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A practical synthesis has been developed for DPD (4,5-dihydroxypentane-2,3-dione), an unstable small molecule that is proposed to be the source of universal signaling agents for quorum sensing in bacteria. The synthesis allows preparation of isotopically labeled DPD and ent-DPD as well as detailed studies of spontaneous binding to borate to give the unusual borate complex 6, the signal for marine bacteria such as Vibrio harveyi. [reaction: see text]

AI-2 is a quorum-sensing signaling molecule proposed to be involved in interspecies communication. In Escherichia coli and Salmonella enterica serovar Typhimurium, extracellular AI-2 accumulates in exponential phase, but the amount decreases drastically upon entry into stationary phase. In S. enterica serovar Typhimurium, the reduction in activity is due to import and processing of AI-2 by the Lsr transporter. We show that the Lsr transporter is functional in E. coli, and screening for mutants defective in AI-2 internalization revealed lsrK and glpD. Unlike the wild type, lsrK and glpD mutants do not activate transcription of the lsr operon in response to AI-2. lsrK encodes the AI-2 kinase, and the lsrK mutant fails to activate lsr expression because it cannot produce phospho-AI-2, which is the lsr operon inducer. glpD encodes the glycerol-3-phosphate (G3P) dehydrogenase, which is involved in glycerol and G3P metabolism. G3P accumulates in the glpD mutant and represses lsr transcription by preventing cyclic AMP (cAMP)-catabolite activator protein (CAP)-dependent activation. Dihydroxyacetone phosphate (DHAP) also accumulates in the glpD mutant, and DHAP represses lsr transcription by a cAMP-CAP-independent mechanism involving LsrR, the lsr operon repressor. The requirement for cAMP-CAP in lsr activation explains why AI-2 persists in culture fluids of bacteria grown in media containing sugars that cause catabolite repression. These findings show that, depending on the prevailing growth conditions, the amount of time that the AI-2 signal is present and, in turn, the time that a given community of bacteria remains exposed to this signal can vary greatly.

Bacteria communicate with one another using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules termed autoinducers . This process, termed quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Most quorum-sensing-controlled processes are unproductive when undertaken by an individual bacterium acting alone but become beneficial when carried out simultaneously by a large number of cells. Thus, quorum sensing confuses the distinction between prokaryotes and eukaryotes because it enables bacteria to act as multicellular organisms. This review focuses on the architectures of bacterial chemical communication networks; how chemical information is integrated, processed, and transduced to control gene expression; how intra- and interspecies cell-cell communication is accomplished; and the intriguing possibility of prokaryote-eukaryote cross-communication.

Quorum sensing is a process that enables bacteria to communicate using secreted signaling molecules called autoinducers. This process enables a population of bacteria to regulate gene expression collectively and, therefore, control behavior on a community-wide scale. Quorum sensing is widespread in the bacterial world and, generally, processes controlled by quorum sensing are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. Cell-cell communication can occur within and between bacterial species, and between bacteria and their eukaryotic hosts, which suggests that the chemical lexicon is complex. Prokaryotic and eukaryotic mechanisms for enhancing and inhibiting quorum sensing have been identified, which suggests that manipulation of quorum-sensing-controlled processes could be common in bacterial-bacterial and bacterial-eukaryotic associations.

In a process called quorum sensing, bacteria communicate using extracellular signal molecules termed autoinducers. Two parallel quorum-sensing systems have been identified in the marine bacterium Vibrio harveyi. System 1 consists of the LuxM-dependent autoinducer HAI-1 and the HAI-1 sensor, LuxN. System 2 consists of the LuxS-dependent autoinducer AI-2 and the AI-2 detector, LuxPQ. The related bacterium, Vibrio cholerae, a human pathogen, possesses System 2 (LuxS, AI-2, and LuxPQ) but does not have obvious homologues of V. harveyi System 1. Rather, System 1 of V. cholerae is made up of the CqsA-dependent autoinducer CAI-1 and a sensor called CqsS. Using a V. cholerae CAI-1 reporter strain we show that many other marine bacteria, including V. harveyi, produce CAI-1 activity. Genetic analysis of V. harveyi reveals cqsA and cqsS, and phenotypic analysis of V. harveyi cqsA and cqsS mutants shows that these functions comprise a third V. harveyi quorum-sensing system that acts in parallel to Systems 1 and 2. Together these communication systems act as a three-way coincidence detector in the regulation of a variety of genes, including those responsible for bioluminescence, type III secretion, and metalloprotease production.

Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence. This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers. While most autoinducers are species specific, autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi, is produced and detected by many Gram-negative and Gram-positive bacteria. The crystal structure of the V. harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester. Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium. We find that LsrB binds a chemically distinct form of the AI-2 signal, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), that lacks boron. Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling., (Copyright 2004 Cell Press)

[reaction: see text] The unstable bacterial metabolic product, DPD, and the related natural product, laurencione, are shown to have a high affinity for borate complexation, through the hydrated analogue. The boron complex of DPD is Vibrio harveyi AI-2, an interspecies quorum sensing signal in bacteria, and an affinity column with a borate resin is effective in providing the first method for concentrating and purifying V. harveyi AI-2 from the biosynthetic product.

Quorum-sensing bacteria communicate with extracellular signal molecules called autoinducers. This process allows community-wide synchronization of gene expression. A screen for additional components of the Vibrio harveyi and Vibrio cholerae quorum-sensing circuits revealed the protein Hfq. Hfq mediates interactions between small, regulatory RNAs (sRNAs) and specific messenger RNA (mRNA) targets. These interactions typically alter the stability of the target transcripts. We show that Hfq mediates the destabilization of the mRNA encoding the quorum-sensing master regulators LuxR (V. harveyi) and HapR (V. cholerae), implicating an sRNA in the circuit. Using a bioinformatics approach to identify putative sRNAs, we identified four candidate sRNAs in V. cholerae. The simultaneous deletion of all four sRNAs is required to stabilize hapR mRNA. We propose that Hfq, together with these sRNAs, creates an ultrasensitive regulatory switch that controls the critical transition into the high cell density, quorum-sensing mode.

In a process known as quorum sensing, bacteria communicate with one another by producing, releasing, detecting, and responding to signal molecules called autoinducers. Vibrio harveyi, a marine pathogen, uses two parallel quorum-sensing circuits, each consisting of an autoinducer-sensor pair, to control the expression of genes required for bioluminescence and a number of other target genes. Genetic screens designed to discover autoinducer-regulated targets in V. harveyi have revealed genes encoding components of a putative type III secretion (TTS) system. Using transcriptional reporter fusions and TTS protein localization studies, we show that the TTS system is indeed functional in V. harveyi and that expression of the genes encoding the secretion machinery requires an intact quorum-sensing signal transduction cascade. The newly completed genome of the closely related marine bacterium Vibrio parahaemolyticus, which is a human pathogen, shows that it possesses the genes encoding both of the V. harveyi-like quorum-sensing signaling circuits and that it also has a TTS system similar to that of V. harveyi. We show that quorum sensing regulates TTS in V. parahaemolyticus. Previous reports connecting quorum sensing to TTS in enterohemorrhagic and enteropathogenic Escherichia coli show that quorum sensing activates TTS at high cell density. Surprisingly, we find that at high cell density (in the presence of autoinducers), quorum sensing represses TTS in V. harveyi and V. parahaemolyticus.

Cell-cell communication in bacteria is accomplished through the exchange of chemical signal molecules called autoinducers. This process, called quorum sensing, allows bacteria to monitor their environment for the presence of other bacteria and to respond to fluctuations in the number and/or species present by altering particular behaviors. Most quorum-sensing systems are species- or group-specific, which presumably prevents confusion in mixed-species environments. However, some quorum-sensing circuits control behaviors that involve interactions among bacterial species. These quorum-sensing circuits can involve both intra- and interspecies communication mechanisms. Finally, anti-quorumsensing strategies are present in both bacteria and eukaryotes, and these are apparently designed to combat bacteria that rely on cell-cell communication for the successful adaptation to particular niches.

Bacterial quorum sensing is mediated by autoinducers, small signaling molecules generated by bacteria. It has been proposed that the LuxS enzyme converts S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2 (AI-2). We report here a chemical synthesis of S-ribosyl-L-homocysteine and its analogue using Mitsunobu coupling. Chemically synthesized ribosylhomocysteine has been confirmed as a substrate for LuxS in both an enzyme assay and a whole cell quorum sensing assay. The chemical entities of products from the LuxS reaction were also established. Several ribosylhomocysteine analogues have been tested as LuxS inhibitors.

