The RNA binding protein CsrA controls cyclic di‐GMP metabolism by directly regulating the expression of GGDEF proteins

Abstract

SummaryThe carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobility shift assays confirmed the direct and specific binding of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH resulted in a more than 20-fold increase in the cellular concentration of the second messenger c-di-GMP, implying that both proteins possess diguanylate cyclase activity. Phenotypic characterization revealed that both proteins are involved in the regulation of motility in a c-di-GMP dependent manner. CsrA was also found to regulate the expression of five additional GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels of c-di-GMP. All together, these data demonstrate a global role for CsrA in the regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins at the post-transcriptional level.

Rapid and robust signaling in the CsrA cascade via RNA–protein interactions and feedback regulation

“…Given that enhanced signaling and overshoot are known to be generated by negative feedback under certain conditions (1) and that native csrD was necessary for these behaviors, our findings are consistent with the reported negativefeedback regulation in which CsrA represses the production of CsrD. In further support of this, we demonstrated that enhanced signaling does not occur with the synthetic csrD gene, which lacks the flanking sequences (including the native csrD promoter and 5′-UTR sequences) that are necessary for CsrA repression of CsrD production (12,25). Instead, we found that the induction of a small, constant amount of CsrD from synthetic csrD resulted in slower signaling than in the control cascade without CsrD induction (Fig.…”

“…RT-PCR that the CsrD mRNA concentration decreases with increased CsrA production (SI Text), which is consistent with negative feedback and previous reports (12,25).…”

“…Specifically, we sought to determine whether putative transcriptional and translational feedback loops described in the native system affect signaling dynamics under our experimental conditions (7,23). Feedback is known to influence signaling dynamics (1) and CsrA has been reported to (i) positively and negatively regulate its own expression via mechanisms that have not been fully elucidated (23), (ii) inhibit its own activity by increasing transcription of CsrB and CsrC (10,24), and (iii) inhibit its own activity by decreasing CsrD production, which increases CsrB (25).…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…The domain requirement is reminiscent of LapD, and it is likely that c-di-GMP-driven conformational change (but not an inside-out mechanism) is associated with CsrD targeting sRNAs for RNase E-mediated degradation (149). The relationship between Csr/Rsm regulation and c-di-GMP signaling has been strengthened by the findings of CsrA regulating the expression of proteins containing the GGDEF, GGDEF-EAL, and EAL domains in E. coli and Salmonella enterica serovar Typhimurium via direct binding to the mRNA leaders of the respective genes (70,71). Although a link between Gac/Rsm regulation and cyclic-di-GMP has recently been established in P. aeruginosa, the relationship remains poorly understood in this organism.…”

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

“…In Escherichia coli, CsrA activates glycolysis and central carbon pathways (7)(8)(9)(10)(11)(12)(13) and motility (14,15). Conversely, it represses gluconeogenesis (7), glycogen biosynthesis (16)(17)(18)(19)(20), biofilm formation (21)(22)(23)(24), the stringent response (25), and expression of genes involved in other stress resistance and stationary-phase processes, e.g., cstA, hfq, cel, sdiA, and nhaR (24,(26)(27)(28)(29)(30). Its effects on pathogenesis are complex.…”