Bacterial Quorum Sensing Signal Molecules Research Articles

AI‐2 biosynthesis module in a magnetic nanofactory alters bacterial response via localized synthesis and delivery

Nanofactories are nano-dimensioned and comprised of modules serving various functions that alter the response of targeted cells when deployed by locally synthesizing and delivering cargo to the surfaces of the targeted cells. In its basic form, a nanofactory consists of a minimum of two functional modules: a cell capture module and a synthesis module. In this work, magnetic nanofactories that alter the response of targeted bacteria by the localized synthesis and delivery of the "universal" bacterial quorum sensing signal molecule autoinducer AI-2 are demonstrated. The magnetic nanofactories consist of a cell capture module (chitosan-mag nanoparticles) and an AI-2 biosynthesis module that contains both AI-2 biosynthetic enzymes Pfs and LuxS on a fusion protein (His-LuxS-Pfs-Tyr, HLPT) assembled together. HLPT is hypothesized to be more efficient than its constituent enzymes (used separately) at conversion of the substrate SAH to product AI-2 on account of the proximity of the two enzymes within the fusion protein. HLPT is demonstrated to be more active than the constituent enzymes, Pfs and LuxS, over a wide range of experimental conditions. The magnetic nanofactories (containing bound HLPT) are also demonstrated to be more active than free, unbound HLPT. They are also shown to elicit an increased response in targeted Escherichia coli cells, due to the localized synthesis and delivery of AI-2, when compared to the response produced by the addition of AI-2 directly to the cells. Studies investigating the universality of AI-2 and unraveling AI-2 based quorum sensing in bacteria using magnetic nanofactories are envisioned. The prospects of using such multi-modular nanofactories in developing the next generation of antimicrobials based on intercepting and interrupting quorum sensing based signaling are discussed.

Microbiological aspects of phyllosoma rearing of the ornate rock lobster Panulirus ornatus

Rock lobsters of the Palinuridae are the most valuable wild fisheries sector in Australia and are currently target aquaculture species. Significant challenges exist however to produce commercial scale quantities of post-larvae due to an extended larval phase which acerbates a high rate of larval attrition caused by inadequate nutrition and a challenging microbial environment. Here we investigate a diverse and varied bacterial community in four compartments of the larval-rearing system: the water column, the biofilm, live feeds and the phyllosomas themselves. External fouling of phyllosoma by filamentous Thiothrix sp. was documented by scanning electron microscopy (SEM) and fluorescence in situ hybridisation (FISH). Internal proliferation of bacteria coinciding with mass mortality of phyllosoma was observed in histopathological analysis and identified as Vibrio sp. by specific labelling of sectioned hepatopancreas tissue using FISH. Of particular interest in relation to larval mortalities was a range of Vibrio species, isolated from the four rearing compartments, closely affiliated with V. alginolyticus, V. parahaemolyticus, and V. harveyi. The presence of bacterial quorum sensing signal molecules within the system was demonstrated in both biofilm and phyllosoma environments during a larval-rearing run. Interestingly, a large increase in quorum sensing signal molecules was detected in phyllosoma corresponding with mass mortality.

Magnetic nanofactories: Localized synthesis and delivery of quorum-sensing signaling molecule autoinducer-2 to bacterial cell surfaces

Magnetic ‘nanofactories’, for localized manufacture and signal-guided delivery of small molecules to targeted cell surfaces, are demonstrated. They recruit nearby raw materials for synthesis, employ magnetic mobility for capture and localization of target cells, and deliver molecules to cells triggering their native phenotypic response, but with user-specified control. Our nanofactories, which synthesize and deliver the “universal” bacterial quorum-sensing signal molecule, autoinducer AI-2, to the surface of Escherichia coli, are assembled by first co-precipitating nanoparticles of iron salts and the biopolymer chitosan. E. coli AI-2 synthases, Pfs and LuxS, constructed with enzymatically activatable “pro-tags”, are then covalently tethered onto the chitosan. These enzymes synthesize AI-2 from metabolite S-adenosylhomocysteine. Chitosan serves as a molecular scaffold and provides cell capture ability; magnetite provides stimuli responsiveness. These magnetic nanofactories are shown to modulate the natural progression of quorum-sensing activity. New prospects for small molecule delivery, based on localized synthesis, are envisioned.

