Antimicrobial properties of pyridine-2,6-dithiocarboxylic acid, a metal chelator produced by Pseudomonas spp.

Pyridine-2,6-dithiocarboxylic acid (pdtc) is a metal chelator produced by Pseudomonas spp. It has been shown to be involved in the biodegradation of carbon tetrachloride; however, little is known about its biological function. In this study, we examined the antimicrobial properties of pdtc and the mechanism of its antibiotic activity. The growth of Pseudomonas stutzeri strain KC, a pdtc-producing strain, was significantly enhanced by 32 microM pdtc. All nonpseudomonads and two strains of P. stutzeri were sensitive to 16 to 32 microM pdtc. In general, fluorescent pseudomonads were resistant to all concentrations tested. In competition experiments, strain KC demonstrated antagonism toward Escherichia coli. This effect was partially alleviated by 100 microM FeCl3. Less antagonism was observed in mutant derivatives of strain KC (CTN1 and KC657) which lack the ability to produce pdtc. A competitive advantage was restored to strain CTN1 by cosmid pT31, which restores pdtc production. pT31 also enhanced the pdtc resistance of all pdtc-sensitive strains, indicating that this plasmid contains elements responsible for resistance to pdtc. The antimicrobial effect of pdtc was reduced by the addition of Fe(III), Co(III), and Cu(II) and enhanced by Zn(II). Analyses by mass spectrometry determined that Cu(I):pdtc and Co(III):pdtc2 form immediately under our experimental conditions. Our results suggest that pdtc is an antagonist and that metal sequestration is the primary mechanism of its antimicrobial activity. It is also possible that Zn(II), if present, may play a role in pdtc toxicity.

Carbon tetrachloride dechlorination by the bacterial transition metal chelator pyridine-2,6-bis(thiocarboxylic acid).

A reaction pathway is proposed to explain the formation of end products during defined chemical reactions between carbon tetrachloride (CCl4) and either metal complexes of pyridine-2,6-bis(thiocarboxylic acid) (PDTC) or pure cultures of Pseudomonas stutzeri KC. The pathway includes one-electron reduction of CCl4 by the Cu(II):PDTC complex, condensation of trichloromethyl and thiyl radicals, and hydrolysis of a labile thioester intermediate. Products detected were carbon dioxide, chloride, carbonyl sulfide, carbon disulfide, and dipicolinic acid. Spin-trapping and electrospray MS/MS experiments gave evidence of trichloromethyl and thiyl radicals generated by reaction of CCl4 with PDTC and copper. Experiments testing the effects of transition metals showed that dechlorination by PDTC requires copper and is inhibited by cobalt but not by iron or nickel. PDTC was shown to react stoichiometrically rather than catalytically without added reducing equivalents. With added reductants, an increased turnover was seen along with increased chloroform production.

Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis(thiocarboxylate)

Pseudomonas stutzeri strain KC was originally characterized as having, under iron-limiting conditions, novel carbon tetrachloride (CCl(4)) dehalogenation activity, specifically, a net conversion of CCl(4) to CO(2). The exact pathway and reaction mechanisms are unknown, but chloroform is not an intermediate and thiophosgene and phosgene have been identified as intermediates in trapping experiments. Previous work by others using cell-free preparations has shown that cell-free culture supernatants that have been passed through a low-molecular-weight cutoff membrane can confer rapid CCl(4) transformation ability upon cultures of bacteria which otherwise show little or no reactivity toward CCl(4). We used a cell-free assay system to monitor the complete purification of compounds showing CCl(4) degradation activity elaborated by iron-limited cultures of strain KC. Electrospray tandem mass spectroscopy, NMR spectroscopy, and comparisons with synthetic material have identified pyridine-2,6-bis(thiocarboxylate) as a metabolite of strain KC which has CCl(4) transformation activity in the presence of chemical reductants, e.g., titanium[III] citrate or dithiothreitiol, or actively growing bacterial cultures.

Optimization of simultaneous chemical and biological mineralization of perchloroethylene.

Optimization of the simultaneous chemical and biological mineralization of perchloroethylene (PCE) by modified Fenton's reagent and Xanthobacter flavus was investigated by using a central composite rotatable experimental design. Concentrations of PCE, hydrogen peroxide, and ferrous iron and the microbial cell number were set as variables. Percent mineralization of PCE to CO2 was investigated as a response. A second-order, quadratic response surface model was generated and fit the data adequately, with a correlation coefficient of 0.72. Analysis of the results showed that the PCE concentration had no significant effect within the tested boundaries of the model, while the other variables, hydrogen peroxide and iron concentrations and cell number, were significant at alpha = 0.05 for the mineralization of PCE. The 14C radiotracer studies showed that the simultaneous chemical and biological reactions increased the extent of mineralization of PCE by more than 10% over stand-alone Fenton reactions.

De novo synthesis of 4,5-dimethoxycatechol and 2, 5-dimethoxyhydroquinone by the brown rot fungus Gloeophyllum trabeum.

