Bioluminescent Bacterium Vibrio Fischeri Research Articles

Bacterial growth dynamics in a rhythmic symbiosis.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for ∼6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage.

Assessing ecotoxicity, synergic and antagonistic actions of commonly used components of polymerization mixture in molecularly imprinted polymers preparation process

Increasingly, the challenge is the optimal process of developing a suitable MIP-type sorptive material (taking into account its final form and application area), which is mainly related to the efficiency/effectiveness of the polymerization process. The lack of reaction or its low efficiency generates additional waste and puts it into the environmental cycle. The main aim of the performed research was the estimation of ecotoxicity, and effect of joint action of commonly used components of polymerization mixture applied in MIPs preparation process. The general purpose of this work is to apply bioluminescent bacteria in order to find any potential interactions taking place in two and three-component polymerization mixtures containing selected MIP polymerization components. According to calculated EC50 values for all tested compounds, the toxicity of MIP components to the bioluminescent bacterium Vibrio fischeri can be arranged in decreasing order as follows: DVB > DCM > EDGMA > MAA > TMSPMA > APTES > MMA > TEOS > TMPTA > AA > ACN. Based on obtained results, it was concluded that EGDMA has a strong influence on the antagonistic interactions among mixture components. An overestimation of the toxicity value was noticed for only eight mixtures, where one of the component was DVB, EGDMA, or TEOS.

Quantification of the capacity of vibrio fischeri to establish symbiosis with Euprymna scolopes.

Most animals establish long-term symbiotic associations with bacteria that are critical for normal host physiology. The symbiosis that forms between the Hawaiian squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri serves as an important model system for investigating the molecular mechanisms that promote animal-bacterial symbioses. E. scolopes hatch from their eggs uncolonized, which has led to the development of squid-colonization assays that are based on introducing culture-grown V. fischeri cells to freshly hatched juvenile squid. Recent studies have revealed that strains often exhibit large differences in how they establish symbiosis. Therefore, we sought to develop a simplified and reproducible protocol that permits researchers to determine appropriate inoculum levels and provides a platform to standardize the assay across different laboratories. In our protocol, we adapt a method commonly used for evaluating the infectivity of pathogens to quantify the symbiotic capacity of V. fischeri strains. The resulting metric, the symbiotic dose-50 (SD50), estimates the inoculum level that is necessary for a specific V. fischeri strain to establish a light-emitting symbiosis. Relative to other protocols, our method requires 2-5-fold fewer animals. Furthermore, the power analysis presented here suggests that the protocol can detect up to a 3-fold change in the SD50 between different strains.

A novel, environmentally friendly dual-signal water toxicity biosensor developed through the continuous release of Fe3+

In this work, a novel, environmentally friendly and simple electrochemical/colorimetric water toxicity biosensor was rationally developed by the continuous release of Fe3+ in a medium. The bioluminescent bacterium Vibrio Fischeri (V. fischeri) was used for the first time as a model bacterium to assess water toxicity for a mediated electrochemical biosensor. The green substance composited by Prussian blue (PB) and yellow K3[Fe(CN)6] was used as the indicator of the colorimetric biosensor. To obtain an ideal electrochemical/colorimetric performance, analytical conditions of the bioassay including NaCl concentration, temperature, concentrations of cells and K3[Fe(CN)6], and incubation time were optimized to 0.5%, 22 oC, 4 (OD600), 10 mM, and 15 min, respectively. The IC50 values of Zn2+, Hg2+, Cd2+ and 3,5-dichlorophenol (3,5-DCP) obtained by electrochemical method were 4.7, 5.0, 17.6 and 10.6 mg/L, respectively. The limits of detection (LODs) of Zn2+, Hg2+, Cd2+ and 3,5-DCP obtained by the naked eye were 6.3, 1.6, 12.5 and 12.5 mg/L, respectively. Two real water samples taken from tap water pipe and the Yitong river were also detected sensitively, and the inhibition ratios obtained were 3.8% and 14.0%, respectively. These results indicate that the V. fischeri-based bioassay is simple, sensitive and inexpensive, which is promising alternative for acute biotoxicity assessment.