Characterization of Pectolytic Erwinias as Highly Sophisticated Pathogens of plants

Erwinia carotovora and Erwinia chrysanthemi are the two most important soft rotting bacteria of commercially-grown plants. They are genetically diverse as is evident from polymorphisms in the pel and recA genes as well as in rrn, the ribsomal gene cluster. Subpopulations grouped into biovars, pathovars, or subspecies associated with various hosts and in different geographic regions suggest specialization in host preference and/or survival in diverse environments. Previous characterization of the pectolytic erwinias as opportunistic pathogens is being replaced by a realization that this group of bacteria exhibits a sophisticated repertoire of pathogenicity and virulence genes and regulators. The presence of an entire hrp gene cluster and associated type III secretion system, and global regulators which regulate virulence determinants such as exoenzyme production and motility, attest to a highly specialized pathogen. The fact that production of extracellular plant cell wall-degrading enzymes are coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone in a population density-dependent manner may explain the occurrence of pectolytic erwinia in asymptomatic plant tissues. Transgenic plants expressing bacterial quorum-sensing signal molecules modulate this sensory system and exhibit resistance to soft rot infection. The pectolytic erwinias, being significant plant pathogens that are neither of quarantine concern nor a human health hazard while readily isolated from field sources, make an ideal model for investigating the genetic basis of plant pathogenesis and environmental fitness.

Haemodynamic effects of the bacterial quorum sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, in conscious, normal and endotoxaemic rats.

N-acylhomoserine lactones (AHLs) are small, diffusible signalling molecules, employed by Gram-negative bacteria to coordinate gene expression with cell population density. Recent in vitro findings indicate that AHLs may function as virulence determinants per se, through modification of cytokine production by eukaryotic cells, and by stimulating the relaxation of blood vessels. In the present study, we assessed the influence of AHLs on cardiovascular function in conscious rats, and draw attention to the ability of the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), a signal molecule produced by P. aeruginosa, to cause marked bradycardia. This bradycardic effect was blocked by atropine and atenolol, and did not occur in vitro. Furthermore, modification of the acyl side chain length resulted in the loss of activity, whereas removal of the homoserine lactone ring, did not. The bradycardic effect of 3-oxo-C12-HSL was also observed in endotoxaemic animals, albeit attenuated. In normal rats, 3-oxo-C12-HSL caused initial mesenteric and hindquarters vasoconstriction, but only slight, and delayed signs of vasodilatation in the renal and mesenteric vascular beds. Furthermore, administration of 3-oxo-C12-HSL (pre-treatment or 2 h post-treatment) together with LPS, did not modify the established regional haemodynamic effects of the LPS, 6 h after the onset of its infusion. Our observations do not provide any clear evidence for an ability of 3-oxo-C12-HSL to modify the haemodynamic responses to LPS infusion. However, they are not inconsistent with the hypothesis that some of the cardiovascular sequelae of bacterial infection may be modulated by an influence of bacterial quorum sensing signalling molecules on the host.

GM plants communicate with bacteria

N-acylhomoserine lactones (AHLs) play an important role in plant–microorganism interactions. The AHLs produced by the transgenic plants are sufficient to induce target gene expression in several recombinant bacterial AHL biosensors, to restore biocontrol activity to an HHL-deficient Pseudomonas aureo-faciens strain and to restore pathogenicity of an Erwinia carotovora strain. The ability to generate bacterial quorum-sensing signaling molecules in plants offers novel opportunities for disease control and for manipu- lating plant–microorganism interactions. Nat. Biotechnol. (1999) 17, 1017–1020.

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.