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QSTR with extended topochemical atom indices. Part 5: Modeling of the acute toxicity of phenylsulfonyl carboxylates to Vibrio fischeri using genetic function approximation.

In continuation of our recent efforts to model the acute toxicity of 56 phenylsulfonyl carboxylates to Vibrio fischeri using principal component factor analysis, the present paper deals with modeling of the same data set with extended topochemical atom (ETA) indices using genetic function approximation (GFA) as the statistical tool. The statistical quality of the best model using ETA descriptors is as follows: n=56, Q2=0.771, Ra2=0.861, R=0.935, F=69.0 (df5,50), s=0.172, AVRES=0.128. This equation was better in statistical quality than that obtained previously using principal component factor analysis as the data-preprocessing step. An attempt was also made to model the data set with different nonETA parameters (topological indices including Wiener, Hosoya Z, molecular connectivity, kappa shape, Balaban J and E-State parameters apart from physicochemical parameters like AlogP98, MolRef and H_bond_acceptor) and the best model showed the following quality: n=56, Q2=0.805, Ra2=0.820, R=0.913, F=53.5 (df4,51), s=0.196, AVRES=0.136. An attempt to use both ETA and nonETA parameters lead to an equation which is marginally better than the best ETA model: n=56, Q2=0.779, Ra2=0.865, R=0.937, F=71.7 (df5,50), s=0.169, AVRES=0.127. Use of the ETA indices suggested negative contributions of steric bulk, functionality of C10, volume of substituents on C10 and functionalities of chloro and nitro substituents at X1 position and positive contributions of functionalities of C4 and C1 and presence of substituents with electronegative atoms, especially at R2 and R3 positions. Furthermore, absence of substituents at R2 and R3 positions decreases toxicity. This study corroborates the results obtained from principal component factor analysis and suggests that ETA parameters are sufficiently rich in chemical information to encode the structural features contributing significantly to the acute toxicity of phenylsulfonyl carboxylates to V. fischeri.

Aliivibrio fischeri↗

Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri.

Most Vibrio fischeri strains isolated from the Euprymna scolopes light organ carry plasmids, often including both a large (>40kb) plasmid, and one or more small (<12kb) plasmids. The large plasmids share homology with pES100, which is the lone plasmid in V. fischeri type strain ES114. pES100 appears to encode a conjugative system similar to that on plasmid R721. The small plasmids lack extensive similarity to pES100, but they almost always occur in cells that also harbor a large plasmid resembling pES100. We found that many or all of these small plasmids share homology with pES213, a plasmid in strain ES213. We determined the 5501-bp pES213 sequence and generated selectable antibiotic resistance encoding pES213 derivatives, which enabled us to examine replication, retention, and transfer in V. fischeri. An 863-bp fragment of pES213 with features characteristic of theta-type replicons conferred replication without requiring any pES213 open reading frame (ORF). We estimated that pES213 derivatives were maintained at 9.4 copies per genome, which corresponds well with a model of random plasmid segregation to daughter cells and the approximately 10(-4) per generation frequency of plasmid loss. pES213 derivatives mobilized between V. fischeri strains at frequencies up to approximately 10(-4) in culture and in the host, apparently by employing the pES100 conjugative apparatus. pES213 carries two homologs of the putative pES100 origin of transfer (oriT), and V. fischeri strains lacking the pES100 conjugative relaxase, including a relaxase mutant, failed to serve as donors for transmission of pES213 derivatives. In other systems, genes directing conjugative transfer can function in trans to oriT, so it was noteworthy that ORFs adjacent to oriT, VFB51 in pES100 and traYZ in pES213, enhanced transfer 100- to 1000-fold when provided in cis. We also identified and disrupted the V. fischeri recA gene. RecA was not required for stable pES213 replication but surprisingly was required in donors for efficient transfer of pES213 derivatives. These studies provide an explanation for the prevalence and co-occurrence of pES100- and pES213-type plasmids, illuminate novel elements of pES213 mobilization, and provide the foundation for new genetic tools in V. fischeri.

Aliivibrio fischeri↗

Variation in, and causes of, toxicity of cigarette butts to a cladoceran and microtox.

