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.     

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.     

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.     

Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate.

Strain Co23, an anaerobic spore-forming microorganism, was enriched and isolated from a compost soil on the basis of its ability to grow with 2,3-dichlorophenol (DCP) as its electron acceptor, ortho chlorines were removed from polysubstituted phenols but not from monohalophenols. Growth by chlororespiration was indicated by a growth yield of 3.24 g of cells per mol of reducing equivalents (as 2[H]) from lactate oxidation to acetate in the presence of 3-chloro-4-hydroxybenzoate but no growth in the absence of the halogenated electron acceptor. Other indicators of chlororespiration were the fraction of electrons from the electron donor used for dechlorination (0.67) and the H2 threshold concentration of < 1.0 ppm. Additional electron donors utilized for reductive dehalogenation were pyruvate, formate, butyrate, crotonate, and H2. Pyruvate supported homoacetogenic growth in the absence of an electron acceptor. Strain Co23 also used sulfite, thiosulfate, and sulfur as electron acceptors for growth, but it did not use sulfate, nitrate or fumarate. The temperature optimum for growth was 37 degrees C; however, the rates of dechlorination were optimum at 45 degrees C and activity persisted to temperatures as high as 55 degrees C. The 16S rRNA sequence was determined, and strain Co23 was found to be related to Desulfitobacterium dehalogenans JW/IU DC1 and Desulfitobacterium strain PCE1, with sequence similarities of 97.2 and 96.8%, respectively. The phylogenetic and physiological properties exhibited by strain Co23 place it into a new species designated Desulfitobacterium chlororespirans.     

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 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     

Studies with 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and related compounds in soils and plants

The inactivation of the herbicide ioxynil by contact with soil has been investigated. Shaking solutions of the sodium salt with acid soils led to a precipitation of the herbicide. With alkaline soils, a small amount of ioxynil became adsorbed on the soil particles. With unsterilized soils, hydrolysis to 3,5-diiodo-4-hydroxybenzoic acid occurred, with 3,5-diiodo-4-hydroxy-benzamide as an intermediate product. Liberation of iodide ion in this system was also demonstrated. The phytotoxicity of ioxynil is enhanced by exposure of treated plants to light. The reduction in chlorophyll level of bean leaf tissue treated with ioxynil and other dihalogenohydroxybenzonitriles when exposed to light has been determined. Evidence is presented showing that although ioxynil is poorly translocated in the dwarf bean plant, its degradation products appear in the shoots of these plants after the herbicide has been supplied through the roots.     

Decomposition of the herbicide bromoxynil in soil and in bacterial cultures

It was found in field, and laboratory experiments that of 50 ppm of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile added to grey forest soil 20-80% were still detected after three months). Bromoxynil did not influence (except for a short-termed stimulation of the number of bacteria) the amount and composition of the basic groups of soil microorganisms. In enrichment cultures of soil microorganisms metabolie products of bromoxynil decomposition (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid) were detected and a stimulating effect of cosubstratos on its decomposition was demonstrated. Bromoxynil concentration, aeration conditions and the presence of cosubstrates (ribose in particular) influenced the rate and degree of the decomposition process inPsevdomonas putida. In addition to the degradation products mentioned above, production of methoxylated and partially dehalogenated aromatic compounds was detected.     

Pure Culture Studies of Erwinia carotovora with 3,5-Diiodo-4-Hydroxybenzonitrile

Interactions of ioxynil (3,5-diiodo-4-hydroxybenzonitrile) with a pure culture of Erwinia carotovora grown in a glucose-simple salts medium were studied. Growth of E. carotovora was inhibited by ioxynil and, to a lesser extent, by its acid form at 25 and 50 mug/ml. Growth was not inhibited by the amide or ester forms of ioxynil or p-hydroxybenzonitrile at the same concentrations. E. carotovora could be trained to grow in 50 mug or higher concentrations of ioxynil per ml by serial transfers of the organism through increasing ioxynil concentrations. No degradation or detoxification of ioxynil was detected. Toxicity tests indicated that, in the adapted culture, cell-free supernatant fluid remained toxic to a nonadapted culture. Adaptation of E. carotovora resulted in a lengthened lag phase, a decreased growth rate, and very few adverse effects on the total population. The adapted resistant culture retained this characteristic only when ioxynil was present. Adaptation was demonstrated to be a physiological variation, not a selection of a mutant or of preexisting resistant cells. Ioxynil slightly stimulated the respiration rate of E. carotovora and moderately inhibited that of an adapted culture. Because the respiration rate of an adapted culture in the absence of ioxynil surpassed that of a parent culture still in the presence of ioxynil, a competition of two alternate routes of electron transport is implied. These data support the conclusion that an alternate growth mechanism is involved in the adaptation mechanism.     

Effects of 3,5-Dihalogenated-4-hydroxybenzonitriles on the Activity of Mitochondria From White Potato Tubers

Effects of the herbicide 3,5-diiodo-4-hydroxybenzonitrile (ioxynil), and its dibromo and dichloro analogs, upon the respiration of mitochondria isolated from white potato tubers (Solanum tuberosum L.) were investigated. Mitochondrial-mediated oxygen uptake was monitored polarographically with a platinum oxygen electrode.     

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.     

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.     

Autodisplay of nitrilase from Klebsiella pneumoniae and whole-cell degradation of oxynil herbicides and related compounds

Using the Autodisplay system, a recombinant Escherichia coli strain displaying the dimeric nitrilase from Klebsiella pneumoniae subsp. ozaenae (NitKp) on the cell surface was constructed. Localization of the nitrilase in the cell envelope of E. coli was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis and surface exposure was verified by its accessibility to externally added protease. The whole-cell biocatalyst obtained converted the substrates analyzed in the following order: chloroxynil?>?bromoxynil?>?ioxynil?>?3-bromo-4-hydroxybenzonitrile (1.67, 0.89, 0.13, and 0.09 mM product formation within 72 h, respectively), indicating the same substrate specificity for the displayed enzyme as for the free enzyme. The whole-cell biocatalyst was also able to convert 3-fluoro-4-hydroxybenzonitrile and 3,5-dimethyl-4-hydroxybenzonitrile to the corresponding carboxylic acids. In contrast, it was not possible to detect any enzyme activity when 4-methoxybenzonitrile was used as substrate. The temperature optimum determined was 45 °C for the surface-displayed enzyme instead of 35 °C for the purified enzyme. In addition, the optimum activity of the displayed nitrilase was shifted to more acidic pH in comparison to the free enzyme.     

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.     

Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.     

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.     

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     

Initial Characterization of a Reductive Dehalogenase from Desulfitobacterium chlororespirans Co23

Desulfitobacterium chlororespirans Co23 is capable of using 3-chloro-4-hydroxybenzoate as terminal electron acceptor for growth. Membrane preparations from cells grown fermentatively on pyruvate in the presence of 3-chloro-4-hydroxybenzoate dechlorinated this compound at a rate of 3.9 nmol min(sup-1) mg of protein(sup-1). Fivefold-greater dechlorination rates were measured with reduced methyl viologen as the artificial electron donor. Reduced benzyl viologen, NADH, NADPH, reduced flavin adenine dinucleotide, and reduced flavin mononucleotide could not substitute for reduced methyl viologen. The maximal initial rate of catalysis was achieved at pH 6.5 and 60(deg)C. The membrane-bound dechlorinating enzyme system was not oxygen sensitive and was stable at 57(deg)C for at least 2 h. Sulfite inhibited dechlorination in cell-free assays, whereas sulfate did not. Several chlorophenols were dehalogenated exclusively in the ortho position by cell extracts.