Diversity and functional analysis of luxS genes in Vibrios from marine sponges Mycale laxissima and Ircinia strobilina

Sponges harbor highly diverse and dense microbial communities, providing an environment in which bacterial signaling may be important. Quorum sensing (QS) is a cell density-dependent signaling process that bacteria employ to coordinate and regulate their gene expression. Previous studies have found that bacteria isolated from sponges are able to produce acyl-homoserine lactones (AHLs), an important class of QS molecules found in proteobacteria. Autoinducer-2 (AI-2) is a second class of QS molecule, and is considered to be an interspecies signal. However, AI-2 signaling has not been reported in sponge bacterial symbionts. In this study, degenerate primers were designed based on known Vibrio luxS sequences to amplify the luxS genes encoding AI-2 synthases of several Vibrio isolates from marine sponges Mycale laxissima and Ircinia strobilina. All the vibrios isolated from these two sponges had luxS genes and were able to produce signals with AI-2 activity as detected using a biological reporter. A novel group of luxS sequences was found, thus extending the known diversity of luxS genes. One isolate was chosen for further analysis of its luxS gene by expression of the gene in Escherichia coli DH5α and by characterization of the profile of AI-2 activity. This work provides the first information about luxS genes and AI-2 activity in sponge-associated bacterial communities.     

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.     

Inhibition of Pseudomonas aeruginosa quorum sensing by AI-2 analogs

Autoinducer-2 (AI-2) has been suggested to serve as a universal interspecies quorum sensing signaling molecule. We have synthesized a set of AI-2 analogs with small incremental changes in alkyl substitution on C-2 and evaluated them for their agonistic and antagonistic potential as quorum sensing (QS) attenuators in two different bacterial species: Pseudomonas aeruginosa and Vibrio harveyi. Unexpectedly, several of the analogs were found to function as synergistic QS agonists in V. harveyi, while two of these analogs inhibit QS in P. aeruginosa.     

Quorum Sensing Signal Production and Microbial Interactions in a Polymicrobial Disease of Corals and the Coral Surface Mucopolysaccharide Layer

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.     

Role of N-acyl homoserine lactone (AHL)-based quorum sensing (QS) in aerobic sludge granulation

N-acyl homoserine lactone (AHL)-based quorum sensing (QS) has been recognized to play an important role in the formation of biofilm. However, aerobic granular sludge is considered as a special biofilm, and its biological implication and role of AHL-based QS still remain unclear. This study investigated the role of AHL-based QS in aerobic granulation. Results showed that AHLs were necessary to the typical aerobic granulation, and AHL-associated coordination of bacteria in sludge aggregation was sludge density dependent only when it reached a threshold of 1.010 g/mL; AHL-based QS was activated to regulate aerobic granulation. Furthermore, a quorum quenching method was firstly adopted to investigate the role of AHLs in aerobic granules. Results showed inhibition of AHL by acylase that reduced the AHL content in aerobic granules and further weakened its attachment potential, which proved that AHLs play an important role in the formation of aerobic granules. Additionally, the assay of quorum quenching not only proved that AHL-based QS could regulate EPS production but also provided additional evidence for the role of AHLs in aerobic granulation by regulating EPS content and its component proportion.     

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.     

Rational design of ‘controller cells’ to manipulate protein and phenotype expression

