Siri Ram Chhabra

Quorum sensing and Chromobacterium violaceum : exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones

Quorum sensing relies upon the interaction of a diffusible signal molecule with a transcriptional activator protein to couple gene expression with cell population density. In Gram-negative bacteria, such signal molecules are usually N-acylhomoserine lactones (AHLs) which differ in the structure of their N-acyl side chains. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein. Previously the authors described a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) in which pigment production can be restored by incubation with supernatants from the wild-type strain. To develop this mutant as a general biosensor for AHLs, the natural C. violaceum AHL molecule was first chemically characterized. By using solvent extraction, HPLC and mass spectrometry, a single AHL, N-hexanoyl-L-homoserine lactone (HHL), was identified in wild-type C. violaceum culture supernatants which was absent from CV026. Since the production of violacein constitutes a simple assay for the detection of AHLs, we explored the ability of CV026 to respond to a series of synthetic AHL and N-acylhomocysteine thiolactone (AHT) analogues. In CV026, violacein is inducible by all the AHL and AHT compounds evaluated with N-acyl side chains from C4 to C8 in length, with varying degrees of sensitivity. Although AHL compounds with N-acyl side chains from C10 to C14 are unable to induce violacein production, if an activating AHL (e.g. HHL) is incorporated into the agar, these long-chain AHLs can be detected by their ability to inhibit violacein production. The versatility of CV026 in facilitating detection of AHL mixtures extracted from culture supernatants and separated by thin-layer chromatography is also demonstrated. These simple bioassays employing CV026 thus greatly extend the ability to detect a wide spectrum of AHL signal molecules.

Quorum‐sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram‐negative bacteria

In cell‐free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N‐acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(ΔAla‐l‐Val) and cyclo(l‐Pro‐l‐Tyr) respectively. These compounds were also found in cell‐free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(ΔAla‐l‐Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(l‐Phe‐l‐Pro). Dose–response curves using a LuxR‐based AHL biosensor indicated that cyclo(ΔAla‐l‐Val), cyclo(l‐Pro‐l‐Tyr) and cyclo(l‐Phe‐l‐Pro) activate the biosensor in a concentration‐dependent manner, albeit at much higher concentrations than the natural activator N‐(3‐oxohexanoyl)‐l‐homoserine lactone (3‐oxo‐C6‐HSL). Competition studies showed that cyclo(ΔAla‐l‐Val), cyclo(l‐Pro‐l‐Tyr) and cyclo(l‐Phe‐l‐Pro) antagonize the 3‐oxo‐C6‐HSL‐mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR‐binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR‐based quorum‐sensing systems, such as the N‐butanoylhomoserine lactone‐dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.

N-Acylhomoserine Lactones Undergo Lactonolysis in a pH-, Temperature-, and Acyl Chain Length-Dependent Manner during Growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa

ABSTRACT In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37°C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.

The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density‐dependency of the quorum sensing hierarchy, regulates rhl‐dependent genes at the onset of stationary phase and can be produced in the absence of LasR

In Pseudomonas aeruginosa, diverse exoproduct virulence determinants are regulated via N‐acylhomoserine lactone‐dependent quorum sensing. Here we show that 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) is also an integral component of the quorum sensing circuitry and is required for the production of rhl‐dependent exoproducts at the onset of stationary phase. Analysis of spent P. aeruginosa culture supernatants revealed that PQS is produced at the end of exponential phase in the parent strain and in the late stationary phase of a lasR mutant. Mutants defective in both PQS production (pqsR‐) and response (pqsE‐) produced substantially reduced levels of exoproducts but retained wild‐type N‐butanoyl homoserine lactone (C4‐HSL) levels. In the wild type, provision of exogenous PQS at the time of inoculation significantly increased PA‐IL lectin, pyocyanin and elastase production during early stationary phase and promoted biofilm formation. Exogenous PQS but not PQS derivatives lacking the 3‐hydroxy group overcame the cell density but not growth phase‐dependent production of exoproducts. PQS also overcame the transcriptional and post‐transcriptional repression of lecA (which codes for the PA‐IL lectin) mediated via the negative regulators MvaT and RsmA respectively. Increased expression of lecA in the presence of exogenous PQS can be explained partially by increases in RhlR, RpoS and C4‐HSL levels. A refined model for quorum sensing in P. aeruginosa is presented.

