Detection of quorum sensing signal molecules in the family Vibrionaceae

Aims: The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N‐acyl‐homoserine lactone (AHL), the autoinducer‐2 (AI‐2) and the cholerae autoinducer‐1‐like (CAI‐1‐like) molecules in 25 Vibrionaceae strains. Methods and Results: The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty‐one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin‐layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI‐2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI‐1‐like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. Conclusions: The results indicated that the QS signal molecules, especially AHL and AI‐2 molecules, were widespread in the family Vibrionaceae. Significance and Impact of the Study: In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.     

Contribution of quorum‐sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

Aims: To investigate roles of quorum‐sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin‐resistant variant (hereinafter DR1R). Methods and Results: The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane‐degradation and biofilm‐formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm‐formation and hexadecane‐biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild‐type cell supernatant and exogenous C₁₂‐AHL. Conclusions: The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. Significance and Impact of the Study: This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.     

Investigating a possible role for the bacterial signal molecules N‐acylhomoserine lactones in Balanus improvisus cyprid settlement

Increased settlement on bacterial biofilms has been demonstrated for a number of marine invertebrate larvae, but the nature of the cue(s) responsible is not well understood. We tested the hypothesis that the bay barnacle Balanus improvisus utilizes the bacterial signal molecules N‐acylhomoserine lactones (AHLs) as a cue for the selection of sites for permanent attachment. Single species biofilms of the AHL‐producing bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 were attractive to settling cypris larvae of B. improvisus. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL‐degrading gene (aiiA), the ability of the bacteria to attract cyprids was abolished. In addition, cyprids actively explored biofilms of E. coli expressing the recombinant AHL synthase genes luxI from Vibrio fischeri (3‐oxo‐C6‐HSL), rhlI from Pseudomonas aeruginosa (C4‐HSL/C6‐HSL), vanI from V. anguillarum (3‐oxo‐C10‐HSL) and sulI from Sulfitobacter sp. BR1 (C4‐HSL, 3‐hydroxy‐C6‐HSL, C8‐HSL and 3‐hydroxy‐C10‐HSL), but not E. coli that did not produce AHLs. Finally, synthetic AHLs (C8‐HSL, 3‐oxo‐C10‐HSL and C12‐HSL) at concentrations similar to those found within natural biofilms (5 μm ) resulted in increased cyprid settlement. Thus, B. improvisus cypris exploration of and settlement on biofilms appears to be mediated by AHL‐signalling bacteria in the laboratory. This adds to our understanding of how quorum sensing inhibition may be used as for biofouling control. Nonetheless, the significance of our results for larvae settling naturally in the field, and the mechanisms that underlay the observed responses to AHLs, is as yet unknown.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.     

SPORE RELEASE IN ACROCHAETIUM SP. (RHODOPHYTA) IS BACTERIALLY CONTROLLED1

The facultative red algal epiphyte Acrochaetium sp. liberated spores preferentially and recruited more successfully in laboratory cultures when its host Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira was present. The same effect was also induced by cell‐free medium from G. chilensis, suggesting it contained a molecular signal. Antibiotics prevented spore release in Acrochaetium sp., even when G. chilensis was present, suggesting a prokaryotic origin of the signal. Simultaneous application of N‐butyl‐homoserine‐lactone (BHL) restored the spore‐release capacity, which demonstrated that spore release was not directly inhibited by the antibiotics and indicated that bacterially generated N‐acyl‐homoserine‐lactones (AHLs) regulate spore release. An involvement of AHL was further indicated by the fact that two different halofuranone inhibitors of AHL receptors also inhibited spore release when they were applied at relatively low concentrations. Of seven different AHLs tested, only BHL induced the effect. However, BHL was only active at relatively high concentrations (100 μM), and it was not detected in spore‐release‐inducing medium of G. chilensis. Another water‐soluble AHL or an AHL structure analog is therefore probably the active compound in G. chilensis cultures. The data presented demonstrate that life cycle completion in Acrochaetium sp. strongly depends on bacteria, which are not always present in sufficient numbers on the alga itself. Exogenous bacteria that are associated with G. chilensis or with other potential substrates may therefore trigger timely spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL is reached. This first finding of AHL perception in a red alga confirms that AHL signalling is more widespread among eukaryotes than was thought until recently. However, spore release of a second red alga, Sahlingia subintegra (Rosenv.) Kornmann, was unaffected by AHL, and the reaction observed is therefore not universal.     

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.     