Coordination between cell populations via prevailing metabolic cues has been noted as a promising approach to connect synthetic devices and drive phenotypic or product outcomes. However, there has been little progress in developing ‘controller cells’ to modulate metabolic cues and guide these systems. In this work, we developed ‘controller cells’ that manipulate the molecular connection between cells by modulating the bacterial signal molecule, autoinducer-2, that is secreted as a quorum sensing (QS) signal by many bacterial species. Specifically, we have engineered Escherichia coli to overexpress components responsible for autoinducer uptake (lsrACDB), phosphorylation (lsrK), and degradation (lsrFG), thereby attenuating cell–cell communication among populations. Further, we developed a simple mathematical model that recapitulates experimental data and characterizes the dynamic balance among the various uptake mechanisms. This study revealed two controller ‘knobs’ that serve to increase AI-2 uptake: overexpression of the AI-2 transporter, LsrACDB, which controls removal of extracellular AI-2, and overexpression of the AI-2 kinase, LsrK, which increases the net uptake rate by limiting secretion of AI-2 back into the extracellular environment. We find that the overexpression of lsrACDBFG results in an extraordinarily high AI-2 uptake rate that is capable of completely silencing QS-mediated gene expression among wild-type cells. We demonstrate utility by modulating naturally occurring processes of chemotaxis and biofilm formation. We envision that ‘controller cells’ that modulate bacterial behavior by manipulating molecular communication, will find use in a variety of applications, particularly those employing natural or synthetic bacterial consortia.     

Detection of Quorum Sensing Signal Molecules in Edwardsiella ictaluri Ei-151

Edwardsiella ictaluri is a Gram-negative pathogenic bacterium in the family Enterobacteriaceae that causes enteric septicemia of catfish, which has become a significant problem in the aquaculture of striped catfish (Pangasianodon hypophthalmus) in Vietnam. In this study, a bacterium designated as Ei-151 was isolated from diseased striped catfish and proved to be virulent. Based on 16S rDNA sequencing and phenotypic tests, the pathogenic bacterium was identified as Edw. ictaluri. The presence of quorum sensing signal molecules in Edw. ictaluri Ei-151 was detected with different biosensor strains. The results showed that Ei-151 produced at least three kinds of acylated homoserine lactone (AHL) signal molecules as detected with the biosensor Agrobacterium tumefaciens KYC55, and the AHLs fingerprint was similar to that of Edw. tarda. During its entire growth, the levels of AHLs and autoinducer-2 produced by Ei-151 peaked at the stationary phase (OD₆₀₀ 1.8), which suggested that both of them may function at the stationary phase. No Cholerae autoinducer-1-like activity (including Edw. ictaluri LMG7860^T) was detected.     

Multiplicity of Quorum Quenching Enzymes: A Potential Mechanism to Limit Quorum Sensing Bacterial Population

Bacteria express certain of their characteristics especially, pathogenicity factors at high cell densities. The process is termed as quorum sensing (QS). QS operates via signal molecules such as acylhomoserine lactones (AHLs). Other bacteria inhibit QS through the inactivation of AHL signals by producing enzymes like AHL-lactonases and -acylases. Comparative genomic analysis has revealed the multiplicity of genes for AHL lactonases (up to 12 copies per genome) among Bacillus spp. and that of AHL-acylases (up to 5 copies per genome) among Pseudomonas spp. This genetic evolution can be envisaged to enable host to withstand the attacks from bacterial population, which regulates its functioning through QS.     

The Kiwifruit Emerging Pathogen Pseudomonas syringae pv. actinidiae Does Not Produce AHLs but Possesses Three LuxR Solos

Pseudomonas syringae pv. actinidiae (Psa) is an emerging phytopathogen causing bacterial canker disease in kiwifruit plants worldwide. Quorum sensing (QS) gene regulation plays important roles in many different bacterial plant pathogens. In this study we analyzed the presence and possible role of N-acyl homoserine lactone (AHL) quorum sensing in Psa. It was established that Psa does not produce AHLs and that a typical complete LuxI/R QS system is absent in Psa strains. Psa however possesses three putative luxR solos designated here as PsaR1, PsaR2 and PsaR3. PsaR2 belongs to the sub-family of LuxR solos present in many plant associated bacteria (PAB) that binds and responds to yet unknown plant signal molecules. PsaR1 and PsaR3 are highly similar to LuxRs which bind AHLs and are part of the canonical LuxI/R AHL QS systems. Mutation in all the three luxR solos of Psa showed reduction of in planta survival and also showed additive effect if more than one solo was inactivated in double mutants. Gene promoter analysis revealed that the three solos are not auto-regulated and investigated their possible role in several bacterial phenotypes.     

