2-Bromoethanesulfonate Affects Bacteria in a Trichloroethene-Dechlorinating Culture

Long-term exposure to 2-bromoethanesulfonate (BES), an agent known to inhibit methanogenesis, altered the bacterial community structure of an anaerobic enrichment culture that reductively dechlorinated trichloroethene (TCE). BES did not hinder the dechlorination of TCE or other chlorinated ethenes as previously reported, although different intermediates and end products were observed.     

Characterization of 2-bromoethanesulfonate as a selective inhibitor of the coenzyme m-dependent pathway and enzymes of bacterial aliphatic epoxide metabolism

Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO(2) fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO(2)-fixing enzyme 2-KPCC.     

2-bromoethanesulfonate (BES) Enhances Sulfate-reducing Bacterial Population and Dichlorodiphenyltrichloroethane (DDT) Dechlorination in an Anaerobic Paddy Soil

2-bromoethanesulfonate (BES) is a structural analogue of 2-mercaptoethanesulfonic acid (coenzyme M) and often used to specifically inhibit methanogenesis. The role of BES and sulfate on the reductive dechlorination of dichlorodiphenyltrichloroethane (DDT) was compared in an anaerobic soil slurry reactor of sulfate-reducing system in this study. The population of soil sulfate-reducing bacteria (SRB) was markedly decreased under DDT condition compared to DDT-free reactor, while greatly increased by sulfate and slightly increased by BES. However, the dechlorination rate of DDT was the highest in the DDT+BES treatment, followed in order by DDT+Sulfate and the control condition. In the DDT+BES treatment, more than 60% of DDT-Cl was cleaved within 16 weeks, which was about 124% and 369% greater than that in the DDT+Sulfate treatment and under the control condition, respectively. The results suggested that the inhibition of methanogenesis by BES was another pathway to improve sulfate-reducing activity and the related dechlorination rate of DDT in waterlogged soils.     

Reactive oxygen species (ROS) generated by cyanobacteria act as an electron acceptor in the biocathode of a bio-electrochemical system

The enhanced electricity generation in a biocathode bio-electrochemical system (BES) with Microcystis aeruginosa IPP as the cathodic microorganism under illumination is investigated. The results show that this cyanobacterium is able to act as a potential cathodic microorganism under illumination. In addition, M. aeruginosa IPP is found to produce reactive oxygen species (ROS) in its growth in the BES. ROS, as more competitive electron acceptors than oxygen, are utilized prior to oxygen. The BES current is substantially reduced when the ROS production is inhibited by mannitol, indicating that the ROS secreted by the cyanobacterium play an important role in the electricity generation of such a biocathode BES. This work demonstrates that the ROS released by cyanobacteria benefit for an enhanced electricity generation of BES.     

Inhibitory effects of 2-bromoethanesulfonate and protection by addition of coenzyme M in hydrogen-oxidizing marine enrichment cultures

Abstract Since bromoethanesulfonate (BES) is an inhibitor of methane production (competitive with methyl-coenzyme M), cells able to accumulate large internal pools of methyl-coenzyme M via uptake of its precursor, HS-CoM, should be protected from BES by addition of HS-CoM to the growth medium. Hydrogen-oxidizing marine methanogen enrichments were prepared from anaerobic sediment samples collected at Sippewisset Salt Marsh and Oyster Bay Inlet near Woods Hole, MA. The three enrichments studied were a mixture of cell types with at least 50% of the culture comprised of methanogens. Methane production was found to be sensitive to BES with half maximal inhibition occurring at 5–20 μM BES depending on the enrichment. For each, half maximal protection against 40 μM BES occurred at a HS-CoM: BES molar ratio of 20: 1 to 40: 1. Since the protected enrichments exhibited normal sensitivity toward BES after removal of HS-CoM, it was concluded that methane production in the presence of both BES and HS-CoM resulted from true protection and not growth of BES-resistant mutants. These results suggest that uptake of HS-CoM may be a general property of methanogens occupying anaerobic marine sediments. It is possible that uptake of this coenzyme is an important nutritional feature of methanogens in their natural habitat.     

Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system.

Mutants of Methanococcus voltae were isolated that were resistant to the coenzyme M (CoM; 2-mercaptoethanesulfonic acid) analog 2-bromoethanesulfonic acid (BES). The mutants displayed a reduced ability to accumulate [35S]BES relative to the sensitive parental strain. BES inhibited methane production from CH3-S-CoM in cell extracts prepared from wild-type sensitive or resistant strains. BES uptake required the presence of both CO2 and H2 and was inhibited by N-ethylmaleimide and several reagents that are known to disrupt energy metabolism. The mutants showed normal uptake of isoleucine and were not cross-resistant to either azaserine or 5-methyltryptophan and, thus, were neither defective in general energy-dependent substrate transport nor envelope permeability. Both HS-CoM and CH3-S-CoM prevented the uptake of BES and protected cells from inhibition by it. We propose that M. voltae has an energy-dependent, carrier-mediated uptake system for HS-CoM and CH3-S-CoM which can also mediate uptake of BES.     

