Effect of glucose and ribose on microbial degradation of the herbicide bromoxynil continuously added to soil

Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was continuously added to chernozem (Haplic typic) soil inoculated with a suspension ofPseudomonas putida capable of cometabolic decomposition of the compound in a heterocontinuous-flow cultivation setup. In the steady state, when glucose or ribose were simultaneously added, 90 and 47% of the added herbicide was degraded per day, respectively. If the saccharides were absent, only 10–27% of the herbicide was decomposed. Addition and removal of glucose feeding resulted in an increase and decrease, respectively, of the degradation intensity, irrespective of the amount of the bacterial decomposers present. Two degradation products, 3,5-dibromo-4-hydroxy-benzamide and 3,5-dibromo-4-hydroxybenzoic acid, were formed during cultivation. The total amount of bromine-containing compounds was reduced only in the presence of glucose. Translated by Č. Novotny     

Primary effect of bromoxynil to induce plant cell death may be cytosol acidification

Bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile, is a commonly used herbicide and is also used as a tool to trigger rapid cell death in basic botany. However, the primary effect inducing cell death is not known. Bromoxynil inhibited the cytoplasmic streaming and killed cells in Chara corallina when it was applied in the acidic external medium. At higher pH, bromoxynil was inert even at high concentrations. It was speculated that bromoxynil in the protonated form enters the cell and acidifies the cytosol by releasing H⁺. Experiments using analogues of bromoxynil supported this possibility. Acidification of the cytosol by bromoxynil was confirmed by experiments using pollen tubes. Based on the acidity of the apoplast, the herbicide action of bromoxynil in higher plants was discussed.     

Degradation of the herbicide bromoxynil inPseudomonas putida

Biological conversion of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was studied in a batch culture ofPseudomonas putida by using HPLC. The process had a cometabolic character and proceeded only in the presence of another, simultaneously metabolizable, carbon and energy source. The intensity of degradation correlated with the growth rate, the degradation stopping when the cosubstrate becomes exhausted or the pH value of the medium falls below 6.5. In a medium with glucose, no lag phase longer than one day was observed concerning growth, sugar and herbicide consumption and formation of metabolic herbicide derivatives (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid). In a medium with ribose, the initial lag of the above processes took 2 d. No formation of other degradation products was detected. Growth inhibition was proportional to the concentration of bromoxynil. Translated by Č. Novotny     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae.

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Degradation of ioxynil and bromoxynil as measured by a modified spectrophotometric method.

A modified spectrophotometric method was developed to estimate ioxynil and bromoxynil residues. The method when compared with a 14C-tracer method was less sensitive but allowed rapid and accurate estimation of the herbicides. A clay loam soil with high organic matter content, which degraded ioxynil completely to CO2, also degraded bromoxynil completely. Bromoxynil degradation proceeded at a faster rate than that of ioxynil. The half-life of degradation was estimated to be 7 days for bromoxynil and 9-10 days for ioxynil. However, soil microorganisms which degraded ioxynil either completely to CO2 or partially did not seem to completely degrade bromoxynil. Degradation products from bromoxynil were detected on thin-layer chromatograms of extracts from pure cultures containing an exogenous carbon source. Complete degradation of bromoxynil and ioxynil in soil could be due to the action of different microorganisms.     

Effects of 3,5-Dibromo-4-Hydroxybenzonitrile (Bromoxynil) on Bioenergetics of Higher Plant Mitochondria (Pisum sativum)

The herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was tested on mitochondria from etiolated pea (Pisum sativum L. cv Alaska) stems. This compound when used at micromolar concentrations ([almost equal to]20 [mu]M) inhibited malate- and succinate-dependent respiration by intact mitochondria but not oxidation of exogenously added NADH. Bromoxynil did not affect the activities of the succinic and the internal NADH dehydrogenases. Analyses of the effects induced by this herbicide on the membrane potential, [delta]pH, matrix Ca2+ movements, and dicarboxylate transport demonstrated that bromoxynil is likely to act as an inhibitor of the dicarboxylate carrier. In addition, bromoxynil caused a mild membrane uncoupling at concentrations [greater than or equal to]20 [mu]M. No effect on the ATPase activity was observed.     