The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.

Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N‐3‐(oxohexanoyl)‐L‐homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes.

Involvement of N‐acyl‐l‐homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens

Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer‐cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high‐performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N‐butanoyl‐l‐homoserine lactone (BHL) and N‐hexanoyl‐l‐homoserine lactone (HHL) in cell‐free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.

Quinolones: from antibiotics to autoinducers

Since quinine was first isolated, animals, plants and microorganisms producing a wide variety of quinolone compounds have been discovered, several of which possess medicinally interesting properties ranging from antiallergenic and anticancer to antimicrobial activities. Over the years, these have served in the development of many synthetic drugs, including the successful fluoroquinolone antibiotics. Pseudomonas aeruginosa and related bacteria produce a number of 2-alkyl-4(1H)-quinolones, some of which exhibit antimicrobial activity. However, quinolones such as the Pseudomonas quinolone signal and 2-heptyl-4-hydroxyquinoline act as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. Here, we review selectively this extensive family of bicyclic compounds, from natural and synthetic antimicrobials to signalling molecules, with a special emphasis on the biology of P. aeruginosa. In particular, we review their nomenclature and biochemistry, their multiple properties as membrane-interacting compounds, inhibitors of the cytochrome bc1 complex and iron chelators, as well as the regulation of their biosynthesis and their integration into the intricate quorum-sensing regulatory networks governing virulence and secondary metabolite gene expression.

A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia

Micro-organisms have evolved complex and diverse mechanisms to sense environmental changes. Activation of a sensory mechanism typically leads to alterations in gene expression facilitating an adaptive response. This may take several forms, but many are mediated by response-regulator proteins. The luxR-encoded protein (LuxR) has previously been characterised as a member of the response-regulator superfamily and is known to respond to the small diffusible autoinducer signal molecule N-(beta-ketocaproyl) homoserine lactone (KHL). Observed previously in only a few marine bacteria, we now report that KHL is in fact produced by a diverse group of terrestrial bacteria. In one of these (Erwinia carotovora), we show that it acts as a molecular control signal for the expression of genes controlling carbapenem antibiotic biosynthesis. This represents the first substantive evidence to support the previous postulate that the lux autoinducer, KHL, is widely involved in bacterial signalling.

A novel strategy for the isolation of luxl homologues: evidence for the widespread distribution of a LuxR:Luxl superfamily in enteric bacteria

The pheromone N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density‐dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid‐based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density‐dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxl. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxl homologue from both E. carotovora (carl) and E. agglomerans (eagl) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri Luxl. Despite this, carl, eagl and luxl are shown to be biologically equivalent. An insertion mutant of eagl demonstrates that this gene is essential for OHHL production in E. agglomerans.

The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase.

Several different species of Pseudomonas: produce N:-acylhomoserine lactones (AHLs), quorum-sensing signal molecules which are involved in the cell-density-dependent control of secondary metabolite and virulence gene expression. When Pseudomonas fluorescens F113 was cross-streaked against AHL biosensors capable of sensitively detecting either short (C(4)-C(8)) or long (C(10)-C(14)) acyl chain AHLs, no activity was detectable. However, by extracting cell-free stationary-phase culture supernatants with dichloromethane followed by reverse-phase HPLC, three distinct fractions were obtained capable of activating the AHL biosensors. Three AHLs were subsequently characterized using high-resolution MS and chemical synthesis. These were (i) N:-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3OH, C(14:1)-HSL), a molecule previously known as the Rhizobium leguminosarum small bacteriocin as a consequence of its growth inhibitory properties, (ii) N:-decanoylhomoserine lactone (C(10)-HSL) and (iii) N:-hexanoylhomoserine lactone (C(6)-HSL). A gene (hdtS) capable of directing synthesis of all three P. fluorescens AHLs in Escherichia coli was cloned and sequenced. In vitro transcription/translation of hdtS yielded a protein of approximately 33 kDa capable of directing the synthesis of 3OH, C(14:1)-HSL, C(10)-HSL and C(6)-HSL in E. coli. HdtS does not belong to either of the known AHL synthase families (LuxI or LuxM) and is related to the lysophosphatidic acid acyltransferase family. HdtS may therefore constitute a member of a third protein family capable of AHL biosynthesis.