Vibrio fischeri Amidase Activity Is Required for Normal Cell Division, Motility, and Symbiotic Competence.

N-Acetylmuramoyl-l-alanine amidases are periplasmic hydrolases that cleave the amide bond between N-acetylmuramic acid and alanine in peptidoglycan (PG). Unlike many Gram-negative bacteria that encode redundant periplasmic amidases, Vibrio fischeri appears to encode a single protein that is homologous to AmiB of Vibrio cholerae We screened a V. fischeri transposon mutant library for strains altered in biofilm production and discovered a biofilm-overproducing strain with an insertion in amiB (VF_2326). Further characterization of biofilm enhancement suggested that this phenotype was due to the overproduction of cellulose, and it was dependent on the bcsA cellulose synthase. Additionally, the amiB mutant was nonmotile, perhaps due to defects in its ability to septate during division. The amidase mutant was unable to compete with the wild type for the colonization of V. fischeri's symbiotic host, the squid Euprymna scolopes In single-strain inoculations, host squid inoculated with the mutant eventually became colonized but with a much lower efficiency than in squid inoculated with the wild type. This observation was consistent with the pleiotropic effects of the amiB mutation and led us to speculate that motile suppressors of the amiB mutant were responsible for the partially restored colonization. In culture, motile suppressor mutants carried point mutations in a single gene (VF_1477), resulting in a partial restoration of wild-type motility. In addition, these point mutations reversed the effect of the amiB mutation on cellulosic biofilm production. These data are consistent with V. fischeri AmiB possessing amidase activity; they also suggest that AmiB suppresses cellulosic biofilm formation but promotes successful host colonization.IMPORTANCE Peptidoglycan (PG) is a critical microbe-associated molecular pattern (MAMP) that is sloughed by cells of V. fischeri during symbiotic colonization of squid. Specifically, this process induces significant remodeling of a specialized symbiotic light organ within the squid mantle cavity. This phenomenon is reminiscent of the loss of ciliated epithelium in patients with whooping cough due to the production of PG monomers by Bordetella pertussis Furthermore, PG processing machinery can influence susceptibility to antimicrobials. In this study, we report roles for the V. fischeri PG amidase AmiB, including the beneficial colonization of squid, underscoring the urgency to more deeply understand PG processing machinery and the downstream consequences of their activities.

Spatially propagating activation of quorum sensing in Vibrio fischeri and the transition to low population density.

Bacteria communicate by secreting and detecting diffusible small molecule signals or pheromones. Using the local concentrations of these signals to regulate gene expression, individual cells can synchronize changes in phenotype population-wide, a behavior known as quorum sensing (QS). In unstirred media, the interplay between diffusion of signals, bacterial growth, and regulatory feedback can generate complex spatial and temporal patterns of expression of QS-controlled genes. Here we identify the parameters that allow a local signal to trigger a self-sustaining, traveling activation of QS behavior. Using the natural bioluminescence of wild-type Vibrio fischeri as a readout of its lux QS system, we measure the induction of a spreading QS response by a localized triggering stimulus in unstirred media. Our data show that a QS response propagates outward, sustained by positive feedback in synthesis of the diffusible signal, and that this response occurs only if the triggering stimulus exceeds a critical threshold. We also test how the autonomous or untriggered activation of the V. fischeri QS pathway changes at very low initial population densities. At the lowest population densities, clusters of cells do not transition to a self-sensing behavior, but rather remain in communication via signal diffusion until they reach sufficiently large size that their own growth slows. Our data, which are reproduced by simple growth and diffusion simulations, indicate that in part owing to bacterial growth behavior, natural QS systems can be characterized by long distance communication through signal diffusion even in very heterogeneous and spatially dispersed populations.

Microencapsulated Aliivibrio fischeri in alginate microspheres for monitoring heavy metal toxicity in environmental waters.