AmiE,a Novel N-Acylhomoserine Lactone Acylase Belonging to the Amidase Family,from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24

Many Gram-negative bacteria use N-acyl-l-homoserine lactones (AHLs) as quorum-sensing signal molecules. We have reported that Acinetobacter strains isolated from activated sludge have AHL-degrading activity. In this study, we cloned the amiE gene as an AHL-degradative gene from the genomic library of Acinetobacter sp. strain Ooi24. High-performance liquid chromatography analysis revealed that AmiE functions as an AHL acylase, which hydrolyzes the amide bond of AHL. AmiE showed a high level of degrading activity against AHLs with long acyl chains but no activity against AHLs with acyl chains shorter than eight carbons. AmiE showed homology with a member of the amidases (EC 3.5.1.4) but not with any known AHL acylase enzymes. An amino acid sequence of AmiE from Ooi24 showed greater than 99% identities with uncharacterized proteins from Acinetobacter ursingii CIP 107286 and Acinetobacter sp. strain CIP 102129, but it was not found in the draft or complete genome sequences of other Acinetobacter strains. The presence of transposase-like genes around the amiE genes of these three Acinetobacter strains suggests that amiE is transferred by a putative transposon. Furthermore, the expression of AmiE in Pseudomonas aeruginosa PAO1 reduced AHL accumulation and elastase activity, which were regulated by AHL-mediated quorum sensing.     

Cross-kingdom signalling: exploitation of bacterial quorum sensing molecules by the green seaweed Ulva

The green seaweed Ulva has been shown to detect signal molecules produced by bacteria. Biofilms that release N-acylhomoserine lactones (AHLs) attract zoospores--the motile reproductive stages of Ulva. The evidence for AHL involvement is based on several independent lines of evidence, including the observation that zoospores are attracted to wild-type bacteria that produce AHLs but are not attracted to mutants that do not produce signal molecules. Synthetic AHL also attracts zoospores and the attraction is lost in the presence of autoinducer inactivation (AiiA) protein. The mechanism of attraction is not chemotactic but involves chemokinesis. When zoospores detect AHLs, the swimming rate is reduced and this results in accumulation of cells at the source of the AHL. It has been demonstrated that the detection of AHLs results in calcium influx into the zoospore. This is the first example of a calcium signalling event in a eukaryote in response to bacterial quorum sensing molecules. The role of AHLs in the ecology of Ulva is discussed. It is probable that AHLs act as cues for the settlement of zoospores, rather than being directly involved as a signalling mechanism.     

The quorum sensing molecule N-acyl homoserine lactone produced by Acinetobacter baumannii displays antibacterial and anticancer properties

Secretory N-acyl homoserine lactones (AHLs) mediate quorum sensing (QS) in bacteria. AHLs are shown to be inhibitory for an unrelated group of bacteria and might mimic host signalling elements, thereby subverting the regulatory events in host cells. This study investigated the AHL produced by Acinetobacter baumannii and analysed its effect on other bacterial species and mammalian cells. Chemically characterized AHL had an m/z value of 325 with a molecular formula C₁₈H₃₁NO₄ and showed its inhibitory potential against Staphylococcus aureus. Molecular docking studies identified D-alanine-D-alanine synthetase A, a cell wall synthesizing enzyme of S. aureus having a strong binding affinity towards AHL. Electron microscopy showed the disruption and sloughing off of the S. aureus cell wall when treated with AHL. In vitro experiments revealed that this bacteriostatic AHL showed time-dependent activity and induced apoptosis in cancer cell lines. This compound could be a potential structural backbone for constructing new AHL analogues against S. aureus. The findings emphasize the need to re-evaluate all previously characterized AHLs for any additional new biological functions other than QS.     

Detection of Homoserine Lactone-Like Quorum Sensing Molecules in Bradyrhizobium Strains

One hundred and forty-two Bradyrhizobium strains were screened for their ability to produce N-acyl homoserine lactone-like molecules (AHLs) by using an Agrobacterium tumefaciens biosensor strain containing a traI-lacZ fusion. Approximately 22% (31 of 142) of the tested strains produced AHLs that induced moderate to elevated β-galactosidase activity levels in the biosensor strain. Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were both shown to produce AHLs. Age of culture, and media composition were each shown to influence production of AHL(s), with greater production occurring in 2 day-old cultures grown in rich media. Reverse-phase high-performance liquid chromatography and thin-layer chromatography analyses indicated that the B. japonicum strain USDA 290 produced at least two types of AHLs. Our results indicate that the production AHL-like autoinducers is widespread among both B. japonicum and B. elkanii strains.     

