Presence of <Emphasis Type="Italic">N</Emphasis>-Acyl Homoserine Lactones in Soil Detected by a Whole-Cell Biosensor and Flow Cytometry

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-P_luxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.     

Visualization of N-acylhomoserine lactone-mediated cell-cell communication between bacteria colonizing the tomato rhizosphere.

Given that a large proportion of the bacteria colonizing the roots of plants is capable of producing N-acyl-L-homoserine lactone (AHL) molecules, it appears likely that these bacterial pheromones may serve as signals for communication between cells of different species. In this study, we have developed and characterized novel Gfp-based monitor strains that allow in situ visualization of AHL-mediated communication between individual cells in the plant rhizosphere. For this purpose, three Gfp-based AHL sensor plasmids that respond to different spectra of AHL molecules were transferred into AHL-negative derivatives of Pseudomonas putida IsoF and Serratia liquefaciens MG1, two strains that are capable of colonizing tomato roots. These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants. Furthermore, we integrated into the chromosome of AHL-negative P. putida strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp. This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces AHL molecules. The results strongly support the view that AHL signal molecules serve as a universal language for communication between the different bacterial populations of the rhizosphere consortium.     

Relevance of N-acyl-L-homoserine lactone production by Yersinia enterocolitica in fresh foods

AIMS: Determination of the food matrix impact on the potential for N-acyl-L-homoserine lactones (AHLs) production by Yersinia enterocolitica. METHODS AND RESULTS: Induction and inhibition of a sensor strain and a fluorescent assay were used to investigate Y. enterocolitica AHL production in artificial media, as well as in different food extracts. All Y. enterocolitica strains tested produced AHLs in artificial media. Thin Layer Chromatography analysis of Y. enterocolitica strains indicated the production of 3-oxo-hexanoyl homoserine lactone and hexanoyl homoserine lactone. Yersinia enterocolitica produced AHL principally in fish and meat extracts. CONCLUSIONS: AHL production by Y. enterocolitica was observed in products of animal origin, but were inhibited by some vegetables extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that quorum sensing systems in Y. enterocolitica is significant in foods but depends upon the type of food. Determination of physiological functions in Y. enterocolitica which are regulated by quorum sensing and their relation to the production of AHLs in foods need to be further assessed.     

Disruption of quorum sensing in seawater abolishes attraction of zoospores of the green alga Ulva to bacterial biofilms

Zoospores of the eukaryotic green seaweed Ulva respond to bacterial N-acylhomoserine lactone (AHL) quorum sensing signal molecules for the selection of surface sites for permanent attachment. In this study we have investigated the production and destruction of AHLs in biofilms of the AHL-producing marine bacterium, Vibrio anguillarum and their stability in seawater. While wild type V. anguillarum NB10 was a strong attractor of zoospores, inactivation of AHL production in this strain by either expressing the recombinant Bacillus lactonase coding gene aiiA, or by mutating the AHL biosynthetic genes, resulted in the abolition of zoospore attraction. In seawater, with a pH of 8.2, the degradation of AHL molecules was temperature-dependent, indicating that the AHLs produced by marine bacterial biofilms have short half-lives. The Ulva zoospores sensed a range of different AHL molecules and in particular more zoospores settled on surfaces releasing AHLs with longer (>six carbons) N-linked acyl chains. However, this finding is likely to be influenced by the differential diffusion rates of AHLs from the experimental surface matrix. Molecules with longer N-acyl chains, such as N-(3-oxodecanoyl)- L-homoserine lactone, diffused more slowly than those with shorter N-acyl chains such as N-(3-hydroxy-hexanoyl)- L-homoserine lactone. Image analysis using GFP-tagged V. anguillarum biofilms revealed that spores settle directly on bacterial cells and in particular on microcolonies which we show are sites of concentrated AHL production.     

The interaction of N-acylhomoserine lactone quorum sensing signaling molecules with biological membranes: implications for inter-kingdom signaling

BACKGROUND: The long chain N-acylhomoserine lactone (AHL) quorum sensing signal molecules released by Pseudomonas aeruginosa have long been known to elicit immunomodulatory effects through a process termed inter-kingdom signaling. However, to date very little is known regarding the exact mechanism of action of these compounds on their eukaryotic targets. METHODOLOGY/PRINCIPAL FINDINGS: The use of the membrane dipole fluorescent sensor di-8-ANEPPS to characterise the interactions of AHL quorum sensing signal molecules, N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL), N-(3-oxododecanoyl)homoserine-L-lactone (3-oxo-C12-HSL) and N-(3-oxodecanoyl) homoserine-L-lactone (3-oxo-C10 HSL) produced by Pseudomonas aeruginosa with model and cellular membranes is reported. The interactions of these AHLs with artificial membranes reveal that each of the compounds is capable of membrane interaction in the micromolar concentration range causing significant modulation of the membrane dipole potential. These interactions fit simple hyperbolic binding models with membrane affinity increasing with acyl chain length. Similar results were obtained with T-lymphocytes providing the evidence that AHLs are capable of direct interaction with the plasma membrane. 3-oxo-C12-HSL interacts with lymphocytes via a cooperative binding model therefore implying the existence of an AHL membrane receptor. The role of cholesterol in the interactions of AHLs with membranes, the significance of modulating cellular dipole potential for receptor conformation and the implications for immune modulation are discussed. CONCLUSIONS/ SIGNIFICANCE: Our observations support previous findings that increasing AHL lipophilicity increases the immunomodulatory activity of these quorum compounds, while providing evidence to suggest membrane interaction plays an important role in quorum sensing and implies a role for membrane microdomains in this process. Finally, our results suggest the existence of a eukaryotic membrane-located system that acts as an AHL receptor.     