In this article a luminescence fiber optic biosensor for the microdetection of heavy metal toxicity in waters based on the marine bacterium Aliivibrio fischeri (A. fischeri) encapsulated in alginate microspheres is described. Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(II) were selected as sample toxic heavy metal ions for evaluation of the performance of this toxicity microbiosensor. The loss of bioluminescence response from immobilized A. fischeri bacterial cells corresponds to changes in the toxicity levels. The inhibition of the luminescent biosensor response collected at excitation and emission wavelengths of 287 ± 2 nm and 487 ± 2 nm, respectively, was found to be reproducible and repeatable within the relative standard deviation (RSD) range of 2.4-5.7% (n = 8). The toxicity biosensor based on alginate micropsheres exhibited a lower limit of detection (LOD) for Cu(II) (6.40 µg/L), Cd(II) (1.56 µg/L), Pb(II) (47 µg/L), Ag(I) (18 µg/L) than Zn(II) (320 µg/L), Cr(VI) (1,000 µg/L), Co(II) (1700 µg/L), Ni(II) (2800 µg/L), and Fe(III) (3100 µg/L). Such LOD values are lower when compared with other previous reported whole cell toxicity biosensors using agar gel, agarose gel and cellulose membrane biomatrices used for the immobilization of bacterial cells. The A. fischeri bacteria microencapsulated in alginate biopolymer could maintain their metabolic activity for a prolonged period of up to six weeks without any noticeable changes in the bioluminescence response. The bioluminescent biosensor could also be used for the determination of antagonistic toxicity levels for toxicant mixtures. A comparison of the results obtained by atomic absorption spectroscopy (AAS) and using the proposed luminescent A. fischeri-based biosensor suggests that the optical toxicity biosensor can be used for quantitative microdetermination of heavy metal toxicity in environmental water samples.

Coordination of the arc regulatory system and pheromone-mediated positive feedback in controlling the Vibrio fischeri lux operon.

Bacterial pheromone signaling is often governed both by environmentally responsive regulators and by positive feedback. This regulatory combination has the potential to coordinate a group response among distinct subpopulations that perceive key environmental stimuli differently. We have explored the interplay between an environmentally responsive regulator and pheromone-mediated positive feedback in intercellular signaling by Vibrio fischeri ES114, a bioluminescent bacterium that colonizes the squid Euprymna scolopes. Bioluminescence in ES114 is controlled in part by N-(3-oxohexanoyl)-L-homoserine lactone (3OC6), a pheromone produced by LuxI that together with LuxR activates transcription of the luxICDABEG operon, initiating a positive feedback loop and inducing luminescence. The lux operon is also regulated by environmentally responsive regulators, including the redox-responsive ArcA/ArcB system, which directly represses lux in culture. Here we show that inactivating arcA leads to increased 3OC6 accumulation to initiate positive feedback. In the absence of positive feedback, arcA-mediated control of luminescence was only ∼2-fold, but luxI-dependent positive feedback contributed more than 100 fold to the net induction of luminescence in the arcA mutant. Consistent with this overriding importance of positive feedback, 3OC6 produced by the arcA mutant induced luminescence in nearby wild-type cells, overcoming their ArcA repression of lux. Similarly, we found that artificially inducing ArcA could effectively repress luminescence before, but not after, positive feedback was initiated. Finally, we show that 3OC6 produced by a subpopulation of symbiotic cells can induce luminescence in other cells co-colonizing the host. Our results suggest that even transient loss of ArcA-mediated regulation in a sub-population of cells can induce luminescence in a wider community. Moreover, they indicate that 3OC6 can communicate information about both cell density and the state of ArcA/ArcB.

Quorum activation at a distance: spatiotemporal patterns of gene regulation from diffusion of an autoinducer signal.

Quorum sensing (QS) bacteria regulate gene expression collectively by exchanging diffusible signal molecules known as autoinducers. Although QS is often studied in well-stirred laboratory cultures, QS bacteria colonize many physically and chemically heterogeneous environments where signal molecules are transported primarily by diffusion. This raises questions of the effective distance range of QS and the degree to which colony behavior can be synchronized over such distances. We have combined experiments and modeling to investigate the spatiotemporal patterns of gene expression that develop in response to a diffusing autoinducer signal. We embedded a QS strain in a narrow agar lane and introduced exogenous autoinducer at one terminus of the lane. We then measured the expression of a QS reporter as a function of space and time as the autoinducer diffused along the lane. The diffusing signal readily activates the reporter over distances of ~1 cm on time scales of ~10 h. However, the patterns of activation are qualitatively unlike the familiar spreading patterns of simple diffusion, as the kinetics of response are surprisingly insensitive to the distance the signal has traveled. We were able to reproduce these patterns with a mathematical model that combines simple diffusion of the signal with logistic growth of the bacteria and cooperative activation of the reporter. In a wild-type QS strain, we also observed the propagation of a unique spatiotemporal excitation. Our results show that a chemical signal transported only by diffusion can be remarkably effective in synchronizing gene expression over macroscopic distances.