D‐dopachrome tautomerase in adipose tissue inflammation and wound repair

D‐dopachrome tautomerase (D‐DT/MIF‐2) is a member of the macrophage migration inhibitory factor (MIF) cytokine superfamily, and a close structural homolog of MIF. MIF and D‐DT have been reported to be involved in obesity, but there is little known about the regulation of D‐DT in adipose tissue inflammation and wound healing. Subcutaneous adipose tissue was collected from 54 healthy donors and 28 donors with acutely inflamed wounds undergoing wound debridement. In addition, epididymal fat pads of mice were injected with lipopolysaccharide to study receptor expression and cell migration in vivo. D‐DT protein levels and mRNA expression were significantly decreased in subcutaneous adipose tissue adjacent to acutely inflamed wounds. D‐DT improved fibroblast viability and increased proliferation in vitro. While D‐DT alone did not have a significant effect on in vitro fibroblast wound healing, simultaneous addition of neutralizing MIF antibody resulted in a significant improvement of fibroblast wound healing. Interestingly, expression of the MIF and D‐DT receptor CD74 was down‐regulated while the MIF receptors CXCR2 and CXCR4 were up‐regulated primarily on macrophages indicating that the MIF‐CXCR2/4 axis may promote recruitment of inflammatory cells into adipose tissue. Our results describe a reciprocal role of D‐DT to MIF in inflamed adipose tissue, and indicate that D‐DT may be beneficial in wound repair by improving fibroblast survival and proliferation.     

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell–cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as β-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound β-glucosidase and leucine-aminopeptidase activities significantly increased in 4%–68% of strains but decreased in 0%–21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.     

Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria

Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N‐acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL‐mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram‐negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid‐type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194‐amino‐acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF‐mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c‐di‐GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens.     

N-acyl-l-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge

The role of intercellular signalling in the regulation of genes and phenotypes in a broad range of bacterial species is now firmly established. In contrast, the impact of intercellular signalling on microbial community parameters, such as species diversity and function, is less well understood. In this study the role of N-acyl-l-homoserine lactones (AHLs) in microbial community dynamics in an industrial wastewater treatment system is addressed. Seven proteobacterial strains producing compounds with AHL-like activity were isolated from the treatment plant. Three of these belong to genera with no previously identified AHL producing species. Addition of AHLs at 2 micro M to sludge samples generated changes in both community function (phenol degradation) and composition as determined by length heterogeneity PCR and denaturing gradient gel electrophoresis. Phenol degradation was more stable as a result of the AHL augmentation. A dominant functional member of the Thauera genus was transiently supplanted by a member of the Comomonas genus in response to AHL addition. This suggests that AHLs can play a role in mediating microbial community parameters and has implications for ecosystem function and industrial wastewater treatment.     

Encapsulated fusion protein confers “sense and respond” activity to chitosan–alginate capsules to manipulate bacterial quorum sensing

We demonstrate that “nanofactory”‐loaded biopolymer capsules placed in the midst of a bacterial population can direct bacterial communication. Quorum sensing (QS) is a process by which bacteria communicate through small‐molecules, such as autoinducer‐2 (AI‐2), leading to collective behaviors such as virulence and biofilm formation. In our approach, a “nanofactory” construct is created, which comprises an antibody complexed with a fusion protein that produces AI‐2. These nanofactories are entrapped within capsules formed by electrostatic complexation of cationic (chitosan) and anionic (sodium alginate) biopolymers. The chitosan capsule shell is crosslinked by tripolyphosphate (TPP) to confer structural integrity. The capsule shell is impermeable to the encapsulated nanofactories, but freely permeable to small molecules. In turn, the capsules are able to take in substrates from the external medium via diffusion, and convert these via the nanofactories into AI‐2, which then diffuses out. The exported AI‐2 is shown to stimulate QS responses in vicinal Escherichia coli. Directing bacterial population behavior has potential applications in next‐generation antimicrobial therapy and pathogen detection. We also envision such capsules to be akin to artificial “cells” that can participate in native biological signaling and communicate in real‐time with the human microbiome. Through such interaction capabilities, these “cells” may sense the health of the microbiome, and direct its function in a desired, host‐friendly manner. Biotechnol. Bioeng. 2013; 110: 552–562. © 2012 Wiley Periodicals, Inc.