Type 2 Quorum Sensing Monitoring,Inhibition and Biofilm Formation in Marine Microrganisms

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 μM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 μM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.     

N-acylhomoserine lactone-degrading bacteria isolated from hatchery bivalve larval cultures

Quorum sensing (QS) systems, which depend on N-acylhomoserine lactone (AHL) signal molecules, mediate the production of virulence factors in many pathogenic microorganisms. One hundred and forty-six bacterial strains, isolated from a bivalve hatchery, were screened for their capacity to degrade five synthetic AHLs [N-butyryl-dl-homoserine lactone (C₄-HSL), N-hexanoyl-dl-homoserine lactone (C₆-HSL), N-octanoyl-dl-homoserine lactone (C₈-HSL), N-decanoyl-dl-homoserine lactone (C₁₀-HSL) and N-dodecanoyl-dl-homoserine lactone (C₁₂-HSL)] using well diffusion agar-plate assays with three biosensors, Chromobacterium violaceum CV026, C. violaceum VIR07 and Agrobacterium tumefaciens NTL4 (pZLR4). The results of these assays led to our choosing four strains (PP2-67, PP2-459, PP2-644 and PP2-663) that were able to degrade all five synthetic AHLs, thus showing a wide spectrum of quorum quenching (QQ) activity. We subsequently confirmed and measured the QQ activity of the four strains by high-performance liquid chromatography plus mass-spectrometry analysis (HPLC–MS). One of the strains which showed the highest AHL-degrading activity, PP2-459, identified as being a member of the genus Thalassomonas was chosen for further study. Finally, using thin-layer chromatography (TLC), we went on to confirm this strain's capacity to degrade the AHLs produced by other non-pathogenic and pathogenic bacteria not taxonomically related.     

细菌群体感应信号分子——酰基高丝氨酸内酯的检测

革兰氏阴性菌根据信号分子N-酰基高丝氨酸内酯(AHLs)的浓度可以监测周围环境中自身或其他细菌的数量变化,当信号分子达到一定浓度阈值时,能启动相关基因的表达来适应环境的变化,这一调控系统被称为细菌的群体感应(quorumsensing,QS)系统。快速简便而有效地检测细菌是否以及产生何种信号分子成为深入研究和了解细菌群体感应的重要手段。现对信号分子AHLs敏感的用于检测不同的信号分子AHLs的微生物传感菌进行综述,并对其检测能力进行了讨论。     

Developing next generation antimicrobials by intercepting AI-2 mediated quorum sensing

Bacteria have been evolving antibiotic resistance since their discovery in the early twentieth century. Most new antibiotics are derivatives of older generations and there are now bacteria that are virtually resistant to almost all antibiotics. This poses a global threat to human health and has been classified as a clinical “super-challenge”, which has necessitated research into new antimicrobials that inhibit bacterial virulence while minimizing selective pressures that lead to the emergence of resistant strains. Quorum sensing (QS), the process of population dependent bacterial cell-cell signaling, can accelerate bacterial virulence and is an increasingly interesting target for developing next generation antimicrobials. Most QS inhibitors target species-specific signals, such as acylhomoserine lactones (AHLs) and oligopeptides. Methodologies for intercepting the cross-species signal, autoinducer-2 (AI-2), have only recently emerged. We review these strategies to prevent the relay of the AI-2 signal amongst pathogens, including Escherichia coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae and Pseudomonas aeruginosa. Inhibition mechanisms are categorized based on the target (i.e., enzymes for signal generation, the signal molecule itself, or the various components of the signal transduction process). The universal nature of the AI-2 signal imparts on its inhibitors the potential for broad spectrum use.     

