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...

SummaryThe carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action. In this paper, we elucidate further the mechanism of CsrA-mediated glgC regulation. Results from gel shift assays demonstrate that several molecules of CsrA can bind to each glgC transcript. RNA footprinting studies indicate that CsrA binds to the glgCAP leader transcript at two positions. One of these sites overlaps the glgC Shine-Dalgarno sequence, whereas the other CsrA target is located further upstream in an RNA hairpin. Results from toeprint and cell-free translation experiments indicate that bound CsrA prevents ribosome binding to the glgC Shine-Dalgarno sequence and that this reduces GlgC synthesis. The effect of two deletions in the upstream binding site was examined. Both of these deletions reduced, but did not eliminate, CsrA binding in vitro and CsrA-dependent regulation in vivo. Our findings establish that bound CsrA inhibits initiation of glgC translation, thereby reducing glycogen biosynthesis. This inhibition of translation probably contributes to destabilization of the glgC transcript that was observed previously. Introduction

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.

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins. eNLs allow five-colour live-cell imaging, as well as detection of single protein complexes and even single molecules. We also develop an eNL-based Ca2+ indicator with a 500% signal change, which can image spontaneous Ca2+ dynamics in cardiomyocyte and neural cell models. These eNL probes facilitate not only multicolour imaging in living cells but also sensitive imaging of a wide repertoire of proteins, even at very low expression levels.

CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia coli genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA-lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5-to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by ⌭ 70 . We investigated the influence of S on cstA expression and found that a S deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA ShineDalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.