ABSTRACTThe plague bacterium,Yersinia pestis, forms a biofilm-mediated blockage in the flea foregut that enhances its transmission by fleabite. Biofilm formation is positively controlled by cyclic di-GMP (c-di-GMP), which is synthesized by the diguanylate cyclases (DGC), HmsD and HmsT. While HmsD primarily promotes biofilm-mediated blockage of fleas, HmsT plays a more minor role in this process. HmsD is a component of the HmsCDE tripartite signaling system. HmsC and HmsE posttranslationally inhibit or activate HmsD, respectively. HmsT-dependent c-di-GMP levels and biofilm formation are positively regulated by the RNA-binding protein CsrA. In this study we determined whether CsrA positively regulates HmsD-dependent biofilm formation through interactions with thehmsEmRNA. Gel mobility shift assays determined that CsrA binds specifically to thehmsEtranscript. RNase T1 footprint assays identified a single CsrA binding site and CsrA-induced structural changes in thehmsEleader region. Translational activation of thehmsEmRNA was confirmedin vivousing plasmid-encoded inducible translational fusion reporters and by HmsE protein expression studies. Furthermore, mutation of the CsrA binding site in thehmsEtranscript significantly reduced HmsD-dependent biofilm formation. These results suggest that CsrA binding leads to structural changes in thehmsEmRNA that enhance its translation to enable increased HmsD-dependent biofilm formation. Given the requisite function of HmsD in biofilm-mediated flea blockage, this CsrA-dependent increase in HmsD activity underscores that complex and conditionally defined modulation of c-di-GMP synthesis within the flea gut is required forY. pestistransmission.IMPORTANCEMutations enhancing c-di-GMP biosynthesis drove the evolution ofY. pestisto flea-borne transmissibility. c-di-GMP-dependent biofilm-mediated blockage of the flea foregut enables regurgitative transmission ofY. pestisby fleabite. TheY. pestisdiguanylate cyclases (DGC), HmsT and HmsD, which synthesize c-di-GMP, play significant roles in transmission. Several regulatory proteins involved in environmental sensing, as well as signal transduction and response regulation, tightly control DGC function. An example is CsrA, a global posttranscriptional regulator that modulates carbon metabolism and biofilm formation. CsrA integrates alternative carbon usage metabolism cues to activate c-di-GMP biosynthesis through HmsT. Here, we demonstrated that CsrA additionally activateshmsEtranslation to promote c-di-GMP biosynthesis through HmsD. This emphasizes that a highly evolved regulatory network controls c-di-GMP synthesis andY. pestistransmission.