Vibrio cholerae transitions between aquatic environmental reservoirs and infection in the gastrointestinal tracts of human hosts. The second-messenger molecule cyclic di-GMP (c-di-GMP) and quorum sensing (QS) are important signaling systems that enable V. cholerae to alternate between these distinct environments by controlling biofilm formation and virulence factor expression. Here we identify a conserved regulatory mechanism in V. cholerae that integrates c-di-GMP and QS to control the expression of two transcriptional regulators: aphA, an activator of virulence gene expression and an important regulator of the quorum-sensing pathway, and vpsT, a transcriptional activator that induces biofilm formation. Surprisingly, aphA expression was induced by c-di-GMP. Activation of both aphA and vpsT by c-di-GMP requires the transcriptional activator VpsR, which binds to c-di-GMP. The VpsR binding site at each of these promoters overlaps with the binding site of HapR, the master QS regulator at high cell densities. Our results suggest that V. cholerae combines information conveyed by QS and c-di-GMP to appropriately respond and adapt to divergent environments by modulating the expression of key transcriptional regulators.Bacteria use multiple signaling pathways to monitor and respond appropriately to changing surroundings. Small-molecule chemical signals convey information about the presence, nature, number, and characteristics of the surrounding bacterial species as well as the composition of the environment. Proper responses to changing environments are vital to the survival of bacteria. Vibrio cholerae, the causative agent of cholera, alternates between a motile, virulent state within the host and a sessile, biofilm state in aquatic environmental reservoirs (15). Quorum sensing (QS) and cyclic di-GMP (c-di-GMP) signaling are two chemical signaling systems that control this transition (19).QS allows bacteria to sense the population density and species composition of the surrounding bacterial consortium through the secretion and detection of chemical signals called autoinducers so as to collectively control behaviors (46). In V. cholerae, in the high-cell-density QS state, both biofilm formation and virulence factor expression are repressed (21, 31). c-di-GMP is a nearly ubiquitous bacterial second messenger that induces biofilm formation and represses motility (19). In contrast to QS, c-di-GMP activates the expression of genes necessary for biofilm formation in V. cholerae (3). However, like QS, c-di-GMP is thought to repress the expression of virulence factors (38, 42).The QS regulatory pathways that control biofilm formation and virulence factor expression have been largely elucidated. HapR, the master high-cell-density regulator of the QS signaling cascade, represses biofilm formation by directly binding to the biofilm activator vpsT and inhibiting its transcription (45). Additionally, HapR production reduces intracellular c-di-GMP levels (45). Inhibition of virulence factor expression by QS is mediated by HapR repre...

Summary Cyclic di-GMP (c-di-GMP) controls the transition between sessility and motility in many bacterial species. This regulation is achieved by a variety of mechanisms including alteration of transcription initiation and inhibition of flagellar function. How c-di-GMP inhibits the motility of Vibrio cholerae has not been determined. FlrA, a homolog of the c-di-GMP binding Pseudomonas aeruginosa motility regulator FleQ, is the master regulator of the V. cholerae flagellar biosynthesis regulon. Here we show that binding of c-di-GMP to FlrA abrogates binding of FlrA to the promoter of the flrBC operon, deactivating expression of the flagellar biosynthesis regulon. FlrA does not regulate expression of extracellular Vibrio polysaccharide (VPS) synthesis genes. Mutation of the FlrA amino acids R135 and R176 to histidine abrogates binding of c-di-GMP to FlrA, rendering FlrA active in the presence of high levels of c-di-GMP. Surprisingly, c-di-GMP still inhibited the motility of V. cholerae only expressing the c-di-GMP blind FlrA(R176H) mutant. We determined that this flagellar transcription-independent inhibition is due to activation of VPS production by c-di-GMP. Therefore, c-di-GMP prevents motility of V. cholerae by two distinct but functionally redundant mechanisms.

Bacteria sense and respond to environmental cues to control important developmental processes. Two widely conserved and important strategies that bacteria employ to sense changes in population density and local environmental conditions are quorum sensing (QS) and cyclic di-GMP (c-di-GMP) signaling, respectively. The importance of these pathways in controlling a broad variety of functions, including virulence, biofilm formation, and motility, has been recognized in many species. Recent research has shown that these pathways are intricately intertwined. Here we review the regulatory connections between QS and c-di-GMP signaling. We propose that the integration of QS with c-di-GMP allows bacteria to assimilate information about the local bacterial population density with other physicochemical environmental signals within the broader c-di-GMP signaling network.

Carboxy-terminal processing proteases (CTPs) occur in all three domains of life. In bacteria, some of them have been associated with virulence. However, the precise roles of bacterial CTPs are poorly understood, and few direct proteolytic substrates have been identified. One bacterial CTP is the CtpA protease of Pseudomonas aeruginosa, which is required for type III secretion system (T3SS) function and for virulence in a mouse model of acute pneumonia. Here, we have investigated the function of CtpA in P. aeruginosa and identified some of the proteins it cleaves. We discovered that CtpA forms a complex with a previously uncharacterized protein, which we have named LbcA (lipoprotein binding partner of CtpA). LbcA is required for CtpA activity in vivo and promotes its activity in vitro. We have also identified four proteolytic substrates of CtpA, all of which are uncharacterized proteins predicted to cleave the peptide cross-links within peptidoglycan. Consistent with this, a ctpA null mutant was found to have fewer peptidoglycan cross-links than the wild type and grew slowly in salt-free medium. Intriguingly, the accumulation of just one of the CtpA substrates was required for some ΔctpA mutant phenotypes, including the defective T3SS. We propose that LbcA-CtpA is a proteolytic complex in the P. aeruginosa cell envelope, which controls the activity of several peptidoglycan cross-link hydrolases by degrading them. Furthermore, based on these and other findings, we suggest that many bacterial CTPs might be similarly controlled by partner proteins as part of a widespread mechanism to control peptidoglycan hydrolase activity.

21Carboxy-terminal processing proteases (CTPs) occur in all domains of life. In bacteria, 22 they have been associated with virulence, but their roles are poorly understood. One is 23 the CtpA protease of Pseudomonas aeruginosa, which is required for type III secretion 24 system function, and for virulence. Here we show that CtpA works with a previously 25 uncharacterized binding partner to degrade four substrates. The accumulation of at 26