The LuxS-dependent autoinducer AI-2 is proposed to function in interspecies cell-cell communication in bacteria. In Salmonella typhimurium, AI-2 is produced and released during exponential growth and is subsequently imported into the bacteria via the Lsr (luxS regulated) ATP binding cassette (ABC) transporter. AI-2 induces transcription of the lsrACDBFGE operon, the first four genes of which encode the Lsr transport apparatus. In this report, we identify and characterize LsrK, a new protein that is required for the regulation of the lsr operon and the AI-2 uptake process. LsrK is a kinase that phosphorylates AI-2 upon entry into the cell. Our data indicate that phosphorylation of AI-2 results in its sequestration in the cytoplasm. We suggest that phospho-AI-2 is the inducer responsible for inactivation of LsrR, the repressor of the lsr operon. We also show that two previously uncharacterized members of the lsr operon, LsrF and LsrG, are necessary for the further processing of phospho-AI-2. Transport and processing of AI-2 could be required for removing the quorum-sensing signal, conveying the signal to an internal detector and/or scavenging boron.

Until recently, bacteria were considered to live rather asocial, reclusive lives. New research shows that, in fact, bacteria have elaborate chemical signaling systems that enable them to communicate within and between species. One signal, termed AI-2, appears to be universal and facilitates interspecies communication. Many processes, including virulence factor production, biofilm formation, and motility, are controlled by AI-2. Strategies that interfere with communication in bacteria are being explored in the biotechnology industry with the aim of developing novel antimicrobials. AI-2 is a particularly attractive candidate for such studies because of its widespread use in the microbial kingdom.

Multiple quorum-sensing circuits function in parallel to control virulence and biofilm formation in Vibrio cholerae. In contrast to other bacterial pathogens that induce virulence factor production and/or biofilm formation at high cell density in the presence of quorum-sensing autoinducers, V. cholerae represses these behaviours at high cell density. Consistent with this, we show here that V. cholerae strains 'locked' in the regulatory state mimicking low cell density are enhanced for biofilm production whereas mutants 'locked' in the regulatory state mimicking high cell density are incapable of producing biofilms. The quorum-sensing cascade we have identified in V. cholerae regulates the transcription of genes involved in exopolysaccharide production (EPS), and variants that produce EPS and form biofilms arise at high frequency from non-EPS, non-biofilm producing strains. Our data show that spontaneous mutation of the transcriptional regulator hapR is responsible for this effect. Several toxigenic strains of V. cholerae possess a naturally occurring frameshift mutation in hapR. Thus, the distinct environments occupied by this aquatic pathogen presumably include niches where cell-cell communication is crucial, as well as ones where loss of quorum sensing via hapR mutation confers a selective advantage. Bacterial biofilms could represent a complex habitat where such differentiation occurs.

Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Quorum sensing allows populations of bacteria to collectively control gene expression, and thus synchronize group behavior. Processes controlled by quorum sensing are typically ones that are unproductive unless many bacteria act together. Most autoinducers enable intraspecies communication; however, a recently discovered autoinducer AI-2 has been proposed to serve as a 'universal signal' for interspecies communication. Studies suggest that AI-2 encodes information in addition to specifics about cell number.

In a process called quorum sensing, bacteria communicate with one another by exchanging chemical signals called autoinducers. In the bioluminescent marine bacterium Vibrio harveyi, two different auto inducers (AI-1 and AI-2) regulate light emission. Detection of and response to the V.harveyi autoinducers are accomplished through two two-component sensory relay systems: AI-1 is detected by the sensor LuxN and AI-2 by LuxPQ. Here we further define the V.harveyi quorum-sensing regulon by identifying 10 new quorum-sensing-controlled target genes. Our examination of signal processing and integration in the V.harveyi quorum-sensing circuit suggests that AI-1 and AI-2 act synergistically, and that the V.harveyi quorum-sensing circuit may function exclusively as a 'coincidence detector' that discriminates between conditions in which both autoinducers are present and all other conditions.

