Assessment of the potential bactericide effect of self‐cleaning floors: a proposed protocol

The antibacterial properties of self‐cleaning coatings are based on bactericide nanoparticles (NPs). Ecotoxicity of these NPs have been assessed mostly in suspension, using standard bioassays. Here a protocol is proposed to test actual coating samples, using the Vibrio fischeri bioluminescence inhibition bioassay. The protocol was designed to test bactericide properties of specially coated PVC floors being used in hospital environments under quasinatural conditions, such as prolonged exposure or room temperature. To take into consideration that the light output of the bacteria under prolonged exposure naturally changes, a correction factor is proposed.     

Ecological Diversification of Vibrio fischeri Serially Passaged for 500 Generations in Novel Squid Host Euprymna tasmanica

Vibrio fischeri isolated from Euprymna scolopes (Cephalopoda: Sepiolidae) was used to create 24 lines that were serially passaged through the non-native host Euprymna tasmanica for 500 generations. These derived lines were characterized for biofilm formation, swarming motility, carbon source utilization, and in vitro bioluminescence. Phenotypic assays were compared between “ES” (E. scolopes) and “ET” (E. tasmanica) V. fischeri wild isolates to determine if convergent evolution was apparent between E. tasmanica evolved lines and ET V. fischeri. Ecological diversification was observed in utilization of most carbon sources examined. Convergent evolution was evident in motility, biofilm formation, and select carbon sources displaying hyperpolymorphic usage in V. fischeri. Convergence in bioluminescence (a 2.5-fold increase in brightness) was collectively evident in the derived lines relative to the ancestor. However, dramatic changes in other properties—time points and cell densities of first light emission and maximal light output and emergence of a lag phase in growth curves of derived lines—suggest that increased light intensity per se was not the only important factor. Convergent evolution implies that gnotobiotic squid light organs subject colonizing V. fischeri to similar selection pressures. Adaptation to novel hosts appears to involve flexible microbial metabolism, establishment of biofilm and swarmer V. fischeri ecotypes, and complex changes in bioluminescence. Our data demonstrate that numerous alternate fitness optima or peaks are available to V. fischeri in host adaptive landscapes, where novel host squids serve as habitat islands. Thus, V. fischeri founder flushes occur during the initiation of light organ colonization that ultimately trigger founder effect diversification.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.     

Photolyase Confers Resistance to UV Light but Does Not Contribute to the Symbiotic Benefit of Bioluminescence in Vibrio fischeri ES114

Recent reports suggest that the selective advantage of bioluminescence for bacteria is mediated by light-dependent stimulation of photolyase to repair DNA lesions. Despite evidence for this model, photolyase mutants have not been characterized in a naturally bioluminescent bacterium, nor has this hypothesis been tested in bioluminescent bacteria under natural conditions. We have now characterized the photolyase encoded by phr in the bioluminescent bacterium Vibrio fischeri ES114. Consistent with Phr possessing photolyase activity, phr conferred light-dependent resistance to UV light. However, upon comparing ES114 to a phr mutant and a dark ΔluxCDABEG mutant, we found that bioluminescence did not detectably affect photolyase-mediated resistance to UV light. Addition of the light-stimulating autoinducer N-3-oxo-hexanoyl homoserine lactone appeared to increase UV resistance, but this was independent of photolyase or bioluminescence. Moreover, although bioluminescence confers an advantage for V. fischeri during colonization of its natural host, Euprymna scolopes, the phr mutant colonized this host to the same level as the wild type. Taken together, our results indicate that at least in V. fischeri strain ES114, the benefits of bioluminescence during symbiotic colonization are not mediated by photolyase, and although some UV resistance mechanism may be coregulated with bioluminescence, we found no evidence that light production benefits cells by stimulating photolyase in this strain.     

Vibrio fischeri σ54 Controls Motility, Biofilm Formation, Luminescence, and Colonization

In this study, we demonstrated that the putative Vibrio fischeri rpoN gene, which encodes σ⁵⁴, controls flagellar biogenesis, biofilm development, and bioluminescence. We also show that rpoN plays a requisite role initiating the symbiotic association of V. fischeri with juveniles of the squid Euprymna scolopes.     