The new dimethoxycatechol 4,5-dimethoxy-1,2-benzenediol (DMC) and the new dimethoxyhydroquinone 2,5-dimethoxy-1,4-benzenediol (DMH) were isolated from stationary cultures of the brown rot fungus Gloeophyllum trabeum growing on a glucose mineral medium protected from light. The structure was elucidated by gas chromatography-mass spectrometry through comparison to a synthetic standard. Further confirmation was obtained by forming a dimethoxyoxazole derivative by condensation of DMC with methylene chloride and through examination of methylated derivatives. DMC and DMH may serve as ferric chelators, oxygen-reducing agents, and redox-cycling molecules, which would include functioning as electron transport carriers to Fenton's reactions. Thus, they appear to be important components of the brown rot decay system of the fungus.

New pathway for degradation of sulfonated azo dyes by microbial peroxidases of Phanerochaete chrysosporium and Streptomyces chromofuscus.

Pathways for the degradation of 3,5-dimethyl-4-hydroxy-azobenzene-4'-sulfonic acid (I) and 3-methoxy-4-hydroxyazobenzene-4'-sulfonamide (II) by the manganese peroxidase and ligninase of Phanerochaete chrysosporium and by the peroxidase of Streptomyces chromofuscus have been proposed. Twelve metabolic products were found, and their mechanisms of formation were explained. Preliminary oxidative activation of the dyes resulted in the formation of cationic species, making the molecules vulnerable to the nucleophilic attack of water. Two types of hydrolytic cleavage were observed. Asymmetric splitting gave rise to quinone and diazene derivatives, while symmetric splitting resulted in the formation of quinone monoimine and nitroso derivatives. These unstable intermediates underwent further redox, oxidation, and hydrolytic transformation, eventually furnishing 11 organic products and ammonia.

Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus.

Five 14C-radiolabeled azo dyes and sulfanilic acid were synthesized and used to examine the relationship between dye substitution patterns and biodegradability (mineralization to CO2) by a white-rot fungus and an actinomycete. 4-Amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid were used as representative compounds having sulfo groups or both sulfo and azo groups. Such compounds are not known to be present in the biosphere as natural products. The introduction of lignin-like fragments into the molecules of 4-amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid by coupling reactions with guaiacol (2-methoxyphenol) resulted in the formation of the dyes 4-(3-methoxy-4-hydroxyphenylazo)-[U-14C]benzenesulfonic acid and 4-(2-sulfo-3'-methoxy-4'-hydroxy-azobenzene-4-azo)-[U-14C]benzenesulf oni c acid, respectively. The synthesis of acid azo dyes 4-(2-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid and 4-(4-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid also allowed the abilities of these microorganisms to mineralize these commercially important compounds to be evaluated. Phanerochaete chrysosporium mineralized all of the sulfonated azo dyes, and the substitution pattern did not significantly influence the susceptibility of the dyes to degradation. In contrast, Streptomyces chromofuscus was unable to mineralize aromatics with sulfo groups and both sulfo and azo groups. However, it mediated the mineralization of modified dyes containing lignin-like substitution patterns. This work showed that lignocellulolytic fungi and bacteria can be used for the biodegradation of anionic azo dyes, which thus far have been considered among the xenobiotic compounds most resistant to biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium.

Twenty-two azo dyes were used to study the influence of substituents on azo dye biodegradability and to explore the possibility of enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. Streptomyces spp. and Phanerochaete chrysosporium were used in the study. None of the actinomycetes (Streptomyces rochei A10, Streptomyces chromofuscus A11, Streptomyces diastaticus A12, S. diastaticus A13, and S. rochei A14) degraded the commercially available Acid Yellow 9. Decolorization of monosulfonated mono azo dye derivatives of azobenzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. The fungus P. chrysosporium attacked Acid Yellow 9 to some extent and extensively decolorized several azo dyes. A different pattern was seen for three mono azo dye derivatives of naphthol. Streptomyces spp. decolorized Orange I but not Acid Orange 12 or Orange II. P. chrysosporium, though able to transform these three azo dyes, decolorized Acid Orange 12 and Orange II more effectively than Orange I. A correlation was observed between the rate of decolorization of dyes by Streptomyces spp. and the rate of oxidative decolorization of dyes by a commercial preparation of horseradish peroxidase type II, extracellular peroxidase preparations of S. chromofuscus A11, or Mn(II) peroxidase from P. chrysosporium. Ligninase of P. chrysosporium showed a dye specificity different from that of the other oxidative enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol.

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.

Selection of amylolytically active Aspergillus niger mutants to 2-deoxy-D-glucose.

As a result of mutagenization and passaging on 2-deoxy-D-glucose containing medium, 10 Aspergillus niger strains resistant to this agent were obtained. These showed (with one exception) an increase in the activity of glucoamylse, the level of which ranged widely in individual cases from several to over 200% in comparison with the parent strain. A weaker rate of glucose accumulation in derepressed strains may account for the fact that the mechanism of their resistance to deoxyglucose is connected with disturbance of the system of glucose transport. However, it is possible that a high activity of acid phosphatase, which the obtained deoxyglucose-resistant cultures showed, may be involved here. Apart from the biochemical character of the catabolic derepression, it seems that it can already be successfully utilized to increase the productivity of industrial mould cultures.