The marine bacterium Vibrio harveyi possesses two quorum sensing systems (System 1 and System 2) that regulate bioluminescence. Although the Vibrio cholerae genome sequence reveals that a V. harveyi-like System 2 exists, it does not predict the existence of a V. harveyi-like System 1 or any obvious quorum sensing-controlled target genes. In this report we identify and characterize the genes encoding an additional V. cholerae autoinducer synthase and its cognate sensor. Analysis of double mutants indicates that a third as yet unidentified sensory circuit exists in V. cholerae. This quorum sensing apparatus is unusually complex, as it is composed of at least three parallel signaling channels. We show that in V. cholerae these communication systems converge to control virulence.

In a process called quorum sensing, groups of bacteria communicate with one another to coordinate their behavior and function like a multicellular organism. A diverse array of secreted chemical signal molecules and signal detection apparatuses facilitate highly productive intra- and interspecies relationships.

The production of virulence factors including cholera toxin and the toxin-coregulated pilus in the human pathogen Vibrio cholerae is strongly influenced by environmental conditions. The well-characterized ToxR signal transduction cascade is responsible for sensing and integrating the environmental information and controlling the virulence regulon. We show here that, in addition to the known components of the ToxR signaling circuit, quorum-sensing regulators are involved in regulation of V. cholerae virulence. We focused on the regulators LuxO and HapR because homologues of these two proteins control quorum sensing in the closely related luminous marine bacterium Vibrio harveyi. Using an infant mouse model, we found that a luxO mutant is severely defective in colonization of the small intestine. Gene arrays were used to profile transcription in the V. cholerae wild type and the luxO mutant. These studies revealed that the ToxR regulon is repressed in the luxO mutant, and that this effect is mediated by another negative regulator, HapR. We show that LuxO represses hapR expression early in log-phase growth, and constitutive expression of hapR blocks ToxR-regulon expression. Additionally, LuxO and HapR regulate a variety of other cellular processes including motility, protease production, and biofilm formation. Together these data suggest a role for quorum sensing in modulating expression of blocks of virulence genes in a reciprocal fashion in vivo.

Cell-cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. Community cooperation probably enhances the effectiveness of processes such as bioluminescence, virulence factor expression, antibiotic production and biofilm development. Unlike other autoinducers, which are specific to a particular species of bacteria, a recently discovered autoinducer (AI-2) is produced by a large number of bacterial species. AI-2 has been proposed to serve as a 'universal' signal for inter-species communication. The chemical identity of AI-2 has, however, proved elusive. Here we present the crystal structure of an AI-2 sensor protein, LuxP, in a complex with autoinducer. The bound ligand is a furanosyl borate diester that bears no resemblance to previously characterized autoinducers. Our findings suggest that addition of naturally occurring borate to an AI-2 precursor generates active AI-2. Furthermore, they indicate a potential biological role for boron, an element required by a number of organisms but for unknown reasons.

In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI-2, originally discovered in the quorum-sensing bacterium Vibrio harveyi, is made by many species of Gram-negative and Gram-positive bacteria. In every case, production of AI-2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI-2 in most of these luxS-containing species of bacteria are not known. Here, we describe the identification and characterization of AI-2-regulated genes in Salmonella typhimurium. We find that LuxS and AI-2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)-type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium. A gene encoding a DNA-binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI-2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI-2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI-2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI-2 into the cells. It is intriguing that an operon regulated by AI-2 encodes functions resembling the ribose transporter, given recent findings that AI-2 is derived from the ribosyl moiety of S-ribosylhomocysteine.

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram-negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram-negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI-1 and AI-2). AI-1 is a homoserine lactone. The structure of AI-2 is not known. We have suggested previously that V. harveyi uses AI-1 for intraspecies communication and AI-2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram-negative and Gram-positive bacteria produce AI-2 and, in every case, production of AI-2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI-2 synthase and that AI-2 is produced from S-adenosylmethionine in three enzymatic steps. The substrate for LuxS is S-ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI-2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI-2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI-2 is a unique, 'universal' signal that could be used by a variety of bacteria for communication among and between species.

Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species. Furthermore, there is mounting data suggesting that bacterial autoinducers elicit specific responses from host organisms. Although the nature of the chemical signals, the signal relay mechanisms, and the target genes controlled by bacterial quorum sensing systems differ, in every case the ability to communicate with one another allows bacteria to coordinate the gene expression, and therefore the behavior, of the entire community. Presumably, this process bestows upon bacteria some of the qualities of higher organisms. The evolution of quorum sensing systems in bacteria could, therefore, have been one of the early steps in the development of multicellularity.

