Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture

An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.     

Enrichment of a microbial culture capable of degrading endosulphate, the toxic metabolite of endosulfan

AIMS: The aim of this study was to isolate a source of enzymes capable of degrading endosulphate (endosulfan sulphate), the toxic metabolite of the pesticide endosulfan. METHODS AND RESULTS: A microbial broth culture capable of degrading endosulphate was enriched from endosulfan-contaminated soil by providing the metabolite as the sole source of sulphur in broth culture. No microbial growth was observed in the absence of endosulphate. In the presence of endosulphate, growth of the culture occurred with the concomitant formation of three chlorine-containing compounds. Thin layer chromatography and gas chromatography--mass spectral analysis identified these metabolites as endosulfan monoaldehyde, 1,2,3,4,7,7-hexachloro-5,6-bis(methylene)bicyclo[2.2.1]-2-heptene and 1,2,3,4,7,7-hexachloro-5-hydroxymethylene-6-methylenebicyclo[2.2.1]-2-heptene. The second and third compounds have not been reported in previous metabolic studies. The enriched culture was also able to utilize alpha- and beta-endosulfan as sulphur sources, each producing the hydrolysis product endosulfan monoaldehyde as the sole chlorine-containing metabolite. Alpha-endosulfan was more readily hydrolysed than the beta-isomer. CONCLUSIONS: This study isolated a mixed microbial culture capable of degrading endosulphate. The products of degradation were characterized as novel endosulfan metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the isolation of a mixed microbial culture that is potentially a valuable source of hydrolysing enzymes for use in enzymatic bioremediation, particularly of endosulphate and alpha-endosulfan residues.     

Biodegradation of endosulfan and endosulfan sulfate by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO₂ from the culture media with endosulfan as sulfur source as compared to MgSO₄ suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.     

Gene cloning and molecular characterization of a two-enzyme system catalyzing the oxidative detoxification of beta-endosulfan

The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent beta-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of beta-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of beta-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either alpha-endosulfan or the metabolite of endosulfan, endosulfan sulfate.     

Isolation and characterization of a Mycobacterium strain that metabolizes the insecticide endosulfan

AIM: The aim of this study was to isolate and characterize a bacterium capable of metabolizing endosulfan. METHODS AND RESULTS: A endosulfan-degrading bacterium (strain ESD) was isolated from soil inoculum after repeated culture with the insecticide as the sole source of sulfur. Analysis of its 16S rRNA gene sequence, and morphological and physiological characteristics revealed it to be a new fast-growing Mycobacterium, closely related to other Mycobacterium species with xenobiotic-degrading capabilities. Degradation of endosulfan by strain ESD involved both oxidative and sulfur-separation reactions. Strain ESD did not degrade endosulfan when sulfite, sulphate or methionine were present in the medium along with the insecticide. Partial degradation occurred when the culture was grown, with endosulfan, in the presence of MOPS (3-(N-morpholino)propane sulphonic acid), DMSO (dimethyl sulfoxide), cysteine or sulphonane and complete degradation occurred in the presence of gutathione. When both beta-endosulfan and low levels of sulphate were provided as the only sources of sulfur, biphasic exponential growth was observed with endosulfan metabolism being restricted to the latter phase of exponential growth. CONCLUSIONS: This study isolated a Mycobacterium strain (strain ESD) capable of metabolizing endosulfan by both oxidative and sulfur-separation reactions. The endosulfan-degrading reactions are a result of the sulfur-starvation response of this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: This describes the isolation of a Mycobacterium strain capable of degrading the insecticide endosulfan. This bacterium is a valuable source of enzymes for use in enzymatic bioremediation of endosulfan residues.     

Metabolic pathways utilized by Phanerochaete chrysosporium for degradation of the cyclodiene pesticide endosulfan.

Recent studies have shown that cultures of white rot fungi not favoring the production of lignin and manganese peroxidases are effective in degrading certain xenobiotics. In this study we have used endosulfan as a model xenobiotic to assess the enzymatic mechanisms of pesticide metabolism under ligninolytic (nutrient-deficient) and nonligninolytic (nutrient-rich) culture conditions. Rapid metabolism of this chlorinated pesticide occurred under each nutrient condition tested. However, the extent of degradation and the nature of the metabolic products differed for nutrient-deficient and nutrient-rich media. The pathways for endosulfan metabolism were characterized by analysis of the fungal metabolites produced. The major endosulfan metabolites were identified by gas chromatography-electron capture detection and gas chromatography-mass spectrometry as endosulfan sulfate, endosulfan diol, endosulfan hydroxyether, and a unknown metabolite tentatively identified as endosulfan dialdehyde. The nature of the metabolites formed indicates that this organism utilizes both oxidative and hydrolytic pathways for metabolism of this pesticide. Piperonyl butoxide, a known cytochrome P-450 inhibitor, significantly inhibited the oxidation of endosulfan to endosulfan sulfate and enhanced hydrolysis of endosulfan to endosulfan diol. We suggest that the metabolism of endosulfan is mediated by two divergent pathways, one hydrolytic and the other oxidative. Judging by the inactivity of extracellular fluid and partially purified lignin peroxidase in metabolizing endosulfan, we conclude that metabolism of this compound does not involve the action of extracellular peroxidases.     