A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm

A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gram-negative bacteria. It is based on the conversion of 3-oxo AHLs to their pentafluorobenzyloxime derivatives followed by gas chromatography-mass spectrometry (electron capture-negative ion). The method used [13C16]-N-3-oxo-dodecanoyl homoserine lactone ([13C16]-OdDHL) as the internal standard, and its validity was tested by spiking the supernatant and cell fractions with three levels of 3-oxo AHLs, i.e. 1, 10 and 100 ng per sample. These showed the method to be both sensitive (S/N ratio >10:1 for 1 ng) and accurate. The assay was applied to the biofilm and effluent of a green fluorescent protein (GFP)-expressing strain of Pseudomonas aeruginosa (6294) culture grown in flow cells. Biofilm volume was determined for three replicate flow cells by confocal scanning laser microscopy. OdDHL was detected in the biofilm at 632 +/- 381 microM and the effluent at 14 +/- 3 nM. The biofilm concentration is the highest level so far reported for an AHL in a wild-type bacterial system. The next most abundant 3-oxo AHL in the biofilm and effluent was N-3-oxo-tetradecanoyl homoserine lactone (OtDHL) at 40 +/- 15 microM and 1.5 +/- 0.7 nM respectively. OtDHL is unreported for P. aeruginosa and has an activity equivalent to OdDHL in a lasR bioassay. Two other 3-oxo AHLs were detected at lower concentrations: N3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 microM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).     

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).     

Functional sites and evolutionary connections of acylhomoserine lactone synthases

Acylhomoserine lactone (AHL) synthases act as chemical communication signals or pheromones in Gram-negative bacteria and regulate diverse physiological events in a cell density-dependent manner. The recent crystal structure determination of EsaI, a key enzyme in this pathway, shows that the AHL synthase superfamily members adopt the fold of the N-acetyltransferase superfamily. We suggest, by the identification of intermediate sequences, that the two superfamilies are evolutionarily related. Evolutionary trace analyses of aligned sequences and docking studies have been used to discuss functionally important residues of EsaI homologues.     

Transgenic tomato plants alter quorum sensing in plant growth-promoting rhizobacteria

Two Gram-negative, plant growth-promoting rhizobacteria (PGPRs), denominated as M12 and M14, were classified by 16S rDNA sequencing as Burkholderia graminis species. Both strains were shown to produce a variety of N-acyl-homoserine lactone (AHL) quorum sensing (QS) signalling molecules. The involvement of these molecules in plant growth promotion and the induction of protection against salt stress was examined. AHL production was evaluated in vitro by thin-layer chromatography using AHL biosensors, and the identity of the AHLs produced was determined by liquid chromatography-tandem mass spectrometry. The in situ production of AHLs by M12 and M14 in the rhizosphere of Arabidopsis thaliana plants was detected by co-inoculation with green fluorescent protein-based biosensor strains and confocal laser scanning microscopy. To determine whether plant growth promotion and protection against salt stress were mediated by QS, these PGPRs were assayed on wild-type tomato plants, as well as their corresponding transgenics expressing YenI (short-chain AHL producers) and LasI (long-chain AHL producers). In wild-type tomato plants, only M12 promoted plant growth, and this effect disappeared in both transgenic lines. In contrast, M14 did not promote growth in wild-type tomatoes, but did so in the LasI transgenic line. Resistance to salt stress was induced by M14 in wild-type tomato, but this effect disappeared in both transgenic lines. The strain M12, however, did not induce salt resistance in wild-type tomato, but did so in LasI tomato plants. These results reveal that AHL QS signalling molecules mediate the ability of both PGPR strains M12 and M14 to promote plant growth and to induce protection against salt stress.     