Helicobacter pylori possesses a homolog of the luxS gene, initially identified by its role in autoinducer production for the quorum-sensing system 2 in Vibrio harveyi. The genomes of several other species of bacteria, notably Escherichia coli, Salmonella enterica serovar Typhimurium, and Vibrio cholerae, also include luxS homologs. All of these bacteria have been shown to produce active autoinducers capable of stimulating the expression of the luciferase operon in V. harveyi. In this report, we demonstrate that H. pylori also synthesizes a functional autoinducer (AI-2) that can specifically activate signaling system 2 in V. harveyi. Maximal activity is produced during early log phase, and the activity is diminished when cells enter stationary phase. We show that AI-2 is not involved in modulating any of the known or putative virulence factors in H. pylori and that a luxS null mutant has a two-dimensional protein profile identical to that of its isogenic parent strain. We discuss the implications of having an AI-2-like quorum-sensing system in H. pylori and suggest possible roles that it may play in H. pylori infection.

The bioluminescent marine bacterium Vibrio harveyi controls light production (lux) by an elaborate quorum-sensing circuit. V. harveyi produces and responds to two different autoinducer signals (AI-1 and AI-2) to modulate the luciferase structural operon (luxCDABEGH) in response to changes in cell-population density. Unlike all other Gram-negative quorum-sensing organisms, V. harveyi regulates quorum sensing using a two-component phosphorylation-dephosphorylation cascade. Each autoinducer is recognized by a cognate hybrid sensor kinase (called LuxN and LuxQ). Both sensors transduce information to a shared phosphorelay protein called LuxU, which in turn conveys the signal to the response regulator protein LuxO. Phospho-LuxO is responsible for repression of luxCDABEGH expression at low cell density. In the present study, we demonstrate that LuxO functions as an activator protein via interaction with the alternative sigma factor, sigma54 (encoded by rpoN). Our results suggest that LuxO, together with sigma54, activates the expression of a negative regulator of luminescence. We also show that phenotypes other than lux are regulated by LuxO and sigma54, demonstrating that in Vibrio harveyi, quorum sensing controls multiple processes.

The bioluminescent marine bacterium Vibrio harveyi controls light production using two parallel quorum-sensing systems. V. harveyi produces two autoinducers (AI-1 and AI-2), which are recognized by cognate membrane-bound two-component hybrid sensor kinases called LuxN and LuxQ respectively. Under conditions of low cell density, in the absence of autoinducer, the hybrid sensors are kinases, and under conditions of high cell density, in the presence of autoinducer, the sensors are phosphatases. These activities allow LuxN and LuxQ to modulate the level of phosphorylation of the response regulator protein LuxO. LuxO, in turn, controls the transcription of the genes encoding luciferase. The phosphorelay protein LuxU is required for signalling to LuxO. In this report, we present a genetic analysis of the activities of the AI-1 sensor LuxN. Point mutations and in frame deletions were constructed in luxN and recombined onto the chromosome of V. harveyi for in vivo phenotypic analysis. We show that the conserved histidine (H471) in the sensor kinase domain of LuxN is required for kinase activity but not for phosphatase activity. In contrast, the conserved aspartate (D771) in the response regulator domain of LuxN is required for both activities. Furthermore, the LuxN phosphatase activity is localized to the response regulator domain. Our results indicate that the LuxN kinase activity is regulated by the presence of AI-1, whereas the LuxN phosphatase activity is constitutive. We also show that signalling from the two V. harveyi quorum-sensing systems is not equivalent. AI-1 and LuxN have a much greater effect on the level of LuxO phosphate and therefore Lux expression than do AI-2 and LuxQ.

Quorum sensing, or the control of gene expression in response to cell density, is used by both gram-negative and gram-positive bacteria to regulate a variety of physiological functions. In all cases, quorum sensing involves the production and detection of extracellular signalling molecules called autoinducers. While universal signalling themes exist, variations in the design of the extracellular signals, the signal detection apparatuses, and the biochemical mechanisms of signal relay have allowed quorum sensing systems to be exquisitely adapted for their varied uses. Recent studies show that quorum sensing modulates both intra- and inter-species cell-cell communication, and it plays a major role in enabling bacteria to architect complex community structures.

