RNA10-2BS was able to compete for CsrA binding without the formation of novel shifted species, while unlabelledphoBRNA did not compete with theSTM3611-CsrA interaction (Fig. (c-di-GMP-inactive) EAL domain protein, recently identified as a negative regulator of flagella gene expression. Here, we demonstrate that CsrA directly downregulates expression of STM1344, which in turn regulatesSTM3611throughfliAand thus reciprocally controls motility and biofilm factors. Altogether, our data reveal that the concerted and complex regulation of several genes encoding GGDEF/EAL domain proteins allows CsrA to control the motility-sessility switch inS.Typhimurium at multiple levels. == Introduction == The survival of bacteria in diverse environments largely depends on their ability to adjust their life style according to the surrounding conditions. An important factor that mediates the choice of an appropriate life style in various bacteria is the signalling molecule (3-5)-cyclic-diguanosine monophosphate (c-di-GMP) (recent reviews byHengge, 2009;Jonas et al., 2009;Romling and Ceftizoxime Simm, 2009). In general, high intracellular levels of Cd248 this second messenger promote sedentary biofilm-associated phenotypes, whereas low concentrations of c-di-GMP favour motility. In several bacteria, c-di-GMP has also been associated with the regulation of virulence and other phenotypes (Cotter and Stibitz, 2007;Tamayo et al., 2007). c-di-GMP is synthesized by diguanylate cyclases (DGCs), which contain catalytically active GGDEF domains, and degradation of the second messenger is mediated by phosphodiesterases (PDE), which harbour either EAL or HD-GYP domains (Paul et al., 2004;Simm et al., 2004;Christen et al., 2005;Ryjenkov et al., 2005;Schmidt et al., 2005;Ryan et al., 2006). Notably, individual bacterial genomes frequently encode numerous GGDEF and EAL/HD-GYP proteins (Galperinet al., 2001;Galperin, 2004), implying that the c-di-GMP network is a highly complex and tightly regulated intracellular signalling system (Jonas et al., 2009). The ability to switch between different life styles is crucial for bacteria such asSalmonellathat alternate between distinct niches in host organisms during infection as well as in the abiotic environment, in which they can persist for weeks in food, soil, water and other habitats.Salmonella entericaserovar Typhimurium contains a c-di-GMP system comprised of 5 GGDEF domain proteins, 7 GGDEF-EAL and 8 EAL domain proteins (Romling, 2005). The characterization of the phenotypes of these proteins has been the subject of several previous studies (Garcia et al., 2004;Kader et al., 2006;Simm et al., 2007;Solano et al., 2009). However, little is known about the regulation of theSalmonellac-di-GMP system by intra- and extracellular factors. In the closely related speciesEscherichia coli, the carbon storage regulator CsrA controls the expression of at least seven of 29 genes encoding GGDEF/EAL domain proteins at the post-transcriptional level (Jonas et al., 2008). Members of the CsrA (RsmA) family are homodimeric RNA-binding proteins (Mercante et al., 2009) that are widely distributed among eubacteria and control various phenotypes including biofilm formation, motility, carbon flux, secondary metabolism, quorum sensing as well as interactions with animal and plant hosts (Romeo, 1998;Babitzke and Romeo, 2007;Lapouge et al., 2008;Lucchetti-Miganeh et al., 2008). In -proteobacteria CsrA proteins are antagonized by small non-coding RNAs (sRNAs) that bind and sequester multiple copies of CsrA (Babitzke and Romeo, 2007;Lapougeet al., 2008). Transcription of the sRNAs is controlled by a two-component system (BarA-UvrY inE. coli, BarA-SirA inS.Typhimurium), permitting the integration of environmental signals into the Csr system (Babitzke and Romeo, 2007;Lapougeet al., Ceftizoxime 2008). By directly binding to target mRNAs CsrA can either down- or upregulate the expression of target genes (Babitzke and Romeo, 2007). CsrA binding to the mRNAs of the GGDEF proteins YcdT and YdeH inE. coliled to a strong downregulation in transcript levels (Jonas et al., 2008). Both proteins encode DGCs, which inhibit motility. YdeH is also involved in the positive regulation of biofilm formation (Boehm et al., 2009). Thus, by regulating the expression of these GGDEF domain proteins CsrA controls motility and biofilm behaviour in a c-di-GMP dependent manner. In addition, CsrA enhances motility and reciprocally inhibits biofilm formation inE. coliby binding to and stabilizing the transcript of the flagella master regulator FlhDC (Weiet al., 2001) and by blocking translation ofpgaAand destabilizingpgaABCDmRNA (Wanget al., 2005), which encodes the synthesis and secretion apparatus of the PGA (poly–1,6-N-acetylglucosamine) biofilm polysaccharide adhesin (Itoh et al., 2008). In contrast toE. coli, the role Ceftizoxime of CsrA in the regulation of the sessility-motility switch is less well understood inSalmonella. S.Typhimurium has a CsrA orthologue that is identical in its amino acid sequence to CsrA inE. coli. However,S.Typhimurium contains neither thepgaoperon nor orthologues ofycdT,ydeHand most of the other GGDEF/EAL genes that are regulated by CsrA inE. coli. Although CsrA regulates genes belonging to the flagella synthesis cascade as well as genes required for virulence (Altier.