Identification of a novel regulatory protein (CsrD) that targets the global regulatory RNAs CsrB and CsrC for degradation by RNase E

Abstract

In Escherichia coli, the global regulatory protein CsrA (carbon store regulator A) binds to leader segments of target mRNAs, affecting their translation and stability. CsrA activity is regulated by two noncoding RNAs, CsrB and CsrC, which act by sequestering multiple CsrA dimers. Here, we describe a protein (CsrD) that controls the degradation of CsrB/C RNAs. The dramatic stabilization of CsrB/C RNAs in a csrD mutant altered the expression of CsrA-controlled genes in a manner predicted from the previously described Csr regulatory circuitry. A deficiency in RNase E, the primary endonuclease involved in mRNA decay, also stabilized CsrB/C, although the half-lives of other RNAs that are substrates for RNase E (rpsO, rpsT, and RyhB) were unaffected by csrD. Analysis of the decay of CsrB RNA, both in vitro and in vivo, suggested that CsrD is not a ribonuclease. Interestingly, the CsrD protein contains GGDEF and EAL domains, yet unlike typical proteins in this large superfamily, its activity in the regulation of CsrB/C decay does not involve cyclic di-GMP metabolism. The two predicted membrane-spanning regions are dispensable for CsrD activity, while HAMP-like, GGDEF, and EAL domains are required. Thus, these studies demonstrate a novel process for the selective targeting of RNA molecules for degradation by RNase E and a novel function for a GGDEF-EAL protein.[Keywords: RNA decay; biofilm formation; Hfq; polynucleotide phosphorylase; degradosome; GGDEF-EAL domain proteins] Supplemental material is available at http://www.genesdev.org. In many species of bacteria, the Csr (carbon storage regulator) and homologous Rsm (repressor of stationary phase metabolites) systems coordinate the expression of diverse genes that facilitate adaptation among major physiological phases of growth; e.g., exponential versus stationary phase, planktonic versus biofilm, and ostensibly acute versus chronic states of infection (Romeo 1998;Wei et al. 2001;Jackson et al. 2002;Goodman et al. 2004;Majdalani et al. 2005). These systems use the RNA-binding protein CsrA (Romeo et al. 1993) to regulate translation and mRNA stability by recognizing specific nucleotide sequences within mRNA leaders. Among the best-studied examples of these are the CsrArepressed glgCAP, cstA, and pgaABCD mRNAs, which are involved in glycogen metabolism, peptide transport, and biofilm formation, respectively Baker et al. 2002;X. Wang et al. 2005), as well as the CsrA-activated flhDC mRNA, which encodes the master regulator of motility and chemotaxis genes (Wei et al. 2001).In Escherichia coli, CsrA protein activity is regulated by the CsrB and CsrC noncoding RNAs, which contain CsrA recognition sequences (18 and 9, respectively) primarily within the loops of predicted stem-loop structures. Interaction of CsrA with these sites leads to its sequestration Weilbacher et al. 2003;Dubey et al. 2005). Thus, this system employs a mechanism distinct from that of other small RNAs such as OxyS and RyhB, which involve RNA-RNA base-pairing (Gottesman 2004). Furthermore, the Csr compon...

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

“…Degradation of CsrB and CsrC requires the protein CsrD, which facilitates endonucleolytic cleavage of these sRNAs by RNase E (42,43). Thus, the effect of CRP on synthesis of CsrB and CsrC under conditions of greatly reduced turnover was examined in a ⌬csrD strain (Fig.…”

“…Transport of glucose by the PTS pathway leads to dephosphorylation of EIIA Glc , which binds to CsrD and activates the decay pathway for CsrB/C in E. coli (43,44). This kind of regulatory pathway may function in most Enterobacteriaceae, Vibrionaceae, and Shewanellaceae species and yet is absent in the majority of gammaproteobacterial families, the members of which lack a csrD homolog (42,43). Furthermore, cAMP-CRP modestly inhibits CsrB/C decay (Fig.…”

“…Transcription of both csrB and csrC (csrB/C) is activated by the BarA-UvrY two-component signal transduction system (TCS) in response to carboxylic acids such as formate and acetate (3,(36)(37)(38)(39)(40)(41). CsrA indirectly activates transcription of CsrB and CsrC through its effects on BarA-UvrY, creating a negative-feedback loop within the Csr circuitry (37,38,42). CsrB/C turnover requires the GGDEF-EAL domain protein CsrD, which is necessary for cleavage by RNase E and turnover (42,43).…”

“…CsrA indirectly activates transcription of CsrB and CsrC through its effects on BarA-UvrY, creating a negative-feedback loop within the Csr circuitry (37,38,42). CsrB/C turnover requires the GGDEF-EAL domain protein CsrD, which is necessary for cleavage by RNase E and turnover (42,43). Therefore, CsrD affects the expression of CsrA-regulated genes and processes.…”

Sticky Situations: Key Components That Control Bacterial Surface Attachment

“…The first indication of an association between a Csr system and c-di-GMP levels came with the characterization of the GGDEF and EAL domain-containing protein CsrD, which targets the sRNAs csrB/csrC for degradation by RNase E (149). While CsrD is not directly involved in c-di-GMP synthesis or turnover, it requires both the GGDEF and the EAL domains for its function (149). 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).…”