The Iron-Dependent Regulator Fur Controls Pheromone Signaling Systems and Luminescence in the Squid Symbiont Vibrio fischeri ES114

Bacteria often use pheromones to coordinate group behaviors in specific environments. While high cell density is required for pheromones to achieve stimulatory levels, environmental cues can also influence pheromone accumulation and signaling. For the squid symbiont Vibrio fischeri ES114, bioluminescence requires pheromone-mediated regulation, and this signaling is induced in the host to a greater extent than in culture, even at an equivalent cell density. Our goal is to better understand this environment-specific control over pheromone signaling and bioluminescence. Previous work with V. fischeri MJ1 showed that iron limitation induces luminescence, and we recently found that ES114 encounters a low-iron environment in its host. Here we show that ES114 induces luminescence at lower cell density and achieves brighter luminescence in low-iron media. This iron-dependent effect on luminescence required ferric uptake regulator (Fur), which we propose influences two pheromone signaling master regulators, LitR and LuxR. Genetic and bioinformatic analyses suggested that under low-iron conditions, Fur-mediated repression of litR is relieved, enabling more LitR to perform its established role as an activator of luxR. Interestingly, Fur may similarly control the LitR homolog SmcR of Vibrio vulnificus. These results reveal an intriguing regulatory link between low-iron conditions, which are often encountered in host tissues, and pheromone-dependent master regulators.     

Breaking the language barrier: experimental evolution of non-native <Emphasis Type="Italic">Vibrio fischeri</Emphasis> in squid tailors luminescence to the host

Although most Vibrio fischeri isolates are capable of symbiosis, the coevolution of certain strains with the Hawaiian bobtail squid, Euprymna scolopes, has led to specific adaptation to this partnership. For instance, strains from different hosts or from a planktonic environment are ineffective squid colonists. Even though bioluminescence is a symbiotic requirement, curiously, symbionts of E. scolopes are dim in culture relative to fish symbionts and free-living isolates. It is unclear whether this dim phenotype is related to the symbiosis or simply coincidental. To further explore the basis of symbiont specificity, we developed an experimental evolution model that utilizes the daily light organ venting behavior of the squid and horizontal acquisition of symbionts for serial passage of cultures. We passaged six populations each derived from the squid-naïve strains of V. fischeri MJ11 (a fish symbiont) and WH1 (a free-living isolate) through a series of juvenile squid light organs. After 15 serially colonized squid for each population, or an estimated 290–360 bacterial generations, we isolated representatives of the light organ populations and characterized their bioluminescence. Multiple evolved lines of both strains produced significantly less bioluminescence both in vitro and in vivo. This reduction in bioluminescence did not correlate with reduced quorum sensing for most isolates tested. The remarkable phenotypic convergence with squid symbionts further emphasizes the importance of bioluminescence in this symbiosis, and suggests that reduced light production is a specific adaptation to the squid.     

Long-term preservation of active luminous bacteria by lyophilization

A simple method for long-term preservation of luminous bacteria is described. Cells of Vibrio fischeri, Photobacterium leiognathi and four strains of P. phosphoreum were suspended in a protective medium of low ionic strength (1% NaCI) supplemented with 15% lactose and 2% soluble starch, and lyophilized. The freeze-dried preparations were sealed under vacuum and stored at 4°C. Luminous bacteria were resuscitated affer six months by adding 2% NaCl up to the original volume. The rehydrated cells exhibited 16-28% of initial bioluminescence so that they could be used for a microbial test of toxicity (the Microtox test). This method is also useful for maintaining luminous bacteria in strain collections.     

Photodynamic antimicrobial chemotherapy in aquaculture: photoinactivation studies of Vibrio fischeri

Background

Photodynamic antimicrobial chemotherapy (PACT) combines light, a light-absorbing molecule that initiates a photochemical or photophysical reaction, and oxygen. The combined action of these three components originates reactive oxygen species that lead to microorganisms'' destruction. The aim was to evaluate the efficiency of PACT on Vibrio fischeri: 1) with buffer solution, varying temperature, pH, salinity and oxygen concentration values; 2) with aquaculture water, to reproduce photoinactivation (PI) conditions in situ.

Methodology/Principal Findings

To monitor the PI kinetics, the bioluminescence of V. fischeri was measured during the experiments. A tricationic meso-substituted porphyrin (Tri-Py⁺-Me-PF) was used as photosensitizer (5 µM in the studies with buffer solution and 10–50 µM in the studies with aquaculture water); artificial white light (4 mW cm⁻²) and solar irradiation (40 mW cm⁻²) were used as light sources; and the bacterial concentration used for all experiments was ≈10⁷ CFU mL⁻¹ (corresponding to a bioluminescence level of 10⁵ relative light units - RLU). The variations in pH (6.5–8.5), temperature (10–25°C), salinity (20–40 g L⁻¹) and oxygen concentration did not significantly affect the PI of V. fischeri, once in all tested conditions the bioluminescent signal decreased to the detection limit of the method (≈7 log reduction). The assays using aquaculture water showed that the efficiency of the process is affected by the suspended matter. Total PI of V. fischeri in aquaculture water was achieved under solar light in the presence of 20 µM of Tri-Py⁺-Me-PF.