Evaluation of flue gas emission factor and toxicity of the PM-bounded PAH from lab-scale waste combustion

Atmospheric particulate matter (PM) has a significant threat not only to human health but also to our environment. In Hungary, 54% of PM10 comes from residential combustion, which also includes the practice of household waste burning. Therefore, this work aims to investigate the quality of combustion through the flue gas concentrations (CO, CO2, O2) and to identify and evaluate the negative impacts of PM and PAHs generated during controlled lab-scale combustion of different mixed wastes (cardboard and glossy paper, polypropylene and polyethylene terephthalate, polyester and cotton). Mixed wastes were burnt in a lab-scale tubular furnace at different temperatures with 180 dm3/h air flow rate. Chemical analyses were coupled with ecotoxicological tests using the bioluminescent bacterium Vibrio fischeri. Ecotoxicity was expressed as toxic unit (TU) values, toxic equivalent factors (TEF) were also presented. During the combustion same amount of O2 enters the reaction, but a different amount CO2 is generated due to the C content of the sample. The waste with highest C-content related to the highest CO2 emission. Increasing the combustion temperature produces more PM-bound PAHs, which remains the same composition in the case of plastic and textile groups. The TU of solid contaminants decreases with increasing combustion temperature and increases with the minerals which are left behind in the water from the solid contaminants.

Photocatalytic and adsorptive performance of polyvinyl alcohol/chitosan/TiO2 composite for antibiotics removal: single- and multi-pollutant conditions.

A polymer-TiO2 macro composite (i.e., PVA-CS-TiO2) was synthesized via chemical precipitation of PVA-CS-TiO2 blend in alkali/solvent medium and applied for the removal of three model antibiotics (i.e., metronidazole (MNZ), ceftiofur (CEF) and tetracycline (TET)), as single compound and multi-compound conditions. The photocatalytic and adsorptive removals of antibiotics (concentrations of 0.1, 1 and 10 mg L-1) by the composite in an UV reactor system (32 W UV-C power, 0.3 g L-1 of composite) was assessed through kinetic models. Antibiotics adsorption followed pseudo-second-order kinetics, and the order of adsorption was MNZ > TET > CEF. On the other hand, the hydrophilic MNZ was degraded faster compared to hydrophobic CEF and TET drugs. Moreover, UV reactor system exhibited antagonistic behavior under multi-compound condition. Micro-toxicity of antibiotics was performed using bioluminescent bacterium Vibrio fischeri and EC50 of CEF, TET and MNZ were found to be 18.25 mg L-1, 173.8 mg L-1, and 668.6 mg L-1, respectively. However, the relative toxicity levels of PVA-CS-TiO2 and treated effluent were well with the limits as inferred from the microtoxicity analysis. Thus, synthesized biocompatible composite exhibited structural stability, consistent performance for three photocatalytic cycles for all antibiotics at a minimal catalyst loading, easily retained using metallic tea strainer and does not exhibit microtoxicity has a scope for real-time applications.

Ecotoxicological assessment of hospital wastewater: analysis of regression models

ABSTRACT This study assessed different regression models to estimate the concentration that inhibits 50% of Allium cepa root growth after exposure to different concentrations (5 to 100%v/v) of hospital wastewater for 72 h. In addition, the mitotic index was also calculated. The results indicate alterations in the normal development of A. cepa owing to the possible presence of toxic substances, identifying a maximum root inhibition of 61.01% and a minimum mitotic index of 2.15%. The Gompertz model was better than other models for its adequate adjustment and low error with a half maximal inhibitory concentration of 57.5% (moderately toxic waters) and a difference of 23% with traditional models such as Probit (80%) or Log-Logistic (80%). Toxicity tests with bioluminescent bacteria (Vibrio fischeri) confirmed the results obtained from A. cepa bioassays.

Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase

Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase–NADH reaction). CTZ reduced from dCTZ in the FRase–NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase–NADH reaction.

A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner.

For more than 30 years, the association between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri has been studied as a model system for understanding the colonization of animal epithelia by symbiotic bacteria. The squid-vibrio light-organ system provides the exquisite resolution only possible with the study of a binary partnership. The impact of this relationship on the partners' biology has been broadly characterized, including their ecology and evolutionary biology as well as the underlying molecular mechanisms of symbiotic dynamics. Much has been learned about thefactors that foster initial light-organ colonization, and more recently about the maturation and long-term maintenance of the association. This Review synthesizes the results of recent research on the light-organ association and also describes the development of new horizons for E. scolopes as a model organism that promises to inform biology and biomedicine about the basic nature of host-microorganism interactions.

A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host.

As our understanding of the human microbiome progresses, so does the need for natural experimental animal models that promote a mechanistic understanding of beneficial microorganism-host interactions. Years of research into the exclusive symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri have permitted a detailed understanding of those bacterial genes underlying signal exchange and rhythmic activities that result in a persistent, beneficial association, as well as glimpses into the evolution of symbiotic competence. Migrating from the ambient seawater to regions deep inside the light-emitting organ of the squid, V. fischeri experiences, recognizes and adjusts to the changing environmental conditions. Here, we review key advances over the past 15 years that are deepening our understanding of these events.

Multiplexed Competition in a Synthetic Squid Light Organ Microbiome Using Barcode-Tagged Gene Deletions.

Beneficial symbioses between microbes and their eukaryotic hosts are ubiquitous and have widespread impacts on host health and development. The binary symbiosis between the bioluminescent bacterium Vibrio fischeri and its squid host Euprymna scolopes serves as a model system to study molecular mechanisms at the microbe-animal interface. To identify colonization factors in this system, our lab previously conducted a global transposon insertion sequencing (INSeq) screen and identified over 300 putative novel squid colonization factors in V. fischeri To pursue mechanistic studies on these candidate genes, we present an approach to quickly generate barcode-tagged gene deletions and perform high-throughput squid competition experiments with detection of the proportion of each strain in the mixture by barcode sequencing (BarSeq). Our deletion approach improves on previous techniques based on splicing by overlap extension PCR (SOE-PCR) and tfoX-based natural transformation by incorporating a randomized barcode that results in unique DNA sequences within each deletion scar. Amplicon sequencing of the pool of barcoded strains before and after colonization faithfully reports on known colonization factors and provides increased sensitivity over colony counting methods. BarSeq enables rapid and sensitive characterization of the molecular factors involved in establishing the Vibrio-squid symbiosis and provides a valuable tool to interrogate the molecular dialogue at microbe-animal host interfaces.IMPORTANCE Beneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Model symbioses are therefore invaluable for studying the mechanisms of beneficial microbe-host interactions. Here, we present a combined barcode-tagged deletion and BarSeq approach to interrogate the molecular dialogue that ensures specific and reproducible colonization of the Hawaiian bobtail squid by Vibrio fischeri The ability to precisely manipulate the bacterial genome, combined with multiplex colonization assays, will accelerate the use of this valuable model system for mechanistic studies of how environmental microbes-both beneficial and pathogenic-colonize specific animal hosts.

A small RNA for a long-lasting relationship.

A recent study found that a small RNA released by the bioluminescent bacterium Vibrio fischeri modulates crucial host responses in the Hawaiian bobtailed squid Euprymna scolopes, revealing a new mode of communication in beneficial animal–bacterial symbioses.

HbtR, a Heterofunctional Homolog of the Virulence Regulator TcpP, Facilitates the Transition between Symbiotic and Planktonic Lifestyles in Vibrio fischeri.

The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the Vibrio cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production.IMPORTANCE TcpP homologs are widespread throughout the Vibrio genus; however, the only protein in this family described thus far is a V. cholerae virulence regulator. Here, we show that HbtR, the TcpP homolog in V. fischeri, has both a biological role and regulatory pathway completely unlike those in V. cholerae Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of the E. scolopes light organ. While LitR becomes activated within the crypts and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion of V. fischeri cells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival of V. fischeri outside its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.

