Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi

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.     

Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis

AIMS: To evaluate the effect of Vibrio harveyi strains on the growth rate of the gnotobiotically cultured rotifer Brachionus plicatilis, and to establish whether quorum sensing is involved in the observed phenomena. METHODS AND RESULTS: Gnotobiotic B. plicatilis sensu strictu, obtained by hatching glutaraldehyde-treated amictic eggs, were used as test organisms. Challenge tests were performed with 11 V. harveyi strains and different quorum sensing mutants derived from the V. harveyi BB120 strain. Brominated furanone [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] as a quorum sensing inhibitor was tested in Brachionus challenge tests. Some V. harveyi strains, such as strain BB120, had a significantly negative effect on the Brachionus growth rate. In the challenge test with MM77, an isogenic strain of BB120 in which the two autoinducers (HAI-1 and AI-2) are both inactivated, no negative effect was observed. The effect of single mutants was the same as that observed in the BB120 strain. This indicates that both systems are responsible for the growth-retarding (GR) effect of the BB120 strain towards Brachionus. Moreover, the addition of an exogenous source of HAI-1 or AI-2 could restore the GR effect in the HAI-1 and AI-2 nonproducing mutant MM77. The addition of brominated furanone at a concentration of 2.5 mg l(-1) could neutralize the GR effect of some strains such as BB120 and VH-014. CONCLUSIONS: Two quorum sensing systems in V. harveyi strain BB120 (namely HAI-1 and AI-2-mediated) are necessary for its GR effect on B. plicatilis. With some other V. harveyi strains, however, growth inhibition towards Brachionus does not seem to be related to quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: Interference with the quorum sensing system might help to counteract the GR effect of some V. harveyi strains on Brachionus. However, further studies are needed to demonstrate the positive effect of halogenated furanone in nongnotobiotic Brachionus cultures and eventually, in other segments of the aquaculture industry.     

The ycf27 genes from cyanobacteria and eukaryotic algae: distribution and implications for chloroplast evolution

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxS(M.h.) was able to complement AI-2 production in the Escherichia coli DH5alpha luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions.     

Oxazaborolidine derivatives inducing autoinducer-2 signal transduction in Vibrio harveyi

The bioluminescence of the marine bacterium Vibrio harveyi is controlled by quorum sensing. This effect is mediated by production, accumulation, and auto-detection of the species-specific autoinducer 1 (AI-1), autoinducer 2 (AI-2), and the V. cholerae autoinducer 1 (CAI-1). The V. harveyi AI-2 was recently identified as furanosyl borate diester. We synthesized several oxazaborolidine derivatives that chemically resemble the structure of AI-2. Five oxazaborolidine derivatives (BNO-1 to BNO-5) were tested, however only BNO-1 (3,4-dimethyl-2,5-diphenyl-1,3,2-oxazaborolidine), and BNO-5 (2-butyl-3,4-dimethyl-5-phenyl-1,3,2-oxazaborolidine) strongly induced V. harveyi bioluminescence in V. harveyi mutant (BB170) lacking sensor 1. A dose-dependent relationship between those oxazaborolidine derivatives and bioluminescence induction was observed with this V. harveyi strain (BB170). BNO-1 and BNO-5 did not affect V. harveyi BB886 lacking sensor 2. Using a mutant strain which produces neither AI-1 nor AI-2 (V. harveyi MM77) we showed that the presence of spent medium containing AI-2 is essential for BNO-1 and BNO-5 activity. This effect was similar when introducing the spent medium and the BNOs together or at a 3-h interval. A comparable induction of bioluminescence was observed when using synthetic DPD (pre-AI-2) in the presence of BNO-1 or BNO-5. The mode of action of BNO-1 and BNO-5 on bioluminescence of V. harveyi is of a co-agonist category. BNO-1 and BNO-5 enhanced AI-2 signal transduction only in the presence of AI-2 and only via sensor 2 cascade. BNO-1 and BNO-5 are the first oxazaborolidines reported to affect AI-2 activity. Those derivatives represent a new class of borates which may become prototypes of novel agonists of quorum sensing mediated by AI-2 in V. harveyi.     

Evidence for a signaling system in Helicobacter pylori: detection of a luxS-encoded autoinducer

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 impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and Vibrio harveyi on their virulence towards gnotobiotically cultured Artemia franciscana

Disruption of quorum sensing, bacterial cell-to-cell communication by means of small signal molecules, has been suggested as a new anti-infective strategy for aquaculture. However, data about the impact of quorum sensing on the virulence of aquatic pathogens are scarce. In this study, a model system using gnotobiotically cultured Artemia franciscana was developed in order to determine the impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and V. harveyi on their virulence. Mutations in the autoinducer 2 (AI-2) synthase gene luxS, the AI-2 receptor gene luxP or the response regulator gene luxO of the dual channel quorum sensing system of V. harveyi abolished virulence of the strain towards Artemia. Moreover, the addition of an exogenous source of AI-2 could restore the virulence of an AI-2 non-producing mutant. In contrast, none of the mutations in either the acylated homoserine lactone (AHL)-mediated component of the V. harveyi system or the quorum sensing systems of Ae. hydrophila and V. anguillarum had an impact on virulence of these bacteria towards Artemia. Our results indicate that disruption of quorum sensing could be a good alternative strategy to combat infections caused by V. harveyi.     

Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54

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.     

Possible quorum sensing in the rumen microbial community: detection of quorum-sensing signal molecules from rumen bacteria

The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria. The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules. Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P. ruminicola subsp. ruminicola and R. flavefaciens. These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen.     

Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression

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.     

Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone

The quorum-sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was found to inhibit the swarming motility of Escherichia coli completely at 13 microg cm-2 (also at 20 microg ml-1) but did not inhibit its growth rate at 13-52 microg cm-2 or from 20 to 100 microg ml-1. Swimming was not inhibited by the furanone at 20-40 microg ml-1. In addition, confocal scanning laser microscopy revealed that this furanone at 60 microg ml-1 inhibited the biofilm formation of E. coli, as it decreased its thickness by 55%, reduced the number of water channels and decreased the percentage of live cells by 87%. This suggests that natural furanone may be used as a new method to control bacterial biofilms that does not involve toxicity. Furanone at 10 microg ml-1 also inhibited by 3300-fold the quorum sensing of Vibrio harveyi via autoinducer 1 (AI-1) and inhibited by 5500-fold that of V. harveyi via of autoinducer 2 (AI-2) as well as inhibited by 26-600-fold the quorum sensing of E. coli via AI-2; hence, this furanone is a non-specific intercellular signal antagonist.     

Presence of LuxS/AI-2 based quorum-sensing system in Vibrio mimicus : luxO controls protease activity

Presence of the quorum-sensing regulation system in Vibrio mimicus was investigated. The culture supernatants of V. mimicus strains were found to possess AI-2 autoinducer like activity, and the strains were found to harbor the genes which are homologous to luxS, luxO, and luxR of V. harveyi. These genes of V. harveyi have been shown to be important components of V. harveyi-like quorum-sensing system. The luxO gene homologue known to encode LuxO, the central component of the regulation system, was disrupted, and effects on protease and hemolysin activity were studied. Disruption of luxO gene resulted in the increased protease activity, but the hemolysin activity did not vary considerably.     

Detection of different quorum-sensing signal molecules in a virulent Edwardsiella tarda strain LTB-4

Aims:  The aim of this study was to elucidate the potential quorum-sensing (QS) signal molecules of an emerging pathogen ( Edwardsiella tarda strain LTB-4) of cultured turbot ( Scophthalmus maximus ).
Methods and Results:  A sensitive and rapid double-layer plate method using biosensor strain Agrobacterium tumefaciens KYC55 was developed to detect the N-acylhomoserine lactone (AHL)-related compounds in bacteria. LTB-4 was found to have two QS systems, one was based on the AHLs and the other was based on the autoinducer-2 (AI-2). The AI-2 activity produced by LTB-4 was growth phase dependent and topped at OD₆₀₀ of 1·0. The protocol to detect cholerae autoinducer 1 (CAI-1) activity in bacteria was modified, lowering the background luminescence of biosensor strain Vibrio harveyi JAF375. CAI-1 activity could not be detected in LTB-4.
Conclusion:  Edwardsiella tarda LTB-4 produced at least four kinds of AHLs during its whole growth phase. In comparison with the AHL-inducing QS, AI-2 may be the first predominant signal, functioning at early exponential phase. LTB-4 did not produce any CAI-1 activity.
Significance and Impact of the Study:  Different QS signal molecules of Edw. tarda LTB-4 were clarified by improved bioassays. In contrast to earlier studies detecting two types of AHLs, strain LTB-4 produced at least four kinds of AHLs, which seemed to be C₄-HSL, C₆-HSL, 3-oxo-C₆-HSL and an uncharacterized AHL molecule.     

Assessment of the roles of LuxS, S-ribosyl homocysteine, and autoinducer 2 in cell attachment during biofilm formation by Listeria monocytogenes EGD-e

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5alpha, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10 degrees C, 15 degrees C, 25 degrees C, and 42 degrees C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.     

长双歧杆菌NCC2705群体感应系统信号分子AI-2的检测

目的:用生物学方法检测长双歧杆菌NCC2705是否产生群体感应系统信号分子AI-2。方法:将长双歧杆菌NCC2705不同时间点的培养上清分别加至AI-2特异报告系统哈氏弧菌BB170中,以空白培养基上清为对照,用荧光光度计对哈氏弧菌发光强度进行计量,推测出长双歧杆菌NCC2705上清中是否含有分泌的AI-2,并由此推断AI-2的活性。结果:通过微孔板检测系统对加入长双歧杆菌NCC2705培养上清的哈氏弧菌BB170进行检测,发现双歧杆菌上清的加入增强了哈氏弧菌BB170发出的荧光强度。结论:长双歧杆菌NCC2705中存在依赖于luxS/AI-2的群体感应系统,并能够分泌有活性的AI-2,为进一步研究长双歧杆菌NCC2705AI-2及luxS基因的功能打下基础。     

A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi

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.     

The LuxS-dependent autoinducer AI-2 controls the expression of an ABC transporter that functions in AI-2 uptake in Salmonella typhimurium

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.     

Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing

Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.