Gordon S. A. B. Stewart

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.

Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the autoinducer molecule N(3‐oxododecanoyl)‐l‐homoserine lactone (OdDHL), the synthesis of which is directed by LasI. Previously we have shown that in PAN067, an elastase‐negative mutant of PAO1, elastase production can be restored to some extent by addition of exogenous N(3‐oxohexanoyl)‐l‐homoserine lactone (OHHL). Here we report that PAN067 is also defective in the production of alkaline protease, haemolysin, cyanide, pyocyanin and autoinducer(s). As neither addition of exogenous OdDHL nor introduction of IasR restored PAN067 to the parental phenotype, we sought to complement PAN067 with PAO1 DNA. From a cosmid library, a 2 kb DNA fragment was identified which re‐established production of autoinducer(s) and exoproducts in PAN067. From the nucleotide sequence of this fragment, two genes termed rhIR and rhII were identified. RhII is responsible for autoinducer synthesis and shares 31% homology with LasI; RhIR has been previously identified in P. aeruginosa strain DSM2659 as a regulator of rhamnolipid biosynthesis and shares 28% identity with LasR. These data provide clear evidence that multiple families of quorum‐sensing modulons interactively regulate gene expression in P. aeruginosa.

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.

Mechanisms of action of disinfectants

Disinfectants and biocides are a chemically diverse group of agents which are generally considered to exhibit poor selective toxicity. This should not be mistaken for poor target specificity, however, and much is now known concerning the damaging interactions which may arise between bacterial cell and disinfectant agent. Critical governing features of these interactions are the physicochemical characteristics of the chemical agent, cell morphology, and the physiological status of the microorganism. Antibacterial events include membrane disruption, macromolecule dysfunction, and metabolic inhibition; the consequential effect is determined by the relative contribution(s) of the target(s) to microbial cell survival and the possible initiation of self-destructive processes. Disinfection kinetics offer a measure to differentiate between physiochemical and chemical interactions. Increasingly demanding disinfectant applications require more sophisticated use of biocidal systems. Approaches include: agents in combination, whereby a knowledge of mechanism of action assists in designing optimal mixtures; intracellular biocide delivery, using cellular transport processes to overcome cellular barriers; and targeted donation of biocide from delivery systems, requiring an understanding of target reactivity. A knowledge of disinfection mechanisms provides a basis from which novel chemistries and synergistic combinations may be developed.

High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells

A procedure has been developed for electroporation-mediated transformation of Listeria monocytogenes with plasmid DNA. The method was optimized for intact cells of L. monocytogenes 23074 by determining the effects of field strength, cell density, and plasmid DNA topology. Transformation efficiencies were dramatically increased when cells were treated with penicillin. Optimum frequencies of transformation (4 x 10(6) transformants/microgram DNA) were obtained when cells were grown in 10 micrograms/ml of penicillin G and electroporated at a field strength of 10 kV/cm. Using this procedure, transformation of relaxed plasmid DNA from ligation reactions provided 1 x 10(4) transformants/microgram DNA, allowing direct molecular cloning of DNA into this organism.

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.

Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp. than among soilborne Pseudomonas spp.

ABSTRACT A total of 137 soilborne and plant-associated bacterial strains belonging to different Pseudomonas species were tested for their ability to synthesize N-acyl-homoserine lactones (NAHL). Fifty-four strains synthesized NAHL. Interestingly, NAHL production appears to be more common among plant-associated than among soilborne Pseudomonas spp. Indeed, 40% of the analyzed Pseudomonas syringae strains produced NAHL which were identified most often as the short-chain NAHL,N-hexanoyl-l-homoserine lactone,N-(3-oxo-hexanoyl)-homoserine lactone, andN-(3-oxo-octanoyl)-l-homoserine lactone (no absolute correlation between genomospecies of P. syringae and their ability to produce NAHL could be found). Six strains of fluorescent pseudomonads, belonging to the species P. chlororaphis, P. fluorescens, and P. putida, isolated from the plant rhizosphere produced different types of NAHL. In contrast, none of the strains isolated from soil samples were shown to produce NAHL. The gene encoding the NAHL synthase in P. syringae pv. maculicola was isolated by complementation of an NAHL-deficient Chromobacteriummutant. Sequence analysis revealed the existence of aluxI homologue that we named psmI. This gene is sufficient to confer NAHL synthesis upon its bacterial host and has strong homology to psyI and ahlI, two genes involved in NAHL production in P. syringae pv. tabaci and P. syringae pv. syringae, respectively. We identified another open reading frame that we termedpsmR, transcribed convergently in relation topsmI and partly overlapping psmI; this gene encodes a putative LuxR regulatory protein. This gene organization, with luxI and luxRhomologues facing each other and overlapping, has been found so far only in the enteric bacteria Erwinia andPantoea and in the related species P. syringae pv. tabaci.

lux genes and the applications of bacterial bioluminescence.

Luminous bacteria constitute some of the most fascinating subjects in microbiology and are much more prevalent than is frequently appreciated. They are found in marine, freshwater and terrestrial environments and can be most easily isolated as saprophytes growing on dead fish or meat. Fig. 1 (a) shows the bioluminescence emission from a fresh fish-market sprat imaged using a photon video camera. This light, which can be obtained from almost any fish, is derived from bioluminescent bacteria. The diversity of these micro-organisms is such that only a very small percentage have been studied biochemically and even fewer have been the subject of genetic analysis. There may, therefore, be much yet to discover about the diverse biochemistry and genetics of bacterial bioluminescence. Nevertheless, through the techniques of genetic engineering, the phenomenon of bacterial bioluminescence can now be captured and applied within any bacterial species from several rather different perspectives. It provides a real-time noninvasive reporter for measuring gene expression, a sensitive marker for bacterial detection and a measure of intracellular biochemical function, i.e. as a holistic determinant of cellular viability. This review aims to provide the necessary background to examine recent developments in these areas and thereby encourage a growing awareness for the multi-faceted nature of in vivo bioluminescence.