An across-species comparison of the sensitivity of different organisms to Pb-based perovskites used in solar cells

Organic–inorganic perovskite solar cells (PSCs) are promising candidates as photovoltaic cells. Recently, they have attracted significant attention due to certified power conversion efficiencies exceeding 23%, low–cost engineering, and superior electrical/optical characteristics. These PSCs extensively utilize a perovskite–structured composite with a hybrid of Pb-based nanomaterials. Operation of them may cause the release of Pb-based nanoparticles. However, limited information is available regarding the potential toxicity of Pb-based PSCs on various organisms. This study conducted a battery of in vitro and in vivo toxicity bioassays for three quintessential Pb-based PSCs (CH3NH3PbI3, NHCHNH3PbBr3, and CH3NH3PbBr3) using progressively more complex forms of life. For all species tested, the three different perovskites had comparable toxicities. The viability of Caco–2/TC7 cells was lower than that of A549 cells in response to Pb-based PSC exposure. Concentration–dependent toxicity was observed for the bioluminescent bacterium Vibrio fischeri, for soil bacterial communities, and for the nematode Caenorhabditis elegans. Neither of the tested Pb-based PSCs particles had apparent toxicity to Pseudomonas putida. Among all tested organisms, V. fischeri showed the highest sensitivity with EC50 values (30 min of exposure) ranging from 1.45 to 2.91 mg L-1. Therefore, this study recommends that V. fischeri should be preferably utilized to assess.PSC toxicity due to its increased sensitivity, low costs, and relatively high throughput in a 96–well format, compared with the other tested organisms. These results highlight that the developed assay can easily predict the toxic potency of PSCs. Consequently, this approach has the potential to promote the implementation of the 3Rs (Replacement, Reduction, and Refinement) principle in toxicology and decrease the dependence on animal testing when determining the safety of novel PSCs.

Analysis of norfloxacin ecotoxicity and the relation with its degradation by means of electrochemical oxidation using different anodes.

In this work, ecotoxicological bioassays based on Lactuca sativa seeds and bioluminescent bacterium (Vibrio fischeri) have been carried out in order to quantify the toxicity of Norfloxacin (NOR) and sodium sulfate solutions, before and after treating them using electrochemical advanced oxidation. The effect of some process variables (anode material, reactor configuration and applied current) on the toxicity evolution of the treated solution has been studied. A NOR solution shows an EC50(5 days) of 336 mg L-1towards Lactuca sativa. This threshold NOR concentration decreases with sodium sulfate concentration, in solutions that contain simultaneously Norfloxacin and sodium sulfate. In every case considered in this work, the electrochemical advanced oxidation process increased the toxicity (towards both Lactuca sativa and Vibrio fischeri) of the solution. This toxicity increase is mainly due to the persulfate formation during the electrochemical treatment. From a final solution toxicity point of view, the best results were obtained using a BDD anode in a divided reactor applying the lowest current intensity.

Adaptation to temperature stress by Vibrio fischeri facilitates this microbe's symbiosis with the Hawaiian bobtail squid (Euprymna scolopes).

For microorganisms cycling between free-living and host-associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether adaptation to stress during the free-living stage can impact microbial fitness in the host. To address this topic, the mutualism between the Hawaiian bobtail squid (Euprymna scolopes) and the marine bioluminescent bacterium Vibrio fischeri was utilized. Using microbial experimental evolution, V. fischeri was selected to low (8°C), high (34°C), and fluctuating temperature stress (8°C/34°C) for 2000 generations. The temperatures 8°C and 34°C were the lower and upper growth limits, respectively. V. fischeri was also selected to benign temperatures (21°C and 28°C) for 2000 generations, which served as controls. V. fischeri demonstrated significant adaptation to low, high, and fluctuating temperature stress. V. fischeri did not display significant adaptation to the benign temperatures. Adaptation to stressful temperatures facilitated V. fischeri's ability to colonize the squid host relative to the ancestral lines. Bioluminescence levels also increased. Evolution to benign temperatures did not manifest these results. In summary, microbial adaptation to stress during the free-living stage can promote coevolution between hosts and microorganisms.