In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows. In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor-autoinducer pair. V. harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E. coli O157:H7, Salmonella typhimurium 14028, and S. typhimurium LT2 produce autoinducers similar or identical to the V. harveyi system 2 autoinducer AI-2. However, the domesticated laboratory strain E. coli DH5alpha does not produce this signal molecule. Here we report the identification and analysis of the gene responsible for AI-2 production in V. harveyi, S. typhimurium, and E. coli. The genes, which we have named luxSV.h., luxSS.t., and luxSE.c. respectively, are highly homologous to one another but not to any other identified gene. E. coli DH5alpha can be complemented to AI-2 production by the introduction of the luxS gene from V. harveyi or E. coli O157:H7. Analysis of the E. coli DH5alpha luxSE.c. gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxSE.c. protein. Our results indicate that the luxS genes define a new family of autoinducer-production genes.

Vibrio harveyi regulates the expression of bioluminescence (lux) in response to cell density, a phenomenon known as quorum sensing. In V. harveyi, two independent quorum-sensing systems exist, and each produces, detects, and responds to a specific cell density-dependent autoinducer signal. The autoinducers are recognized by two-component hybrid sensor kinases called LuxN and LuxQ, and sensory information from both systems is transduced by a phosphorelay mechanism to the response regulator protein LuxO. Genetic evidence suggests that LuxO-phosphate negatively regulates the expression of luminescence at low cell density in the absence of autoinducers. At high cell density, interaction of the sensors with their cognate autoinducers results in dephosphorylation and inactivation of the LuxO repressor. In the present report, we show that LuxN and LuxQ channel sensory information to LuxO via a newly identified phosphorelay protein that we have named LuxU. LuxU shows sequence similarity to other described phosphorelay proteins, including BvgS, ArcB, and Ypd1. A critical His residue (His 58) of LuxU is required for phosphorelay function.

Two independent quorum-sensing systems control the expression of bioluminescence (lux) in the marine bacterium Vibrio harveyi. Each system is composed of an autoinducer (AI-1 or AI-2) and its cognate sensor (LuxN or LuxQ). The sensors are two-component hybrid kinases, containing both sensor kinase domains and response regulator domains. Sensory information from the two systems is relayed by a phosphotransfer mechanism to a shared integrator protein called LuxO. LuxO is a member of the response regulator class of the two-component family of signal transduction proteins, and LuxO acts negatively to control luminescence. In this report, missense and in frame deletion mutations were constructed in luxO that encoded proteins mimicking either the phosphorylated or the unphosphorylated form, and these mutations were introduced into the V. harveyi chromosome at the luxO locus. Phenotypical analyses of the resulting mutant V. harveyi strains indicate that the phosphorylated form of LuxO is the repressor, and that the unphosphorylated form of the protein is inactive. Analysis of the lux phenotypes of V. harveyi strains containing single and double luxN and luxQ mutations indicate that LuxN and LuxQ have two activities on LuxO. They act as LuxO protein kinases at low cell density in the absence of autoinducers, and they switch to LuxO protein phosphatases at high cell density in the presence of autoinducers. Furthermore, the timing and potency of inputs from the two systems into regulation of quorum sensing are different.

Salmonella typhimurium strain LT2 secretes an organic signalling molecule that can be assayed by its ability to activate one of two specific quorum-sensing systems in Vibrio harveyi. Maximal activity is produced during mid- to late exponential phase when S. typhimurium is grown in the presence of glucose or other preferred carbohydrates. The signal is degraded by the onset of stationary phase or when the carbohydrate is depleted from the medium. Presumably, quorum sensing in S. typhimurium is operational during periods of rapid, nutrient-rich growth. Protein synthesis is required for degradation of the activity, suggesting that a complex regulatory circuitry controls signal production and detection in S. typhimurium. Increased signalling activity is observed if, after growth in the presence of glucose, S. typhimurium is transferred to a high-osmolarity (0.4 M NaCl) or to a low-pH (pH 5.0) environment. Degradation of the signal is induced by conditions of low osmolarity (0.1 M NaCl). High osmolarity and low pH are two conditions encountered by S. typhimurium cells when they undergo the transition to a pathogenic existence inside a host organism, suggesting that quorum sensing may have a role in the regulation of virulence in S. typhimurium.