Degradation of Bromoxynil Octanoate by Strain Acinetobacter sp. XB2 Isolated from Contaminated Soil

Bromoxynil octanoate (BOO), the most widespread herbicide applied to maize, is potentially toxic to both animals and humans. In this article, a highly effective BOO-degrading bacterial strain, XB2, was isolated from the soil of a herbicide factory. The strain was identified as an Acinetobacter sp. based on its 16S rRNA gene sequence analysis, morphological, physiological, and biochemical properties. This strain could use BOO as its sole carbon source and could degrade 100?mg?l(-1) BOO to non-detectable levels in 72?h (h). The optimal pH and temperature for strain XB2's growth and degradation of BOO in MSM are 7.0 and 30°C, respectively. We propose the following pathway of BOO degradation by strain XB2: the first step is the scission of the ester bond to form bromoxynil, bromoxynil then transformed to 3,5-dibromo-4-hydroxybenzoic acid?due to the hydrolysis of nitriles, and debromination finally results in the formation of 3-bromo-4-hydroxybenzoic acid. Inoculating BOO-treated soil samples with strain XB2 resulted in a higher rate of BOO degradation than in non-inoculated soil, regardless of whether the soil had previously been sterilized.     

Cloning and expression in Escherichia coli of a Klebsiella ozaenae plasmid-borne gene encoding a nitrilase specific for the herbicide bromoxynil.

An enzyme (nitrilase) that converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to its metabolite 3,5-dibromo-4-hydroxybenzoic acid was shown to be plasmid encoded in the natural soil isolate Klebsiella ozaenae. The bromoxynil-specific nitrilase was expressed in Escherichia coli by direct transfer and stable maintenance in E. coli of a naturally occurring 82-kilobase K. ozaenae plasmid. Irreversible loss of the ability to metabolize bromoxynil both in E. coli and K. ozaenae was associated with the conversion of the 82-kilobase plasmid to a 68-kilobase species. In E. coli this conversion was the result of a host recA+-dependent recombinational event. A gene, designated bxn, encoding the bromoxynil-specific nitrilase was constitutively expressed in K. ozaenae and E. coli and subcloned on a 2.6-kilobase PstI DNA segment. The polarity and the location of the gene were determined by assaying hybrid constructs of the bromoxynil-specific nitrilase gene fused with the heterologous lac promoter.     

Dehalogenation of the herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodino-4-hydroxybenzonitrile) by Desulfitobacterium chlororespirans

Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 microM) and DBHB (250 to 260 microM) were used as electron acceptors for growth. Doubling times for growth (means +/- standard deviations for triplicate cultures) on bromoxynil (18.4 +/- 5.2 h) and DBHB (11.9 +/- 1.4 h), determined by rate of [14C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.     

Dehalogenation of the Herbicides Bromoxynil (3,5-Dibromo-4-Hydroxybenzonitrile) and Ioxynil (3,5-Diiodino-4-Hydroxybenzonitrile) by Desulfitobacterium chlororespirans

Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 μM) and DBHB (250 to 260 μM) were used as electron acceptors for growth. Doubling times for growth (means ± standard deviations for triplicate cultures) on bromoxynil (18.4 ± 5.2 h) and DBHB (11.9 ± 1.4 h), determined by rate of [¹⁴C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.     

Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1–1.9 U mg^?1 dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30°C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile ? chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92–100, 70–90 and 30–51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.     

The degradation of the herbicide bromoxynil and its impact on bacterial diversity in a top soil

Aims: To study how repeated applications of an herbicide bromoxynil to a soil, mimicking the regime used in the field, affected the degradation of the compound and whether such affects were reflected by changes in the indigenous bacterial community present. Methods and Results: Bromoxynil degradation was monitored in soil microcosms using HPLC. Its impact on the bacterial community was determined using denaturing gradient gel electrophoresis (DGGE) and quantitative PCR of five bacterial taxa (Pseudomonads, Actinobacteria, α‐Proteobacteria, Acidobacteria and nitrifying bacteria). Three applications of 10 mg kg^?1 of bromoxynil at 28‐day intervals resulted in rapid degradation, the time for removal of 50% of the compound decreasing from 6·4 days on the first application to 4·9 days by the third. Bacterial population profiles showed significant similarity throughout the experiment. With the addition of 50 mg kg^?1 bromoxynil to soil, the degradation was preceded by a lag phase and the time for 50% of the compound to be degraded increased from 7 days to 28 days by the third application. The bacterial population showed significant differences 7 days after the final application of bromoxynil that correlated with an inhibition of degradation during the same period. Conclusions: These analyses highlighted that the addition of bromoxynil gave rise to significant shifts in the community diversity and its structure as measured by four abundant taxa, when compared with the control microcosm. These changes persisted even after bromoxynil had been degraded. Significance and Impact of the Study: Here we show that bromoxynil can exert an inhibitory effect on the bacterial population that results in decreased rates of degradation and increased persistence of the compound. In addition, we demonstrate that molecular approaches can identify statistically significant changes in microbial communities that occur in conjunction with changes in the rate of degradation of the compound in the soil.     

