Specificity of acyl-homoserine lactone synthases examined by mass spectrometry

Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.     

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.     

Plants genetically modified to produce N-acylhomoserine lactones communicate with bacteria.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.     

Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors

Many Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. AHL QS has been the subject of extensive investigation in the last decade and has become a paradigm for bacterial intercellular signaling. Research in AHL QS has been considerably aided by simple methods devised to detect AHLs using bacterial biosensors that phenotypically respond when exposed to exogenous AHLs. This article reviews and discusses the currently available bacterial biosensors which can be used in detecting and studying the different AHLs.     

A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence

In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae ( Xoo ), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri . Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.     

A quorum-quenching approach to investigate the conservation of quorum-sensing-regulated functions within the Burkholderia cepacia complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.     

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.     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

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.     

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.     

Profiling acylated homoserine lactones in Yersinia ruckeri and influence of exogenous acyl homoserine lactones and known quorum-sensing inhibitors on protease production

AIMS: To profile the quorum-sensing (QS) signals in Yersinia ruckeri and to examine the possible regulatory link between QS signals and a typical QS-regulated virulence phenotype, a protease. METHODS AND RESULTS: Liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) showed that Y. ruckeri produced at least eight different acylated homoserine lactones (AHLs) with N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL) being the dominant molecule. Also, some uncommon AHL, N-(3-oxoheptanoyl)-L-homoserine lactone (3-oxo-C7-HSL) and N-(3-oxononanoyl)-L-homoserine lactone (3-oxo-C9-HSL), were produced. 3-oxo-C8-HSL was detected in organs from fish infected with Y. ruckeri. Protease production was significantly lower at temperatures above 23 degrees C than below although growth was faster at the higher temperatures. Neither addition of sterile filtered high-density Y. ruckeri culture supernatant nor the addition of pure exogenous AHLs induced protease production. Furthermore, three QS inhibitors (QSIs), sulfur-containing AHL analogues, did not inhibit protease production in Y. ruckeri. CONCLUSIONS: Exogenous AHL or sulfur-containing AHL analogues did not influence the protease production indicating that protease production may not be QS regulated in Y. ruckeri. SIGNIFICANCE AND IMPACT OF THE STUDY: The array of different AHLs produced indicates that the QS system of Y. ruckeri is complex and could involve several regulatory systems. In this case, neither AHLs nor QSI would be likely to directly affect a QS-regulated phenotype.     

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.     

The presence, type and role of N-acyl homoserine lactone quorum sensing in fluorescent Pseudomonas originally isolated from rice rhizospheres are unpredictable

In Gram-negative bacteria, a typical quorum-sensing (QS) system involves the production and response to N-acyl homoserine lactones (AHLs). It still remains unclear as to how pivotal and conserved AHL QS is in root-colonizing rhizosphere Pseudomonas. We, therefore, performed a systematic study of AHL QS on a set of 50 rice rhizosphere Pseudomonas isolates. We also isolated the AHL QS genes in two representative strains and analyzed the role of AHL QS regulation of various phenotypes. Our results are discussed with the current knowledge of AHL QS of rhizosphere Pseudomonas, implicating a lack of conservation and an unpredictable role played by AHL QS in this group of bacteria.     

Synthesis of multiple N-acylhomoserine lactones is wide-spread among the members of the Burkholderia cepacia complex

Seventy strains of the Burkholderia cepacia complex, which currently comprises six genomic species, were tested for their ability to produce N-acylhomoserine lactone (AHL) signal molecules. Using thin layer chromatography in conjunction with a range of AHL biosensors, we show that most strains primarily produce two AHLs, namely N-octanoylhomoserine lactone (C8-HSL) and N-hexanoylhomoserine lactone (C6-HSL). Furthermore, some strains belonging to B. vietnamiensis (genomovar V) produce additional long chain AHL molecules with acyl chains ranging from C10 to C14. For B. vietnamiensis R-921 the structure of the most abundant long chain AHL was confirmed as N-decanoylhomoserine lactone (C10-HSL) by liquid chromatography-mass spectrometry (LC-MS) in combination with total chemical synthesis. Interestingly, a number of strains, most notably all representatives of B. multivorans (genomovar II), did not produce AHLs at least under the growth conditions used in this study. All strains were also screened for the production of extracellular lipase, chitinase, protease, and siderophores. However, no correlation between the AHL production and the synthesis of these exoproducts was apparent. Southern blot analysis showed that all the B. cepacia complex strains investigated, including the AHL-negative strains, possess genes homologous to the C8-HSL synthase cepI and to cepR, which encodes the cognate receptor protein. The nucleotide sequence of the cepI and cepR genes from one representative strain from each of the six genomovars was determined. Furthermore, the cepI genes from the different genomovars were expressed in Escherichia coli and it is demonstrated that all genes encode functional proteins that direct the synthesis of C8-HSL and C6-HSL. Given that cepI from the B. multivorans strain encodes a functional AHL synthase, yet detectable levels of AHLs were not produced by the wild-type, this suggests that additional regulatory functions may be present in members of this genomovar that negatively affect expression of cepI.