, which is known to reduce mRNA <i>csgD</i> mRNA translation RprA via its binding to the 5'-untranslated region (UTR) of <i>csgD</i>. These findings indicate that, in <i>E. coli</i>, the RcsBCD system suppresses <i>csgD</i> expression through both direct transcriptional repression by the regulator RcsB and translational repression by the sRNA RprA.