Characterization of the adaptive response to trichloroethylene-mediated stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Bronchial epithelial spheroids: an alternative culture model to investigate epithelium inflammation-mediated COPD

Background

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal lung inflammation that exceeds the protective response. Various culture models using epithelial cell lines or primary cells have been used to investigate the contribution of bronchial epithelium in the exaggerated inflammation of COPD. However, these models do not mimic in vivo situations for several reasons (e.g, transformed epithelial cells, protease-mediated dissociation of primary cells, etc.). To circumvent these concerns, we developed a new epithelial cell culture model.

Methods

Using non transformed non dissociated bronchial epithelium obtained by bronchial brushings from COPD and non-COPD smokers, we developed a 3-dimensional culture model, bronchial epithelial spheroids (BES). BES were analyzed by videomicroscopy, light microscopy, immunofluorescence, and transmission electron microscopy. We also compared the inflammatory responses of COPD and non-COPD BES. In our study, we chose to stimulate BES with lipopolycaccharide (LPS) and measured the release of the pro-inflammatory mediators interleukin-8 (IL-8) and leukotriene B4 (LTB4) and the anti-inflammatory mediator prostaglandin E2 (PGE2).

Results

BES obtained from both COPD and non-COPD patients were characterized by a polarized bronchial epithelium with tight junctions and ciliary beating, composed of basal cells, secretory cells and ciliated cells. The ciliary beat frequency of ciliated cells was not significantly different between the two groups. Of interest, BES retained their characteristic features in culture up to 8 days. BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS. Interestingly, LPS induced a higher release of IL-8, but not PGE2 and LTB4 in COPD BES (p < 0.001) which correlated with lung function changes.

Conclusion

This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium. More importantly, BES represent an attractive culture model to investigate the mechanisms of injuring agents that mediate epithelial cell inflammation and its contribution to COPD pathogenesis.     

Effect of trichloroethylene (TCE) and toluene concentrations on TCE and toluene biodegradation and the population density of TCE and toluene degraders in soil.

Toluene is one of several cosubstrates able to support the cometabolism of trichloroethylene (TCE) by soil microbial communities. Indigenous microbial populations in soil degraded TCE in the presence, but not the absence, of toluene after a 60- to 80-h lag period. Initial populations of toluene and TCE degraders ranged from 0.2 x 10(3) to 4 x 10(3) cells per g of soil and increased by more than 4 orders of magnitude after the addition of 20 micrograms of toluene and 1 microgram of TCE per ml of soil solution. The numbers of TCE and toluene degraders and the percent removal of TCE increased with an increase in initial toluene concentration. As the initial TCE concentration was increased from 1 to 20 micrograms/ml, the numbers of toluene and TCE degraders and the rate of toluene degradation decreased, and no TCE degradation occurred. No toluene or TCE degradation occurred at a TCE concentration of 50 micrograms/ml.     

Characterization of the Adaptive Response to Trichloroethylene-Mediated Stresses in Ralstonia pickettii PKO1

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.     

Strategies to suppress hydrogen-consuming microorganisms affect macro and micro scale structure and microbiology of granular sludge

Treatment of anaerobic granules with heat and two chemical treatments, contacting with 2-bromoethanesulfonate (BES) and with BES + Chloroform, were applied to suppress hydrogen-consuming microorganisms. Three mesophilic expanded granular sludge bed (EGSB) reactors-R(Heat), R(BES), and R(BES + Chlo)--were inoculated with the treated sludges and fed with synthetic sugar-based wastewater (5 g(COD) L(-1), HRT 20-12 h). Morphological integrity of granules and bacterial communities were assessed by quantitative image analysis and 16S rRNA gene based techniques, respectively. Hydrogen production in R(Heat) was under 300 mL H(2) L(-1) day(-1), with a transient peak of 1,000 mL H(2) L(-1) day(-1) after decreasing HRT. In R(BES + Chlo) hydrogen production rate did not exceed 300 mL H(2) L(-1) day(-1) and there was granule fragmentation, release of free filaments from aggregates, and decrease of granule density. In R(BES), there was an initial period with unstable hydrogen production, but a pulse of BES triggered its production rate to 700 ± 200 mL H(2) L(-1) day(-1). This strategy did not affect granules structure significantly. Bacteria branching within Clostridiaceae and Ruminococcaceae were present in this sludge. This work demonstrates that, methods applied to suppress H(2)-consuming microorganisms can cause changes in the macro- and microstructure of granular sludge, which can be incompatible with the operation of high-rate reactors.     