Multiple N-Acyl Homoserine Lactone Signals of Rhizobium leguminosarum Are Synthesized in a Distinct Temporal Pattern

A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites. The nitrogen-fixing symbiont Rhizobium leguminosarum reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules. We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by R. leguminosarum cells with and without the symbiosis plasmid pRL1JI. Cells containing pRL1JI were found to produce three predominant signals. In decreasing order of abundance, these were N-(3-oxo)octanoyl homoserine lactone [(3-O)C(8)HSL], N-octanoyl homoserine lactone, and N-hexanoyl homoserine lactone. Cells without pRL1JI produced only two major signals, N-(3-hydroxy-7-cis)tetradecanoyl homoserine lactone [(3-OH)C(14:1)HSL] and (3-O)C(8)HSL. Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms. While (3-O)C(8)HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRL1JI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid. None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C(14:1)HSL did activate synthesis of the three AHLs made by cells containing pRL1JI. These results indicate that the synthesis of multiple AHLs in R. leguminosarum is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C(14:1)HSL can supersede these controls in pRL1JI-containing cells. This work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems.     

Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.     

Type 1 and type 2 responses to Leishmania major

Pseudomonas aeruginosa and Burkholderia cepacia cause destructive lung disease in cystic fibrosis (CF) patients. Both pathogens employ 'quorum sensing', i.e. cell-to-cell communication, via diffusible N-acyl-L-homoserine lactone (AHL) signal molecules, to regulate the production of a number of virulence determinants in vitro. However, to date, evidence that quorum sensing systems are functional and play a role in vivo is lacking. This study presents the first direct evidence for the presence of AHLs in CF sputum. A total of 42 samples from 25 CF patients were analysed using lux-based Escherichia coli AHL biosensors. AHLs were detected in sputum from patients colonised by P. aeruginosa or B. cepacia but not Staphylococcus aureus. Furthermore, using liquid chromatography-mass spectrometry and thin layer chromatography, we confirmed the presence of N-hexanoylhomoserine lactone and N-(3-oxododecanoyl)homoserine lactone respectively in sputum samples from patients colonised by P. aeruginosa.     

N‐ACYL HOMOSERINE LACTONe LACTONASE,AiiA, INACTIVATION OF QUORUM‐SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE1

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N‐acyl homoserine lactone (AHL) bacterial quorum‐sensing (QS) signals and alter QS‐regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal‐mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR‐stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal‐mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL‐producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.     

华癸根瘤菌中自体诱导物的初步研究

群体感应 (Quorumsensing)是细菌通过产生可扩散的小分子量自体诱导物信号分子感知细胞群体密度变化 ,进行基因表达调控的生理行为。将根癌土壤杆菌 (Agrobacteriumtumefaciens)构建为超量表达群体感应调节蛋白TraR的检测菌株JZA1,试验证明该检测菌株能检测纳摩尔浓度的自体诱导物 ,利用该菌株对 3株不同华癸根瘤菌(Mesorhizobiumhuakuii)进行自体诱导物活性检测 ,发现该 3株华癸根瘤菌均能产生自体诱导物 ,其表达量与菌体密度成正相关 ,但 3株菌在相同培养条件下自体诱导物的表达量存在差异 ,结果表明自体诱导物在种内水平上存在一定的多样性 ;同时发现高pH条件能大大降低自体诱导物的稳定性 ,为进一步研究群体感应调节在共生固氮上的作用提供理论及实践依据     

Antibody catalyzed hydrolysis of a quorum sensing signal found in Gram-negative bacteria

N-(3-Oxo-acyl) homoserine lactones are used by Gram-negative bacteria to signal the establishment of specific population densities and coordinate population-wide gene expression. Herein we report the antibody-catalyzed hydrolysis of N-(3-oxo-acyl) homoserine lactone (AHL) using a reactive immunization strategy with a squaric monoester monoamide hapten. Kinetic analysis of the most efficient antibody revealed a modest k(cat), with AHL hydrolysis competitively inhibited by original squaric monoester monoamide hapten. These studies suggest that antibody catalysis could provide a new avenue for blocking quorum sensing in bacteria.     

Use of a Whole-Cell Biosensor and Flow Cytometry to Detect AHL Production by an Indigenous Soil Community During Decomposition of Litter

Quorum sensing, mediated by acylated homoserine lactones (AHLs), is well described for pure culture bacteria, but few studies report detection of AHL compounds in natural bacterial habitats. In this study, we detect AHL production during a degradation process in soil by use of whole-cell biosensor technology and flow cytometry analysis. An indigenous soil bacterium, belonging to the family of Enterobacteriaceae, was isolated and transformed with a low-copy plasmid harboring a gene encoding an unstable variant of the green fluorescent protein (gfpASV) fused to the AHL-regulated P_luxI promoter originating from Vibrio fischeri. This resulted in a whole-cell biosensor, responding to the presence of AHL compounds. The biosensor was introduced to compost soil microcosms amended with nettle leaves. After 3 days of incubation, cells were extracted and analyzed by flow cytometry. All microcosms contained induced biosensors. From these microcosms, AHL producers were isolated and further identified as species previously shown to produce AHLs. The results demonstrate that AHL compounds are produced during degradation of litter in soil, indicating the presence of AHL-mediated quorum sensing in this environment.