Effect of endosulfan on growth, alpha amylase activity and plasmids amplification in Bacillus subtilis

Endosulfan, a chlorinated hydrocarbon insecticide of cyclodiene subgroup acts as a contact poison in a wide variety of organisms. In the present study, the effect of endosulfan on the growth, alpha amylase activity and plasmid amplification was investigated in Bacillus subtilis system. The bacteria were grown in medium, incubated with different concentrations (32, 48, 64 and 80 microg/mL) of endosulfan. The bacterial growth was gradually seen after 1st day at up to 48 microg/L endosulfan. The 48 microg/L endosulfan inhibited approximately 50% of the bacterial growth. No growth was observed at and after 64 microg/L endosulfan, for all days (1-5). Also, no alpha amylase activity was found in the supernatant of the culture medium containing 64 and 80 microg/L endosulfan, whereas slight activity was observed with 32 and 48 microg/L endosulfan concentration. The amount of plasmid increased up to 50% in the presence of 32 microg/L endosulfan. Endosulfan had no effect on the alpha amylase activity in vitro.     

Pathways of microbial metabolism of parathion.

A mixed bacterial culture, consisting of a minimum of nine isolates, was adapted to growth on technical parathion (PAR) as a sole carbon and energy source. The primary oxidative pathway for PAR metabolism involved an initial hydrolysis to yield diethylthiophosphoric acid and p-nitrophenol. A secondary oxidative pathway involved the oxidation of PAR to paraoxon and then hydrolysis to yield p-nitrophenol and diethylphosphoric acid. Under low oxgen conditions PAR was reduced via a third pathway to p-aminoparathion and subsequently hydrolyzed to p-aminophenol and diethylthiophosphoric acid. PAR hydrolase, an enzyme produced by an isolate from the mixed culture, rapidly hydrolyzed PAR and paraoxon (6.0 mumol/mg per min). This enzyme was inducible and stable at room temperature and retained 100% of its activity when heated for 55 C for 10 min.     

Pathways of microbial metabolism of parathion.

A mixed bacterial culture, consisting of a minimum of nine isolates, was adapted to growth on technical parathion (PAR) as a sole carbon and energy source. The primary oxidative pathway for PAR metabolism involved an initial hydrolysis to yield diethylthiophosphoric acid and p-nitrophenol. A secondary oxidative pathway involved the oxidation of PAR to paraoxon and then hydrolysis to yield p-nitrophenol and diethylphosphoric acid. Under low oxgen conditions PAR was reduced via a third pathway to p-aminoparathion and subsequently hydrolyzed to p-aminophenol and diethylthiophosphoric acid. PAR hydrolase, an enzyme produced by an isolate from the mixed culture, rapidly hydrolyzed PAR and paraoxon (6.0 mumol/mg per min). This enzyme was inducible and stable at room temperature and retained 100% of its activity when heated for 55 C for 10 min.     

Klebsiella pneumoniae KE-1 degrades endosulfan without formation of the toxic metabolite,endosulfan sulfate

For bioremediation of toxic endosulfan, endosulfan degradation bacteria, which do not form toxic endosulfan sulfate, were isolated from various soil samples using endosulfan as sole carbon and energy source. Among the 40 isolated bacteria, strain KE-1, which was identified as Klebsiella pneumoniae by physiological and 16S rDNA sequence analysis, showed superior endosulfan degradation activity. Analysis of culture pH, growth, free sulfate and endosulfan and its metabolites demonstrated that KE-1 biologically degrades 8.72 microg endosulfan ml(-1) day(-1) when incubated with 93.9 microg ml(-1) endosulfan for 10 days without formation of toxic endosulfan sulfate. Our results suggest that K. pneumoniae KE-1 degraded endosulfan by a non-oxidative pathway and that strain KE-1 has potential as a biocatalyst for endosulfan bioremediation.     