Escherichia coli and Salmonella typhimurium strains grown in Luria-Bertani medium containing glucose secrete a small soluble heat labile organic molecule that is involved in intercellular communication. The factor is not produced when the strains are grown in Luria-Bertani medium in the absence of glucose. Maximal secretion of the substance occurs in midexponential phase, and the extracellular activity is degraded as the glucose is depleted from the medium or by the onset of stationary phase. Destruction of the signaling molecule in stationary phase indicates that, in contrast to other quorum-sensing systems, quorum sensing in E. coli and S. typhimurium is critical for regulating behavior in the prestationary phase of growth. Our results further suggest that the signaling factor produced by E. coli and S. typhimurium is used to communicate both the cell density and the metabolic potential of the environment. Several laboratory and clinical strains of E. coli and S. typhimurium were screened for production of the signaling molecule, and most strains make it under conditions similar to those shown here for E. coli AB1157 and S. typhimurium LT2. However, we also show that E. coli strain DH5alpha does not make the soluble factor, indicating that this highly domesticated strain has lost the gene(s) or biosynthetic machinery necessary to produce the signaling substance. Implications for the involvement of quorum sensing in pathogenesis are discussed.

Different species of bacteria were tested for production of extracellular autoinducer-like activities that could stimulate the expression of the luminescence genes in Vibrio harveyi. Several species of bacteria, including the pathogens Vibrio cholerae and Vibrio parahaemolyticus, were found to produce such activities. Possible physiological roles for the two V. harveyi detection-response systems and their joint regulation are discussed.

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably beta-hydroxybutyl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyi.

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density-dependent response. Transposon Tn5 insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild-type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins. The constitutive phenotype of LuxO- mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal-response system controlling density-dependent expression of luminescence remains to be identified.

Immense quantities of chitin are catabolized by marine bacteria, and this process involves at least three signal transduction systems in Vibrio furnissii. One system, chemotaxis to chitin oligosaccharides, is probably used to colonize chitin particles. But how do the first few cells find this highly insoluble polysaccharide? The following hypothesis is proposed to answer this question: the bacteria respond to soluble chemo-attractants in exudates from injured organisms. Virtually all chitin-producing organisms also contain glucose and/or trehalose, often at high concentrations such as trehalose in insect hemolymph. Chemotaxis of V. furnissii was therefore studied with a variety of sugars. Fructose, ribose, and glycerol are catabolites but not attractants. The cells exhibit weak constitutive taxis to Glc and GlcNAc. After induction, they show a weak response to galactose but are strongly attracted to the following substrates of the phosphoenolpyruvate:glycose phosphotransferase system (PTS): GlcNAc, trehalose, glucose, sucrose, mannose, and mannitol. There is a rough qualitative but no quantitative correlation between the rate of phosphorylation and the chemotactic response to PTS sugars. Trehalose is especially noteworthy because it is phosphorylated at a very rapid rate by uninduced cells but is not an attractant until the cells are induced. We suggest that unidentified inducible factors link the PTS to chemotaxis.

Chitin turnover is essential for recycling carbon and nitrogen in marine ecosystems. A key step in this process is the adhesion of marine bacteria to chitin-containing particulates. Vibrio species were therefore surveyed for their ability to bind to immobilized carbohydrates, and one, Vibrio furnissii, adhered to glycosides of three sugars, N-acetylglucosamine (the preferred ligand), D-mannose, and D-glucose. A single Ca(2+)-requiring lectin is responsible for binding to the three sugars. Cells adhering to the chitin analogue divided at the same rate as cells in liquid culture. The first progeny of adherent cells continued to bind to the beads, but the population gradually shifted to a large fraction of free swimming cells, a process that may be necessary for colonization. Metabolic energy is required for cell adhesion to the glycosides, and transient (or no) adhesion occurred in incomplete growth media. The results were explained by studying met and pro mutants. Both the initiation and maintenance of lectin-mediated adhesion requires continuous protein synthesis; expression of lectin activity is a major priority of these cells and functions under conditions adequate for minimal protein synthesis, but insufficient to support cell growth. The adhesion/deadhesion apparatus is apparently used to continuously monitor the nutrient status of the environment, i.e. as a nutrient sensorium. In incomplete medium, cells deadhere, presumably to migrate to a more favorable environment.