Cigarette butts are the most numerically frequent form of litter in the world. In Australia alone, 24-32 billion cigarette butts are littered annually. Despite this littering, few studies have been undertaken to explore the toxicity of cigarette butts in aquatic ecosystems. The acute toxicity of 19 filtered cigarette types to Ceriodaphnia cf. dubia (48-hr EC50 (immobilization)) and Vibrio fischeri (30-min EC50 (bioluminescence)) was determined using leachates from artificially smoked cigarette butts. There was a 2.9- and 8-fold difference in toxicity between the least and most toxic cigarette butts to C. cf. dubia and V. fischeri, respectively. Overall, C. cf. dubia was more inherently sensitive than V. fischeri by a factor of approximately 15.4, and the interspecies relationship between C. cf. dubia and V. fischeri was poor (R(2) = 0.07). This poor relationship indicates that toxicity data for cigarette butts for one species could not predict or model the toxicity of cigarette butts to the other species. However, the order of the toxicity of leachates can be predicted. It was determined that organic compounds caused the majority of toxicity in the cigarette butt leachates. Of the 14 organic compounds identified, nicotine and ethylphenol were suspected to be the main causative toxicants. There was a strong relationship between toxicity and tar content and between toxicity and nicotine content for two of the three brands of cigarettes (R(2 )> 0.70) for C. cf. dubia and one brand for V. fischeri. However, when the cigarettes were pooled, the relationship was weak (R(2) < 0.40) for both test species. Brand affected the toxicity to both species but more so for V. fischeri.

Aliivibrio fischeri↗

Bioluminescence of Vibrio fischeri in continuous culture: optimal conditions for stability and intensity of photoemission.

Continuous cultivation of the bioluminescent bacterium, Vibrio fischeri NRRL-B-11177, in a fermenter provided a constant supply of bacteria in exponential growth phase. These bacteria may be used as stable bioindicators to measure perturbation of the metabolic state by physical and chemical agents. However, optimization of light emission necessitates careful choice of growth medium and culture operating conditions. Optimized conditions that supported long-term cultivation of V. fischeri NRRL-B-11177 with stable intense bioluminescence were: a dilution rate of 0.08-0.09h(-1), air supply of 600mlmin(-1), stirring of 300-350rpm at a constant incubation temperature within the range of 20 to 26 degrees C and pH7.8. These conditions were as successful in a 500-ml as in a 10-ml fermenter. This system provided a reliable long-term (more than 1 month) continuous culture facility for the reproducible measurement of perturbation of V. fischeri by monitoring changes in luminescence.

Aliivibrio fischeri↗

Comparative toxic effects of formulated simazine on Vibrio fischeri and gilthead seabream (Sparus aurata L.) larvae.

The use of Early Life Stage (ELS) tests is a useful tool in risk assessment. The purpose of this study was to compare the sensitivity of the seabream (Sparus aurata) larvae with the extensively used Microtox test on a commercial formulation containing simazine, an s-triazine herbicide. To this end, survival, growth and histopathological changes displayed by seabream yolk sac larvae exposed during 72 h post-hatching to nominal concentrations of the commercial preparation up to its saturating concentration in water, and bioluminescence of the marine bacteria Vibrio fischeri (Microtox) were studied. Survival of larvae after three days of exposure was significantly reduced in the highest (4.5 mg/l) concentration, but no effects on growth were found in any of the simazine treatments. The 72 h LC50 value for yolk sac larvae was estimated as 4.19 mg/l. Commercial grade simazine did not exert any significant toxicity to the marine bacterium V. fischeri at the concentrations tested.

Aliivibrio fischeri↗

Influence of cyclodextrin complexation on the Vibrio fischeri toxicity of phenylsulfonyl carboxylates.

This paper describes the research about the beta -cyclodextrin influence on Vibrio fischeri toxicity of 10 phenylsulfonyl carboxylates. It is illustrated that the formation of 1:1 cyclodextrin-solute inclusion complexes dominates the toxicity influence of phenylsulfonyl carboxylates to Vibrio fischeri and greatly weakens the toxicity level. Using the octanol-water partition coefficients lg KOW, the grouped electrotopological state (E-state) index S(> SO2) and the stability constants of inclusion complexes lg KS, three models with high squared correlation coefficients r2adj at 0.908, 0.951 and 0.917, respectively were constructed to evaluate the beta-cyclodextrin influence on Vibrio fischeri toxicity. For the type of phenylsulfonyl carboxylates, the inclusion stability constants lg KS can reliably model and predict the beta-cyclodextrin influence on Vibrio fischeri toxicity (TI).

Aliivibrio fischeri↗

Diguanylate cyclases control magnesium-dependent motility of Vibrio fischeri.

Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB, respectively, for magnesium-dependent induction of flagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we introduced the wild-type genes on high-copy plasmids into Escherichia coli. We assayed for the production of cyclic di-GMP using two-dimensional thin-layer chromatography and found that strains carrying these plasmids produced a small but reproducible spot that migrated with an R(f) value consistent with cyclic di-GMP that was not produced by strains carrying the vector control. Disruptions of mifA or mifB increased flagellin levels, while multicopy expression decreased them. Semiquantitative reverse transcription-PCR experiments revealed no significant difference in the amount of flagellin transcripts produced in either the presence or absence of Mg(2+) by either vector control or mifA-overexpressing cells, indicating that the impact of magnesium and cyclic-di-GMP primarily acts following transcription. Finally, we present a model for the roles of magnesium and cyclic di-GMP in the control of motility of V. fischeri.

Aliivibrio fischeri↗

A novel RBF neural network training methodology to predict toxicity to Vibrio fischeri.

This work introduces a neural network methodology for developing QSTR predictors of toxicity to Vibrio fischeri. The method adopts the Radial Basis Function (RBF) architecture and the fuzzy means training strategy, which is fast and repetitive, in contrast to most traditional training techniques. The data set that was utilized consisted of 39 organic compounds and their corresponding toxicity values to Vibrio fischeri, while lipophilicity, equalized electronegativity and one topological index were used to provide input information to the models. The performance and predictive ability of the RBF model were illustrated through external validation and various statistical tests. The proposed methodology can be used to successfully model toxicity to Vibrio fischeri for a heterogeneous set of compounds.

Aliivibrio fischeri↗

Tropical ecotoxicity testing with Ceriodaphnia cornuta.

The Sai Gon-Dong Nai river system in southern Vietnam is of great social and economic importance yet receives a large amount of industrial, domestic, and agricultural discharges. Toxicity assessment has started to become an issue in Vietnam, and it is important to employ a test system that is appropriate for typical Vietnamese conditions with a species that is representative of the invertebrates living in its aquatic ecosystems. The aim of this study was to develop and to validate an ecotoxicity test with an autochthonous organism. The microcrustacean Ceriodaphnia cornuta (Cladocera) was isolated from the Sai Gon River. A protocol was developed for the culturing of the organisms, and satisfactory results were obtained for long-term reproduction of C. cornuta. Quality control criteria were established. The toxicity of two relevant pesticides (diazinon and methyl parathion) and two metals (chromium and mercury) to C. cornuta was evaluated and compared to the standard organisms for ecotoxicological testing: Daphnia magna and Vibrio fischeri. Nonlinear regression models were applied to estimate such parameters as EC(50). The results of acute toxicity tests showed that C. cornuta was less tolerant than D. magna and V. fischeri to salinity and that C. cornuta was more sensitive than D. magna and V. fischeri to potassium dichromate, diazinon, methyl parathion, and mercury.

Aliivibrio fischeri↗

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 approximately 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.

Acyltransferases↗

Evaluation of antibacterial activity of extracts of five species of wood-colonizing fungi.

Screening new organisms for antibacterial activity and searching for new antibacterial drugs is important due to the constant generation of new antibiotic-resistant strains of pathogenic bacteria. An E. coli broth microdilution test was used to evaluate the results of the Vibrio fischeri bioluminescence test in five of the most antibacterially active species of wood-colonizing fungi. Serpula lacrymans was found to be a potential source of thermostable antibiotic(s) and the Vibrio fischeri bioluminescence test was confirmed to be a useful method for screening for antibacterial activity.

Aliivibrio fischeri↗

Diethylaminoethyl-cellulose clean-up of a large volume naphthenic acid extract.

The Athabasca oil sands of Alberta, Canada contain an estimated 174 billion barrels of bitumen. During oil sands refining processes, an extraction tailings mixture is produced that has been reported as toxic to aquatic organisms and is therefore collected in settling ponds on site. Investigation into the toxicity of these tailings pond waters has identified naphthenic acids (NAs) and their sodium salts as the major toxic components, and a multi-year study has been initiated to identify the principal toxic components within NA mixtures. Future toxicity studies require a large volume of a NA mixture, however, a well-defined bulk extraction technique is not available. This study investigated the use of a weak anion exchanger, diethylaminoethyl-cellulose (DEAE-cellulose), to remove humic-like material present after collecting the organic acid fraction of oil sands tailings pond water. The NA extraction and clean-up procedure proved to be a fast and efficient method to process large volumes of tailings pond water, providing an extraction efficiency of 41.2%. The resulting concentrated NA solution had a composition that differed somewhat from oil sands fresh tailings, with a reduction in the abundance of lower molecular weight NAs being the most significant difference. This reduction was mainly due to the initial acidification of tailings pond water. The DEAE-cellulose treatment had only a minor effect on the NA concentration, no noticeable effect on the NA fingerprint, and no significant effect on the mixture toxicity towards Vibrio fischeri.

Aliivibrio fischeri↗