Synthesis Of N-acyl Homoserine Lactones Research Articles

Arachis hypogaea L. produces mimic and inhibitory quorum sensing like molecules

A wide variety of plant-associated soil bacteria (rhizobacteria) communicate with each other by quorum sensing (QS). Plants are able to detect and produce mimics and inhibitor molecules of the QS bacterial communicative process. Arachis hypogaea L. (peanut) establishes a nitrogen-fixing symbiosis with rhizobia belonging to the genus Bradyrhizobium. These bacteria use a QS mechanism dependent on the synthesis of N-acyl homoserine lactones (AHLs). Given the relevance that plant-rhizobacteria interactions have at the ecological level, this work addresses the involvement of peanut in taking part in the QS mechanism. By using biosensor bacterial strains capable of detecting AHLs, a series of standard and original bioassays were performed in order to determine both (i) the production of QS-like molecules in vegetal materials and (ii) the expression of the QS mechanism throughout plant-bacteria interaction. Mimic QS-like molecules (mQS) linked to AHLs with long acyl chains (lac-AHL), and inhibitor QS-like molecules (iQS) linked to AHLs with short acyl chains (sac-AHL) were detected in seed and root exudates. The results revealed that synthesis of specific signaling molecules by the plant (such as mQS and iQS) probably modulates the function and composition of the bacterial community established in its rhizosphere. Novel bioassays of QS detection during peanut-Bradyrhizobium interaction showed an intense production of QS signals in the contact zone between root and bacteria. It is demonstrated that root exudates stimulate the root colonization and synthesis of lac-AHL by Bradyrhizobium strains in the plant rhizosphere, which leads to the early stages of the development of beneficial plant-bacteria interactions.

Transcriptome of the quorum-sensing signal-degrading Rhodococcus erythropolis responds differentially to virulent and avirulent Pectobacterium atrosepticum.

Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.

GacS-dependent regulation of enzymic and antifungal activities and synthesis of N-acylhomoserine lactones in rhizospheric strain Pseudomonas chlororaphis 449

Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30-84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G(+) bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS (-) mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.

Synthesis of N-acyl homoserine lactones and phenazines, some enzymatic activities, and fungicidal activity in the cells of Pseudomonas chlororaphis 449 with inactivated rpoS gene

The inactivation of the rpoS gene, which encodes the sigma S subunit of RNA polymerase of rhizospheric strain Pseudomonas chlororaphis 449 results in a drastic (5–8-fold) decrease in the synthesis of phenazine antibiotics, a decline in the acid and alkaline phosphatase activities (pH 2.5 and 8.8, respectively), and the antagonistic activity of this strain against phytopathogenic fungus Rhizoctonia solani. A mutation in the rpoS gene also causes a minor decrease of lipase and proteolytic activity in the supernatants of Pseudomonas chlororaphis 449 and has no substantial effect on the synthesis of three types of N-acyl homoserine lactones, the signal molecules of quarum sensing regulation, and the ability of bacteria to migrate over the medium surface (swarming).

The Role of RsmA in the Regulation of Swarming Motility in Serratia marcescens

Swarming motility is a multicellular phenomenon comprising population migration across surfaces by specially differentiated cells. In Serratia marcescens, a network exists in which the flhDC flagellar regulatory master operon, temperature, nutrient status, and quorum sensing all contribute to the regulation of swarming motility. In this study, the rsmA (repressor of secondary metabolites) gene (hereafter rsmA_Sm) was cloned from S. marcescens. The presence of multicopy, plasmid-encoded rsmA_Sm expressed from its native promoter in S. marcescens inhibits swarming. Synthesis of N-acylhomoserine lactones, presumably by the product of smaI (a luxI homolog isolated from S. marcescens), was also inhibited. Knockout of rsmA_Sm on the S. marcescens chromosome shortens the time before swarming motility begins after inoculation to an agar surface. A single copy of the chromosomal PrsmA_Sm::luxAB reporter of rsmA_Smtranscription was constructed. Using this reporter, the roles of the flhDC flagellar regulatory master operon, temperature and autoregulation in the control of rsmA_Sm expression were determined. Our findings indicate that RsmA_Sm is a component of the complex regulatory network that controls swarming.

The role of RsmA in the regulation of swarming motility in Serratia marcescens.

Swarming motility is a multicellular phenomenon comprising population migration across surfaces by specially differentiated cells. In Serratia marcescens, a network exists in which the flhDC flagellar regulatory master operon, temperature, nutrient status, and quorum sensing all contribute to the regulation of swarming motility. In this study, the rsmA (repressor of secondary metabolites) gene (hereafter rsmA(Sm)) was cloned from S. marcescens. The presence of multicopy, plasmid-encoded rsmA(Sm) expressed from its native promoter in S. marcescens inhibits swarming. Synthesis of N-acylhomoserine lactones, presumably by the product of smaI (a luxI homolog isolated from S. marcescens), was also inhibited. Knockout of rsmA(Sm) on the S. marcescens chromosome shortens the time before swarming motility begins after inoculation to an agar surface. A single copy of the chromosomal PrsmA(Sm)::luxAB reporter of rsmA(Sm) transcription was constructed. Using this reporter, the roles of the flhDC flagellar regulatory master operon, temperature and autoregulation in the control of rsmA(Sm) expression were determined. Our findings indicate that RsmA(Sm) is a component of the complex regulatory network that controls swarming.

Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control.

Serratia sp. ATCC 39006 produces the carbapenem antibiotic, carbapen-2-em-3-carboxylic acid and the red pigment, prodigiosin. We have previously reported the characterization of a gene, carR, controlling production of carbapenem in this strain. We now describe further characterization of the carR locus to locate the genes encoding carbapenem biosynthetic and resistance functions. A novel family of diverse proteins showing sequence similarity to the C-terminal domain of CarF (required for carbapenem resistance) is described. We also report the isolation of the locus involved in the biosynthesis of the red pigment, prodigiosin. A cosmid containing approximately 35 kb of the Serratia chromosome encodes synthesis of the pigment in the heterologous host, Erwinia carotovora, demonstrating, for the first time, that the complete prodigiosin biosynthetic gene cluster had been cloned and functionally expressed. We report the isolation of a third locus in Serratia, containing convergently transcribed genes, smaI and smaR, encoding LuxI and LuxR homologues respectively. SmaI directs the synthesis of N-acyl homoserine lactones involved in the quorum sensing process. We demonstrate that biosynthesis of the two secondary metabolites, carbapenem antibiotic and prodigiosin pigment, is under pheromone-mediated transcriptional regulation in this bacterium. Finally, we describe a new prodigiosin-based bioassay for detection of some N-acyl homoserine lactones.

Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules.

Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high-performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with approximately 20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5' transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N-acylhomoserine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two-dimensional SDS-PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.