Bioluminescence of Vibrio fischeri: bacteria respond quickly and sensitively to pulsed microwave electric (but not magnetic) fields.

.Biological systems with intrinsic luminescent properties serve as powerful and noninvasive bioreporters for real-time and label-free monitoring of cell physiology. This study employs the bioluminescent marine bacterium Vibrio fischeri to investigate the effects of separated microwave electric (E) and magnetic (H) fields. Using a cylindrical mode aluminum resonant cavity, designed to spatially separate E and H fields of a pulsed microwave (2.45 GHz) input, we sampled at 100-ms intervals the 490-nm emission of bioluminescence from suspensions of the V. fischeri. E-field exposure (at 4.24 and 13.4 kV/m) results in rapid and sensitive responses to 100-ms pulses. H-field excitation elicits no measurable responses, even at 100-fold higher power input levels (equivalent to 183 A/m). The observed effects on bacterial light output partially correlate with measured E-field-induced temperature increases. In conclusion, the endogenous bioluminescence of V. fischeri provides a sensitive and noninvasive method to assess the biological effects of microwave fields.

Should they stay or should they go? Nitric oxide and the clash of regulators governing Vibrio fischeri biofilm formation.

A key regulatory decision for many bacteria is the switch between biofilm formation and motile dispersal, and this dynamic is well illustrated in the light-organ symbiosis between the bioluminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid. Biofilm formation mediated by the syp gene cluster helps V. fischeri transition from a dispersed planktonic lifestyle to a robust aggregate on the surface of the nascent symbiotic organ. However, the bacteria must then swim to pores and down into the deeper crypt tissues that they ultimately colonize. A number of positive and negative regulators control syp expression and biofilm formation, but until recently the environmental inputs controlling this clash between opposing regulatory mechanisms have been unclear. Thompson et al. have now shown that Syp-mediated biofilms can be repressed by a well-known host-derived molecule: nitric oxide. This regulation is accomplished by the NO sensor HnoX exerting control over the biofilm regulator HahK. The discoveries reported here by Thompson et al. cast new light on a critical early stage of symbiotic initiation in the V. fischeri-squid model symbiosis, and more broadly it adds to a growing understanding of the role(s) that NO and HnoX play in biofilm regulation by many bacteria.

Disposable luciferase-based microfluidic chip for rapid assay of water pollution.

In the present study, we demonstrate the use of a disposable luciferase-based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3-dihydroxybenzene and 1,4-benzoquinone for the proposed microfluidic biosensor measured 3μM, 15mM, and 2μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.

Advanced oxidation processes on doxycycline degradation: monitoring of antimicrobial activity and toxicity.

Advanced oxidation processes (AOPs) have been highly efficient in degrading contaminants of emerging concern (CEC). This study investigated the efficiency of photolysis, peroxidation, photoperoxidation, and ozonation at different pH values to degrade doxycycline (DC) in three aqueous matrices: fountain, tap, and ultrapure water. More than 99.6% of DC degradation resulted from the UV/H2O2 and ozonation processes. Also, to evaluate the toxicity of the original solution and throughout the degradation time, antimicrobial activity tests were conducted using Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria, and acute toxicity test using the bioluminescent marine bacterium (Vibrio fischeri). Antimicrobial activity reduced as the drug degradation increased in UV/H2O2 and ozonation processes, wherein the first process only 6min was required to reduce 100% of both bacteria activity. In ozonation, 27.7mgL-1 of ozone was responsible for reducing 100% of the antimicrobial activity. When applied the photoperoxidation process, an increase in the toxicity occurred as the high levels of degradation were achieved; it means that toxic intermediates were formed. The ozonated solutions did not present toxicity.