Sensor kinase RscS induces the production of antigenically distinct outer membrane vesicles that depend on the symbiosis polysaccharide locus in Vibrio fischeri.

Robust biofilm formation by Vibrio fischeri depends upon activation of the symbiosis polysaccharide (syp) locus, which is achieved by overexpressing the RscS sensor kinase (RscS(+)). Other than the Syp polysaccharide, however, little is known about V. fischeri biofilm matrix components. In other bacteria, biofilms contain polysaccharides, secreted proteins, and outer membrane vesicles (OMVs). Here, we asked whether OMVs are part of V. fischeri biofilms. Transmission electron microscopy revealed OMV-like particles between cells within colonies. In addition, OMVs could be purified from culture supernatants of both RscS(+) and control cells, with the former releasing 2- to 3-fold more OMVs. The increase depended upon the presence of an intact syp locus, as an RscS(+) strain deleted for sypK, which encodes a putative oligosaccharide translocase, exhibited reduced production of OMVs; it also showed a severe defect in biofilm formation. Western immunoblot analyses revealed that the RscS(+) strain, but not the control strain or the RscS(+) sypK mutant, produced a distinct set of nonproteinaceous molecules that could be detected in whole-cell extracts, OMV preparations, and lipopolysaccharide (LPS) extracts. Finally, deletion of degP, which in other bacteria influences OMV production, decreased OMV production and reduced the ability of the cells to form biofilms. We conclude that overexpression of RscS induces OMV production in a manner that depends on the presence of the syp locus and that OMVs produced under these conditions contain antigenically distinct molecules, possibly representing a modified form of lipopolysaccharide (LPS). Finally, our data indicate a correlation between OMV production and biofilm formation by V. fischeri.

Evaluation of bacterial surfactant toxicity towards petroleum degrading microorganisms.

The acute toxicity of bacterial surfactants LBBMA111A, LBBMA155, LBBMA168, LBBMA191 and LBBMA201 and the synthetic surfactant sodium dodecyl sulfate (SDS) on the bioluminescent bacterium Vibrio fischeri was evaluated by measuring the reduction of light emission (EC(20)) by this microorganism when exposed to different surfactant concentrations. Moreover, the toxic effects of different concentrations of biological and synthetic surfactants on the growth of pure cultures of isolates Acinetobacter baumannii LBBMA04, Acinetobacter junni LBBMA36, Pseudomonas sp. LBBMA101B and Acinetobacter baumanni LBBMAES11 were evaluated in mineral medium supplemented with glucose. The EC(20) values obtained confirmed that the biosurfactants have a significantly lower toxicity to V. fischeri than the SDS. After 30 min of exposure, bacterial luminescence was almost completely inhibited by SDS at a concentration of 4710 mg L(-1). Growth reduction of pure bacterial cultures caused by the addition of biosurfactants to the growth medium was lower than that caused by SDS.

Quorum sensing in bacterial species that use degenerate autoinducers can be tuned by using structurally identical non-native ligands.

