Homologues of csrC are apparent in several Enterobacteriaceae. The regulatory and evolutionary implications of these findings are discussed.

SummaryThe RNA-binding protein CsrA represses biofilm formation, while the non-coding RNAs CsrB and CsrC activate this process by sequestering CsrA. We now provide evidence that the pgaABCD transcript,

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA-lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.

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...

The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation in Escherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activates csrB transcription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreased csrB transcription but did not affect csrA-lacZ expression. The uvrY defect was severalfold more severe than that of barA. Both csrA and uvrY were required for optimal barA expression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression of uvrY suppressed the csrB-lacZ expression defects caused by uvrY, csrA, or barA mutations; csrA suppressed csrA or barA defects; and barA complemented only the barA mutation. Purified UvrY protein stimulated csrB-lacZ expression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY on csrB transcription. Disruption of sdiA, which encodes a LuxR homologue, decreased the expression of uvrY-lacZ and csrB-lacZ fusions but did not affect csrA-lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression of uvrY stimulated biofilm formation by a csrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate that uvrY resides downstream from csrA in a signaling pathway for csrB and that CsrA stimulates UvrY-dependent activation of csrB expression by BarA-dependent and -independent mechanisms.