Transformations of arylpropane lignin model compounds by a lignin peroxidase of the white-rot fungus Phanerochaete chrysosporium.

Various lignin model compounds of the O-arylpropane type were oxidized with purified lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium, and oxidation products were identified by gas-chromatography/mass-spectroscopy procedures. Our results are in accord with the theory that lignin peroxidase catalyzes one-electron oxidations of its substrates with formation of cation radicals, and that these radicals undergo degradative reactions that are predictable from a knowledge of cation radical and oxygen chemistry. Cation radicals formed from O-arylpropane model compounds appeared to undergo the following types of degradative transformations: addition of water to ring-centered radicals, followed by proton loss yielding quinones and alcohols; nucleophilic attack by hydroxy functions on propanoid moieties giving cyclic ketals as intermediates which decompose to yield side chain migration products; transfer of the charge of a radical from a ring to the associated alkyl moiety through an ether bond, with loss of a proton from the latter, forming a new carbon-centered radical. The new alkyl-centered radicals apparently were able to abduct dioxygen to form peroxyl radicals which decomposed giving a variety of oxidation products and probably superoxide anion. Specific examples of the above transformations are presented, and their relevance to lignin degradation is discussed.

Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium.

Ligninase-I (Mr 42,000-43,000; carbohydrate, 21%) and peroxidase-M2 (Mr 45,000-47,000; carbohydrate, 17%), two representative, hydrogen peroxide-dependent extracellular enzymes produced by ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium BKM-F-1767, were purified and their properties compared. Spectroscopic studies showed that both native enzymes are heme proteins containing protoporphyrin IX. EPR spectroscopy indicated that iron ions are coordinated with the enzymes' prosthetic groups as high-spin ferriheme complexes. We confirmed reports of others that the ligninase-hydrogen peroxide complex (activated enzyme) reverts to its native state on addition of dithionite or one of the enzyme's substrates (e.g., veratryl alcohol); however, we found that the peroxidase-M2-hydrogen peroxide complex required Mn2+ ions to accomplish a similar cycle. The peroxidase oxidized Mn2+ to a higher oxidation state, and the oxidized Mn acted as a diffusible catalyst able to oxidize numerous organic substrates. Unlike ligninase-I which is found free extracellularly, peroxidase-M2 appears to be associated closely with the fungal mycelium. In its peroxidatic reactions, ligninase-I oxidizes a variety of nonphenolic and phenolic lignin model compounds. In the presence of Mn2+, peroxidase-M2 oxidizes numerous phenolic compounds, especially syringyl (3,5-dimethoxy-4-hydroxyphenyl) and vinyl side-chain substituted substrates. Also, the peroxidase-Mn2+ system (without hydrogen peroxide) expresses oxidase activity against NADPH, GSH, dithiothreitol, and dihydroxymaleic acid, forming hydrogen peroxide at the expense of oxygen. Both enzymes were believed to play roles in lignin degradation, and these are discussed.

The effect of proteases on the synthesis of glucoamylase by mutants of Aspergillus niger C.

Out of 1230 cultures grown from conidia of the strain Aspergillus niger C, which were previously treated with mutagens, 6 mutants were selected, 5 of which were characterized by considerably lower or lack of proteolytic activity than the parent strain. It was observed that glucoamylase activity and protein synthesis in the mutants examined were directly proportional to the rate of their proteolytic activity. From electrophoretic analysis it was found that in some protease-free mutants or with strongly reduced activity of this enzyme one or two glucoamylase fractions out of four occurring in the parent strain were absent.

Keratin and polyamide-coated inorganic matrices as supports for glucoamylase immobilization.

Previously solubilized feather keratin and polyamide were used for coating sand, glass beads and silica gel. These new seven supports were employed for comparative studies on pure glucoamylase / EC 3.2.1.3 / immobilization. The immobilization yield of glucoamylase on keratin and polyamide coated supports was comparable with conventional matrices used earlier. The highest activity per 1 g of support was shown by the enzyme bound to polyamide-coated CPG, and the bests operational stability by the enzyme immobilized on polyamide-coated CPG with keratin subsequently deposited on it.

An affinity-column procedure for the purification of veratrate O-demethylase from fungi.

An affinity column procedure is reported for purifying veratrate O-demethylase from higher fungi. The procedure is based on the affinity of the fungal demethylases for veratrate, which was coupled to AH-Sepharose 4B. An over 300-fold purification of the enzyme from an Ascomycete (Chaetomium piluliferum), and a lower degree of purification (20-fold) from a Basidiomycete (Xerocomus badius), were obtained. The O-demethylases from higher fungi require NADH and oxygen. The enzyme activity is sensitive to exposure to oxygen. The pH optima are 5 for enzyme from Chaetomium, and 7 for demethylase from Xerocomus, respectively. The enzymes are not specific for veratrate. They also demethylate p- and m-anisate and 3,4-dimethoxycinnamate, but to a lower degree.