Further electrophysiological studies on cellular effect of herbicide, bromoxynil, using characean cells

In the previous paper, I reported that 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil) depolarizes the plasma membrane by inhibiting the electrogenic proton pump and discussed that the inhibition is caused by cytosol acidification due to influx of protonated bromoxynil and following release of proton (Shimmen in J Plant Res 123:715–722, 2010). However, a possibility of direct inhibition of the proton pump by bromoxynil flowed into the cell could not be excluded. In the present study, the direct effect of bromoxynil on the proton pump was unequivocally excluded.     

Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.     

The application of the herbicide bromoxynil to a model soil-derived bacterial community: impact on degradation and community structure

AIMS: Bromoxynil degradation by soil micro-organisms has been shown to be co-oxidative in character. In this study, we investigate both the impact of the application of increasing bromoxynil concentrations on soil-derived bacterial communities and how these changes are reflected in the degradation of the compound. Our aim was to test the hypothesis that the addition of bromoxynil to a soil-derived bacterial community, and the availability of a readily utilizable carbon source would have an impact on bromoxynil degradation, and that would be reflected in the bacteria present in the soil community. METHODS AND RESULTS: Degradation of bromoxynil was observed in soil-derived communities containing 15 mg l(-1), but not 50 mg l(-1) of the compound, unless glucose was added. This suggests that the addition of carbon stimulates co-oxidative bromoxynil degradation by the members of the bacterial community. Measurable changes in the bacterial community indicated that the addition of bromoxynil led to deterministic selection on the bacterial population, i.e. the communities observed arise through the selection of specific micro-organisms that are best adapted to the conditions in the soil. The addition of bromoxynil was also shown to have a negative impact on the presence of alpha and gamma-proteobacteria in the soil community. CONCLUSION: Bromoxynil degradation is significantly inhibited in bacterial soil communities in the absence of readily accessible carbon. The application of bromoxynil appears to exert deterministic selection on the bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effects of increasing bromoxynil concentrations on a model bacterial population derived from soil. Soil communities show qualitative and quantitative differences to bromoxynil application depending on the availability of organic carbon. These findings might have implications for the persistence of bromoxynil in agricultural soils.     

Unique cellular effect of the herbicide bromoxynil revealed by electrophysiological studies using characean cells

In a previous paper, we proposed that the primary action of the herbicide bromoxynil (BX; 3,5-dibromo-4-hydroxybenzonitrile) is cytosol acidification, based on the fact that bromoxynil induced the inhibition of cytoplasmic streaming and cell death of Chara corallina in acidic external medium (Morimoto and Shimmen in J Plant Res 121:227–233, 2008). In the present study, electrophysiological analysis of the BX effect was carried out in internodal cells of C. corallina. Upon addition of BX, a large and rapid pH-dependent depolarization was induced, supporting our hypothesis. Ioxynil, which belongs to the same group as bromoxynil, also induced a large and rapid membrane depolarization in a pH-dependent manner. On the other hand, four herbicides belonging to other groups of herbicides did not induce such a membrane depolarization. Thus, BX has a unique cellular effect. The decrease in the electro-chemical potential gradient for H⁺ across the plasma membrane appears to result in inhibition of cell growth and disturbance of intracellular homeostasis in the presence of BX.     

Degradation of bromoxynil byStreptomyces felleus in soil

The completein vivo degradation of the herbicide bromoxynil byStreptomyces felleus and soil microorganisms was investigated. Little breakdown occurred in sterile soil. TLC techniques were used to detect two degradation products in non-sterile soil. Authors are obliged to Mrs. E. Chrastinová for technical assistance.     

Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene

A Klebsiella ozaenae nitrilase which converts the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) to 3,5-dibromo-4-hydroxybenzoic acid has been expressed at 5-10% of the total protein in Escherichia coli from a cloned K. ozaenae DNA segment and purified 10.3-fold to homogeneity. The purified polypeptide is molecular weight 37,000 in size, but the active form of the enzyme is composed of two identical subunits. The purified enzyme exhibits a pH optimum of 9.2 and a temperature optimum of 35 degrees C. The purified enzyme is also quite sensitive to thiol-specific reagents. The nitrilase is highly specific for bromoxynil as substrate with a Km of 0.31 mM and Vmax of 15 mumol of NH3 released/min/mg protein. Analysis of bromoxynil-related substrates indicates the enzyme exhibits preference for compounds containing two meta-positioned halogen atoms. Nucleotide sequence analysis of a 1,212-base pair PstI-HincII DNA segment containing the locus (bxn) encoding the bromoxynil-specific nitrilase reveals a single open reading frame encoding a polypeptide 349 amino acids in length. The predicted sequence of the purified enzyme was derived from the nucleotide sequence of the bxn gene.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.