Trichloroethylene metabolism in the rat ovary reduces oocyte fertilizability

Exposure to trichloroethylene (TCE, an environmental toxicant) reduced oocyte fertilizability in the rat. In vivo, TCE may be metabolized by cytochrome P450 dependent oxidation or glutathione conjugation in the liver or kidneys, respectively. Cytochrome P450 dependent oxidation is the higher affinity pathway. The primary isoform of cytochrome P450 to metabolize TCE in the liver, cytochrome P450 2E1, is present in the rodent ovary. Ovarian metabolism of TCE by the oxidative pathway and the production of reactive oxygen species (ROS) may occur given the presence of the metabolizing enzyme. The objectives of this study were to define the sensitive interval of oocyte growth to TCE exposure, and to determine if TCE exposure resulted in the formation of ovarian protein carbonyls, an indicator of oxidative damage. Rats were exposed to TCE in drinking water (0.45% TCE (v/v) in 3% Tween) or 3% Tween (vehicle control) during three 4-5 day intervals of oocyte development preceding ovulation. Oocytes from TCE-exposed females were less fertilizable compared with vehicle-control oocytes. Immunohistochemical labeling of ovaries and Western blotting of ovarian proteins demonstrated TCE treatment induced a greater incidence of protein carbonyls compared with vehicle controls. Protein carbonyl formation in the ovary is consistent with TCE metabolism by the cytochrome P450 pathway. Oxidative damage following ovarian TCE metabolism or the presence of TCE metabolites may contribute to reduced oocyte fertilizability. In summary, these results indicate maturing oocytes are susceptible to very short in vivo exposures to TCE.     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Characterization of a deep-coverage carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) BAC library and initial analysis of BAC-end sequences

Carrot is the most economically important member of the Apiaceae family and a major source of provitamin A carotenoids in the human diet. However, carrot molecular resources are relatively underdeveloped, hampering a number of genetic studies. Here, we report on the synthesis and characterization of a bacterial artificial chromosome (BAC) library of carrot. The library is 17.3-fold redundant and consists of 92,160 clones with an average insert size of 121 kb. To provide an overview of the composition and organization of the carrot nuclear genome we generated and analyzed 2,696 BAC-end sequences (BES) from nearly 2,000 BACs, totaling 1.74 Mb of BES. This analysis revealed that 14% of the BES consists of known repetitive elements, with transposable elements representing more than 80% of this fraction. Eleven novel carrot repetitive elements were identified, covering 8.5% of the BES. Analysis of microsatellites showed a comparably low frequency for these elements in the carrot BES. Comparisons of the translated BES with protein databases indicated that approximately 10% of the carrot genome represents coding sequences. Moreover, among eight dicot species used for comparison purposes, carrot BES had highest homology to protein-coding sequences from tomato. This deep-coverage library will aid carrot breeding and genetics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers FJ147695–FJ150390.     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture.

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Recovery of trichloroethylene removal efficiency through short-term toluene feeding in a biofilter enriched withPseudomonas putida F1

Trichloroethylene (TCE) is an environmental contaminant provoking genetic mutation and damages to liver and central nerve system even at low concentrations. A practical scheme is reported using toluene as a primary substrate to revitalize the biofilter column for an extended period of TCE degradation. The rate of trichloroethylene (TCE) degradation byPseudomonas putida F1 at 25°C decreased exponentially with time, without toluene feeding to a biofilter column (11 cm I.D.×95 cm height). The rate of decrease was 2.5 times faster at a TCE concentration of 970 μg/L compared to a TCE concentration of 110 μg/L. The TCE itself was not toxic to the cells, but the metabolic intermediates of the TCE degradation were apparently responsible for the decrease in the TCE degradation rate. A short-term (2 h) supply of toluene (2,200 μg/L) at an empty bed residence time (EBRT) of 6.4 min recovered the relative column activity by 43% when the TCE removal efficiency at the time of toluene feeding was 58%. The recovery of the TCE removal efficiency increased at higher incoming toluene concentrations and longer toluene supply durations according to the Monod type of kinetic expression. A longer duration (1.4∼2.4 times) of toluene supply increased the recovery of the TCE removal efficieny by 20% for the same toluene load.