The adhesion/deadhesion apparatus of the marine bacterium Vibrio furnissii (Yu, C., Lee, A., Bassler, B. L., and Roseman, S. (1991) J. Biol. Chem. 266, 24260-24267) probably catalyzes the first step in colonizing chitin. Evidence is presented here for a second step, chemotaxis to chitin hydrolysis products. V. furnissii swarms toward chitin oligomers (GlcNAc)n, n = 1-6, at initial concentrations as low as 10 microM. A modified capillary assay was used for quantitation; the cells exhibit low level constitutive taxis to GlcNAc but not to the oligosaccharides. A mutant defective in the GlcNAc receptor (IINag of the phosphotransferase system) showed inducible taxis to the oligosaccharides. Two (or more) independently inducible receptors with overlapping specificities recognize (GlcNAc)n, n = 2-4. (GlcNAc)5 and (GlcNAc)6 were inactive in the capillary assay; expression of this receptor(s) apparently require special induction conditions. The (GlcNAc)n, n = 1-4, chemoreceptors of V. furnissii may be the most potent reported for bacteria. L-Amino acids were weak, constitutive attractants; glutamine, not known to be an attractant in other bacteria, was the most effective amino acid. The most potent receptor in Escherichia coli, Tar (aspartate), is not expressed in V. furnissii. The chemotactic responses were greatly affected by growth and induction conditions and the presence of nutrients in the assay media. Taxis to GlcNAc and GlcNAc oligomers was optimally induced by growth in lactate medium containing 0.6 mM sugar, while growth on the sugar per se resulted in poor taxis. Chemotaxis to the sugars increased 2- to 3-fold when the cells were starved. Nutrients in the assay medium, especially compounds that feed into or are part of the Krebs cycle, were potent inhibitors of taxis to the sugars and Gln. With the exception of isocitrate, inhibition of taxis correlated with the rate of oxidation of these compounds. The results suggest a link between catabolism and taxis in this organism, i.e. interactions or "cross-talk" between systems that are regulated by protein phosphorylation (Stock, J. A., Ninfa, A. J., and Stock, A. M. (1989) Microbiol. Rev. 53, 450-490).

Chemotaxis of the marine bacterium Vibrio furnissii to chitin oligosaccharides has been described (Bassler, B. L., Gibbons, P. J., Yu, C., and Roseman, S. (1991) J. Biol. Chem. 266, 24268-24275). Some steps in catabolism of the oligosaccharides are reported here. GlcNAc, (GlcNAc)2, and (GlcNAc)3 are very rapidly consumed by intact cells, about 320 nmol of GlcNAc equivalents/min/mg of protein. (GlcNAc)4 is utilized somewhat more slowly. During these processes, there is virtually no release of hydrolysis products by the cells. The oligosaccharides enter the periplasmic space (via specific porins?) and are hydrolyzed by a unique membrane-bound endoenzyme (chitodextrinase) and an exoenzyme (N-acetyl-beta-glucosaminidase; beta-Glc-NAcidase). The genes encoding these enzymes have been cloned and expressed in Escherichia coli. The chitodextrinase cleaves soluble oligomers, but not chitin, to the di- and trisaccharides, while the periplasmic beta-GlcNAcidase hydrolyzes the GlcNAc termini from the oligomers. The end products in the periplasm, GlcNAc and (GlcNAc)2 (possibly (GlcNAc)3) are catabolized as follows. (a) Disaccharide pathway, A (GlcNAc)2 permease is apparently expressed by Vibrio furnissii. Translocated (GlcNAc)2 is rapidly hydrolyzed by a soluble, cytosolic beta-GlcNAcidase, and the GlcNAc is phosphorylated by an ATP-dependent, constitutive kinase to GlcNAc-6-P. (b) Monosaccharide pathway, Periplasmic GlcNAc is taken up by Enzyme IINag of the phosphoenolpyruvate:glycose phosphotransferase system, yielding GlcNAc-6-P, the common intermediate for both pathways. Finally, GlcNAc-6-P----Ac- + GlcNH2-6-P----Fru-6-P + NH3. (GlcNAc)2 is probably the "true" inducer of the chitin degradative enzymes described in this report and, depending on its concentration in the growth medium, differentially induces the periplasmic and cytosolic beta-GlcNAcidases. The disaccharide pathway appears to be the most important when the cells are confronted with low concentrations of the oligomers (e.g. in chemotaxis swarm plates). The relative activities of the induced enzymes suggest that the rate-limiting steps in oligosaccharide catabolism are the glycosidase activities in the periplasm.