Biodegradation of α and β endosulfan in broth medium and soil microcosm by bacterial strain Bordetella sp. B9

Bacterial strains were isolated from endosulfan treated soil to study the microbial degradation of this pesticide in broth medium and soil microcosm. The isolates were grown in minimal medium and screened for endosulfan degradation. The strain, which utilized endosulfan and showed maximum growth, was selected for detail studies. Maximum degrading capability in shake flask culture was shown by Bordetella sp. B9 which degraded 80% of α endosulfan and 86% of β endosulfan in 18 days. Soil microcosm study was also carried out using this strain in six different treatments. Endosulfan ether and endosulfan lactone were the main metabolites in broth culture, while in soil microcosm endosulfan sulfate was also found along with endosulfan ether and endosulfan lactone. This bacterial strain has a potential to be used for bioremediation of the contaminated sites.     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Atropine Metabolism by Pseudomonas sp. Strain AT3: Evidence for Nortropine as an Intermediate in Tropine Breakdown and Reactions Leading to Succinate

Pseudomonas strain AT3, isolated by elective culture with atropine, hydrolyzed atropine and grew diauxically, first on the tropic acid and then on the tropine. Tropine was also used as a sole carbon and energy source. The methyl group of tropine was eliminated as formaldehyde, and the nortropine thus formed was a precursor of 6-hydroxycyclohepta-1,4-dione. Ammonia was detected as a product of nitrogen elimination. 6-Hydroxycyclohepta-1,4-dione was oxidized to cyclohepta-1,3,5-trione by an induced NAD(sup+)-specific dehydrogenase. Although cyclohepta-1,3,5-trione is a (beta)-diketone with two potential hydrolytic cleavage sites, an induced hydrolase was specific for one of these sites, with 4,6-dioxoheptanoate as the only hydrolysis product. Unlike the alternative cleavage product (3,6-dioxoheptanoate), this compound is also a (beta)-diketone, and a second hydrolytic cleavage formed succinate and acetone. Although Pseudomonas strain AT3 was not capable of growth with acetone, the compound was not detected in the culture medium and may have been lost to the atmosphere. Exhaustive experimentation with a wide range of conditions did not result in detection of the enzymes required for cleavage of the carbon-nitrogen bonds leading to the formation of nortropine and 6-hydroxycyclohepta-1,4-dione.     

Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida     

Biodegradation and bioremediation of endosulfan contaminated soil

Among the three mixed bacterial culture AE, BE, and CE, developed by enrichment technique with endosulfan as sole carbon source, consortium CE was found to be the most efficient with 72% and 87% degradation of alpha-endosulfan and beta-endosulfan, respectively, in 20 days. In soil microcosm, consortium AE, BE and CE degraded alpha-endosulfan by 57%, 88% and 91%, respectively, whereas beta-endosulfan was degraded by 4%, 60% and 67% after 30 days. Ochrobacterum sp., Arthrobacter sp., and Burkholderia sp., isolated and identified on the basis of 16s rDNA gene sequence, individually showed in situ biodegradation of alpha-endosulfan in contaminated soil microcosm by 61, 73, and 74, respectively, whereas degradation of beta-endosulfan was 63, 75, and 62, respectively, after 6 weeks of incubation over the control which showed 26% and 23 % degradation of alpha-endosulfan and beta-endosulfan, respectively. Population survival of Ochrobacterum sp., Arthrobacter sp., and Burkholderia sp., by plate count on Luria Broth with carbenicillin showed 75-88% survival of these isolates as compared to 36-48% of survival obtained from PCR fingerprinting. Arthrobacter sp. oxidized endosulfan to endosulfan sulfate which was further metabolized but no known metabolite of endosulfan sulfate was detected.     

Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method.

A crude cell extract from a mixed bacterial culture growing on parathion, an organophosphate insecticide, hydrolyzed parathion (21 C) at a rate of 416 nmol/min per mg of protein. This rate of enzymatic hydrolysis, when compared with chemical hydrolysis by 0.1 N sodium hydroxide at 40 C, was 2, 450 times faster. Eight of 12 commonly used organophosphate insecticides were enzymatically hydrolyzed with this enzyme preparation at rates ranging from 12 to 1,360 nmol/min per mg of protein. Seven pesticides were hydrolyzed at rates significantly higher (40 to 1,005 times faster) than chemical hydrolysis. The pH optimum for enzymatic hydrolysis of the eight pesticides ranged from 8.5 to 9.5, with less than 50% of maximal activity expressed at pH 7.0. Maximal enzyme activity occurred at 35 C. The crude extract lost its activity at the rate of only 0.75%/day when stored at 6 C. Eight organic solvents, ranging from methanol to hexane, at low concentrations stimulated enzymatic hydrolysis by 3 to 20%, whereas at higher concentrations (1,000 mg/liter) they inhibited the reaction (9 to 50%). Parathion metabolites p-nitrophenol, hydroquinone, and diethylthiophosphoric acid, at up to 100-mg/liter concentrations, did not significantly influence enzyme activity.     

Endosulfan decreases cell growth and apoptosis in human HaCaT keratinocytes: partial ROS-dependent ERK1/2 mechanism

Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen-activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 microM endosulfan led to persistent extracellular signal-regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan-treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N-acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS-dependent mechanism. Therefore, to assess the putative pro-apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP-ribose)-polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine-induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp.

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.