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.     

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.     

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.     

嗜水气单胞菌群体感应信号分子AI-2的细胞外生物 合成及活性检测

【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对LuxS、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的LuxS分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏LuxS一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了LuxS、MtnN-1和MtnN-2蛋白,细胞外LuxS和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,LuxS和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与LuxS共同催化AI-2的细胞外生物合成。     

Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi

Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes were designed. Structural resemblances can be found between thiazolidinediones and well-known furanone type quorum sensing (QS) inhibitors such as N-acylaminofuranones, and/or acyl-homoserine lactone signaling molecules, while dioxazaborocanes structurally resemble previously reported oxazaborolidine derivatives which antagonized autoinducer 2 (AI-2) binding to its receptor. Because of this, we hypothesized that these compounds could affect AI-2 QS in Vibrio harveyi. Although all compounds blocked QS, the thiazolidinediones were the most active AI-2 QS inhibitors, with EC₅₀ values in the low micromolar range. Their mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of V. harveyi QS mutants and by DNA-binding assays with purified LuxR protein. The active compounds neither affected bioluminescence as such nor the production of AI-2. Instead, our results indicate that the thiazolidinediones blocked AI-2 QS in V. harveyi by decreasing the DNA-binding ability of LuxR. In addition, several dioxazaborocanes were found to block AI-2 QS by targeting LuxPQ.     

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.     

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 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.     

Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2

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.     

Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae

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.     

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.     

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。     

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.     

禽致病性大肠杆菌安徽分离株luxS和pfs基因的克隆、表达与细胞外合成AI-2 活性检测

【目的】LuxS/AI-2型密度感应系统存在于革兰氏阴性和阳性菌中,可产生用于细菌种间交流的通用自诱导信号分子AI-2(Autoinducer-2,AI-2),细菌许多生理功能都受此系统的调节。本研究开展对禽致病性大肠杆菌(Avian Pathogenic Escherichia coli,APEC)自诱导信号分子AI-2的检测和建立体外合成、定量的方法,为进一步研究APEC的AI-2调控作用奠定基础。【方法】利用哈维弧菌BB170(Vibrio harveyi BB170)开展对APEC AI-2的检测;利用表达、纯化的LuxS和Pfs在体外催化S-腺苷同型半胱氨酸(Sadenosylhomocysteine,SAH),进行AI-2的体外合成。【结果】APEC能产生自诱导信号分子AI-2;成功表达可用于AI-2合成的可溶性重组蛋白LuxS和Pfs;纯化的重组蛋白LuxS和Pfs与SAH同时作用后,合成了浓度为300μmol/L的AI-2;运用哈维弧菌BB170对合成的AI-2活性检测表明,其活性是阴性对照的700倍。【结论】APEC存在LuxS/AI-2型密度感应系统,APEC的LuxS和Pfs可以在体外催化SAH生成有活性的AI-2分子。本研究为进一步研究APEC的AI-2的调控作用奠定基础。     

Autoinducer-2 bioassay is a qualitative, not quantitative method influenced by glucose

Autoinducer-2 (AI-2) is a cell-to-cell signaling molecule which is thought to be utilized for quorum sensing processes by a variety of prokaryotic species. This molecule is usually detected using a so-called autoinducer bioassay, which relies on the ability of a Vibrio harveyi reporter strain to produce light in response to AI-2. However, as previously reported, the presence of glucose in the sample can inhibit the bioluminescence of the reporter strain, a fact that is often ignored by investigators. Our data suggest that the presence of glucose in concentrations below that required for the inhibition of bioluminescence may lead to incorrect AI-2 readings and produce misleading (false-positive) results. Our findings also suggest that even if all the limitations of this bioassay are considered, the large standard deviation of the method allows only for a qualitative and not quantitative interpretation of the obtained results.     

N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures

Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.