Production of N-acyl Homoserine Lactones and Virulence Factors of Waterborne Aeromonas hydrophila

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules and some virulence factors, including hemolysins, proteases, extracellular nucleases production and cytotoxicity by waterborne Aeromonas hydrophila. A total of 24 strains isolated from fresh-water or diseased fish were used in the study. The majority A.hydrophila strains produce two AHL molecules (21/24), one is N-butanoyl homoserine lactone (BHL), and the other is N-hexanoyl homoserine lactone (HHL) according to thin-layer chromatography analysis. Among the virulence factors tested, more than 83 % of the isolates produced β haemolysin when inoculated on sheep blood agar, only 50 % of the isolates displayed DNase activity, 75 % of the isolates shown proteolytic activity on skimmed milk plate, and cytotoxic activity was detected in 20 of 24 of the isolates. The strains producing AHLs possessed one or more virulence factors. In conclusion, the production of quorum sensing signal molecules is common among the strains that we examined, and there seems to some relationships between quorum sensing signal production and virulence factors in A. hydrophila.     

Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

Background

Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors.

Methods and Results

Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C₁₂HSL and C₄HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C₄HSL. F7 also showed antagonistic activity against 3-oxo-C₁₂HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract.

Conclusions

This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.     

The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules

Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C₁₂-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.     

Quorum sensing by <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones is not required for <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during growth with organic particles in lake water microcosms

It was investigated whether quorum sensing (QS) mediated by N-acylhomoserine lactones (AHLs) was important for heterotrophic bacteria from the littoral zone of the oligotrophic Lake Constance for growth with organic particles. More than 900 colonies from lake water microcosms with artificial organic aggregates consisting of autoclaved unicellular algae embedded in agarose beads were screened for AHL-production. AHL-producing bacteria of the genus Aeromonas enriched in the microcosms but AHLs could not be detected in any microcosm. To test for a potential function of AHL-mediated QS, growth experiments with the wild type and an AHL-deficient mutant of Aeromonas hydrophila in lake water microcosms were performed. Growth of both strains did not differ in single cultures and showed no mutual influence in co-cultures. In co-cultures with a competitor bacterium belonging to the Cytophaga–Flavobacterium group, growth of both A. hydrophila strains was reduced while growth of the competitor bacterium was not affected. Exogenous AHL-addition did not influence growth of the Aeromonas strains in any microcosm experiment. These results showed that AHL-mediated QS was not required for A. hydrophila during colonization and degradation of organic particles in lake water microcosms, suggesting that cell–cell signalling of heterotrophic bacteria in oligotrophic waters relies on novel signal molecules.     

Molecular evidence of digestion and absorption of epibiotic bacterial community by deep-sea crab Shinkaia crosnieri

The hydrothermal vent crab Shinkaia crosnieri is considered to obtain nutrition from the epibiotic bacteria found on the setae, but previous studies have not shown how nutrients can be transferred from the epibionts to the host. In this study, microscopic observations of S. crosnieri intestinal components detected autofluorescent setae fragments and pigmentation derived from the digestion of epibionts in a dye-stained epibiont tracer experiment. An in vitro digestion experiment with epibiotic populations using an intestinal extract demonstrated the degradation of epibiotic cells by digestive enzymes. A phylogenetic analysis showed that many of the bacterial 16S ribosomal RNA gene sequences obtained from the intestine were closely related to the sequences of the epibionts, thus they were probably derived from the epibionts. A stable isotope tracer experiment also indicated that ¹³C assimilated by the epibionts provided a carbon (nutrition) source for the host. Both activity measurements and isotope studies showed that chemosynthetic metabolism by the gut microbial components were inactive. Together with the feeding behaviour of living S. crosnieri, these results indicate that S. crosnieri ingests the epibionts using maxillipeds and assimilates them via its digestive organs as a nutrient source. The results of this study elucidate the mechanism of nutritional transfer in ectosymbiosis between chemosynthetic bacteria and deep-sea invertebrates.