Conclusions/Significance

If PACT is to be used in environmental applications, the matrix containing target microbial communities should be previously characterized in order to establish an efficient protocol having into account the photosensitizer concentration, the light source and the total light dose delivered. The possibility of using solar light in PACT to treat aquaculture water makes this technology cost-effective and attractive.     

Experimental design of a simple medium for the bioluminescence of Aliivibrio fischeri and mathematical modelling for growth estimation

Increasing numbers of studies are using Aliivibrio fischeri (A. fischeri), a marine bioluminescent bacterium as a model, however the culture medium used for its growth are complex and expensive. The objectives of this study were: (1) to evaluate the effect of yeast extract, tryptone, and NaCl to select a simple and inexpensive culture medium suitable for A. fischeri growth and bioluminescence induction; and (2) to compare the performance of mathematical models to predict the growth of A. fischeri. A fractional factorial design was performed to evaluate the effect of yeast extract, tryptone, and sodium chloride on the luminescence of A. fischeri. The result showed that sodium chloride is the most important factor, congruent with its inducer role in bioluminescence. The best medium for bioluminescence induction was selected through an optimization plot, this medium is inexpensive, and generates the same luminescence as commercial formulations. The estimation of A. fischeri growth at OD₆₀₀ measurement was statistically analyzed. All evaluated models fitted the data adequately (r²  > 0.96). The nonlinear models Gompertz, Richards and logistic provided a lower variation and a better fit of the growth estimation (r² >0.99), showing that these mathematical models can be used for the accurate growth prediction of A. fischeri.     

Combination ecotoxicity and testing of common chemical discharges to sewer using the<Emphasis Type="Italic"> Vibrio fischeri</Emphasis> luminescence bioassay

In order to investigate possible synergistic or antagonistic (more or less than additive) toxicity effects, mixtures of chemicals were tested in water using a microbial bioassay. Ten toxicants (3,4-dichloroaniline, 3,5-dichlorophenol, cadmium, chromium, copper, Lindane, linear alkylbenzene sulphonate, pentachlorophenol, toluene, zinc) were chosen on the basis of their common occurrence in industrial effluents within local waste water treatment plants. These toxicants also cover a wide range of modes of toxic action, namely, polar and non-polar narcosis, membrane disruption, respiratory disruption, uncouplers of oxidative phosphorylation, biochemical disruption and enzyme inhibition. Efficient screening for possible combination toxicity between toxicants involved testing the chemicals both singly and in triplet combinations. The triplets were based on four replicates of a balanced incomplete block design (BIB). A standardised Vibrio fischeri rapid toxicity bioluminescence assay was used. The combinations tested showed that only one mixture was found to be significantly more toxic than expected from the pure single-toxicant results. Two triplets were significantly less toxic. Further tests on the more toxic triplet showed that the effect was due to only one of the 45 pairs originally screened. It is concluded that synergistic effects in combinations of toxicants are rather rare in bioluminescence systems utilising common effluents discharged to sewer. Electronic Publication     

The cytochromes of luminous bacteria and their coupling to bioluminescence

Vibrio fischeri andV. harveyi possess cytochromes a, b, and c, whereasPhotobacterium leiognathi andP. phosphoreum also contain cytochrome d. In all, cytochrome a as well as some of c binds carbon monoxide. Carbon monoxide does not inhibit bioluminescence (in vivo or in vitro), but carbonyl cyanidem-chlorophenylhydrazone inhibits only in vivo bioluminescence. This inhibition is due to dissipation of the proton motive force which indirectly inhibits bioluminescence by interruption of aldehyde recycling. Bioluminescence is thereby indirectly coupled to the proton motive force.     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.     

New rfp- and pES213-Derived Tools for Analyzing Symbiotic Vibrio fischeri Reveal Patterns of Infection and lux Expression In Situ

Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by ~99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.     

Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m⁻²) and solar irradiation (≈620 W m⁻²). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py⁺-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.     

Detection of bioluminescence from individual bacterial cells: a comparison of two different low-light imaging systems

Detection of very low light levels arising from individual cells of the naturally bioluminescent bacterium Vibrio fischeri as well as from a luminescence-marked Pseudomonas putida strain was achieved by the aid of two different camera systems. Using a liquid nitrogen-cooled slow-scan CCD (charge-coupled device) camera we were able to detect single-cell bioluminescence within 1 min, and the pictures obtained were of good resolution. In contrast, employing a photon-counting video camera we were able to detect bioluminescent cells within 10 seconds, but at the expense of spatial resolution. This study demonstrates the feasibility of microscopic single cell analysis employing bioluminescence as reporter system. © 1997 John Wiley & Sons, Ltd.