Many bacteria use quorum sensing (QS) to regulate cell-density dependent phenotypes that play critical roles in the maintenance of their associations with eukaryotic hosts. In Gram-negative bacteria, QS is primarily controlled by N-acylated L-homoserine lactone (AHL) signals and their cognate LuxR-type receptors. AHL-LuxR-type receptor binding regulates the expression of target genes necessary for QS phenotypes. We recently identified a series of non-native AHLs capable of intercepting AHL-LuxR binding in the marine symbiont Vibrio fischeri, and thereby strongly promoting or inhibiting QS in this organism. V. fischeri utilizes N-(3-oxo)-hexanoyl L-HL (OHHL) as its primary QS signal, and OHHL is also used by several other bacterial species for QS. Such signal degeneracy is common among bacteria, and we sought to determine if our non-native LuxR agonists and antagonists, which are active in V. fischeri, would also modulate QS phenotypes in other bacteria that use OHHL. Herein, we report investigations into the activity of a set of synthetic LuxR modulators in the plant pathogen Pectobacterium carotovora subsp. carotovora Ecc71. This pathogen uses OHHL and two closely related LuxR-type receptors, ExpR1 and ExpR2, to control virulence, and we evaluated their responses to synthetic ligands by quantifying virulence factor production. Our results suggest an overall conservation in the activity trends of the ligands between the ExpR receptors in P. carotovora Ecc71 and LuxR in V. fischeri, and indicate that these compounds could be used as tools to study QS in an expanded set of bacteria. Notable differences in activity were apparent for certain compounds, however, and suggest that it might be possible to selectively regulate QS in bacteria that utilize degenerate AHLs.

Efficient synthesis and evaluation of quorum-sensing modulators using small molecule macroarrays.

A method for the synthesis of small molecule macroarrays of N-acylated L-homoserine lactones (AHLs) is reported. A focused library of AHLs was constructed, and the macroarray platform was found to be compatible with both solution and agar-overlay assays using quorum-sensing (QS) reporter strains. Several QS antagonists were discovered and serve to showcase the macroarray as a straightforward technique for QS research.

Reactivity of hydroxyl radicals with neonicotinoid insecticides: mechanism and changes in toxicity.

The reactivity of hydroxyl radicals (HO ) towards three neonicotonoid insecticides, namely imidacloprid, thiacloprid and acetamiprid was investigated. These radicals were generated by photolysis of H(2)O(2) solutions. Flash photolysis experiments were used to determine the rate constants of 5.5 x 10(10) M(-1)s(-1), 6 x 10(10) M(-1)s(-1), and 7.5 x 10(10) M(-1)s(-1), for the reactions of HO with acetamiprid, imidacloprid, and thiacloprid, respectively. Continuous irradiation experiments in the absence and presence of H(2)O(2) allowed the identification and toxicity evaluation of the primary photo- and oxidation products of the insecticides. In all cases, the less toxic 6-chloronicotinic acid was found to be the major product at higher degrees of oxidation. The results reported here indicate that the half life of the insecticides due to their reaction with HO radicals in natural aquatic reservoirs may vary between 5 h and 19 days, and therefore the hydroxyl radical-mediated oxidation may be a significant abiotic elimination route. However, elimination of the insecticide under such conditions might not improve the quality of the contaminated water, as the primary products of degradation still show considerable toxicity to Vibrio fischeri assays.

Bioluminescence color modulation of Vibrio fischeri strain Y1 coupled with alterable levels of endogenous yellow fluorescent protein and its fluorescence imaging.

Bioluminescence (BL) (lambda(max) approximately 535 nm) of Vibrio fischeri strain Y1 has been previously characterized in terms of the fluctuation in intracellular levels of yellow fluorescent protein (YFP). In this study fluorescence microscopic analysis has revealed that yellow fluorescence, as well as blue fluorescence attributable to a luciferase intermediate, is localized to the periphery of V. fischeri Y1 cells. This finding indicates that both YFP and the luciferase are present in the vicinity of the cell membrane. By using cyanide to enhance yellow BL, it has been shown that BL modulation is coupled with the fluctuations in the intracellular levels of YFP and the primary emitter. On the basis of the BL characterization, combined with results of a sedimentation experiment, it has been shown that larger cells produce a relatively stronger yellow BL. Two-dimensional gel electrophoresis of cell-protein extracts has shown that the YFP level is more alterable than the luciferase level. It is postulated that the yellow BL modulation takes place in connection with cell growth.

Effect of Nannochloris sp. on the toxicity of four algae.

The effect of Nannochloris sp. on the toxicity of cultures of four algae was evaluated using a Microtox 500 that measures the effect on a light-producing bacterium, Vibrio fischeri. Cultures of four algae produced a toxic effect, but in the presence of Nannochloris sp., the effect was reversed, and a stimulatory effect was observed. The effect was tested for Microcystis aeroginosa, Cyclotella menengheniana, Scenedesmus dimorphis, and Lyngbya sp. using cultures obtained from the University of Texas Culture Collection of Algae.