The effect of pH on the selection of carbaryl-degrading bacteria from garden soil

At an alkaline pH and in an aqueous solution carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Soil perfusion column experiments indicated that the rate of carbaryl degradation at pH 6.0 to 7.0 was limited by the rate of chemical hydrolysis. Bacterial communities of at least 12 and 14 members were selected in continuous cultures using carbaryl as the sole carbon and nitrogen source at pH 6.0. These communities were supported by the slow formation of hydrolysis products and a carbaryl-degrading bacterium was not selected after > 2000 h. A bacterial community of at least eight members was selected using equimolar 1-naphthol and methylamine as its sole carbon and nitrogen source. In contrast, after a lag of between 10 and 50 days, soil perfusion column and continuous culture enrichments at pH 5.2 and 5.0, respectively, led to the selection of a Pseudomonas sp. which could utilize carbaryl as its sole carbon and nitrogen source.     

The effect of pH on the selection of carbaryl-degrading bacteria from garden soil

At an alkaline pH and in an aqueous solution carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Soil perfusion column experiments indicated that the rate of carbaryl degradation at pH 6.0 to 7.0 was limited by the rate of chemical hydrolysis. Bacterial communities of at least 12 and 14 members were selected in continuous cultures using carbaryl as the sole carbon and nitrogen source at pH 6.0. These communities were supported by the slow formation of hydrolysis products and a carbaryl-degrading bacterium was not selected after greater than 2000 h. A bacterial community of at least eight members was selected using equimolar 1-naphthol and methylamine as its sole carbon and nitrogen source. In contrast, after a lag of between 10 and 50 days, soil perfusion column and continuous culture enrichments at pH 5.2 and 5.0, respectively, led to the selection of a Pseudomonas sp. which could utilize carbaryl as its sole carbon and nitrogen source.     

Effects on drug metabolism of carbaryl and 1-naphthol in the mouse

The effects of carbyl and 1-naphthol on hepatic microsomal drug-metabolizing enzyme systems were investigated. The agents were fed at a level of 25 mmol/kg of feed to groups of young male Swiss-Webster mice for 14-day periods. Body weight was depressed by carbaryl, but not by 1-naphthol. The rates of $\text{in vitro}$ metabolism of aniline and benzphetamine were greater than control rates in livers of mice fed carbaryl, but the rate of $\text{in vivo}$ hydrolysis of the carbamate insecticide Zectran was decreased by carbaryl feeding. Administration of 1-naphthol did not change the rates of $\text{in vitro}$ metabolism of either aniline or benzphetamine. Hepatic microsomal concentrations of cytochromes P-450 and b₅ were increased by carbaryl, but feeding of 1-naphthol did not affect levels of either cytochrome. Radiolabeled pentobarbital disappeared from the blood of carbaryl-fed mice more rapidly than from the blood of control animals, and carbaryl-fed mice slept a shorter period of time than controls following pentobarbital administration. The LD50 of an acute oral dose of carbaryl was increased two-fold by feeding carbaryl for 14 days. It was concluded that carbaryl is a weak inducer of hepatic microsomal drug-metabolizing activity, and that the effects observed are not likely due to 1-naphthol.     

Metabolism of carbaryl via 1,2-dihydroxynaphthalene by soil isolates Pseudomonas sp. strains C4, C5, and C6

Pseudomonas sp. strains C4, C5, and C6 utilize carbaryl as the sole source of carbon and energy. Identification of 1-naphthol, salicylate, and gentisate in the spent media; whole-cell O2 uptake on 1-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate, and gentisate; and detection of key enzymes, viz, carbaryl hydrolase, 1-naphthol hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, and gentisate dioxygenase, in the cell extract suggest that carbaryl is metabolized via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. Here, we demonstrate 1-naphthol hydroxylase and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbaryl-grown cells. 1-Naphthol hydroxylase is present in the membrane-free cytosolic fraction, requires NAD(P)H and flavin adenine dinucleotide, and has optimum activity in the pH range 7.5 to 8.0. Carbaryl-degrading enzymes are inducible, and maximum induction was observed with carbaryl. Based on these results, the proposed metabolic pathway is carbaryl --> 1-naphthol --> 1,2-dihydroxynaphthalene --> salicylaldehyde --> salicylate --> gentisate --> maleylpyruvate.     

Physiological and morphological alterations induced by carbaryl and 1-naphthol combinations inNostoc linckia isolated from soil

Carbaryl and its major hydrolysis product, 1-naphthol, were tested singly and together at concentrations ranging from 2 to 20 g/ml towards photosynthesis, in vivo nitrate reductase activity and nitrogen fixation, and ultrastructure of a diazotrophic cyanobacterium,Nostoc linckia, isolated from soil. All three physiological processes tested were drastically affected and correlated well with the alterations in the ultrastructure of the organism. Combination of carbaryl and 1-naphthol, at different concentrations, interacted significantly, yielding additive, antagonistic, and synergistic responses.     

Toxicity of carbaryl and 1-naphthol to four species of freshwater fish

The toxicity of the carbamate insecticide carbaryl (Seven√) and its metabolite, 1-naphthol, to four species of fish was studied. The calculated 96 h LC 60 values of carbaryl forCatla catla (Ham.), Anabas testudineus (Bloch),Mystus cavasius (Ham.) andMystus vittatus (Bloch) are 6.4, 6.6, 4.6 and 2.4 ppm respectively and that of 1-naphthol are 4.3,3, 0.33 and 1.1 ppm respectively. The degradation product of the insecticide was found to be more toxic than the parent compound, to all the four species studied.     

Metabolism of Carbaryl via 1,2-Dihydroxynaphthalene by Soil Isolates Pseudomonas sp. Strains C4, C5, and C6

Pseudomonas sp. strains C4, C5, and C6 utilize carbaryl as the sole source of carbon and energy. Identification of 1-naphthol, salicylate, and gentisate in the spent media; whole-cell O₂ uptake on 1-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate, and gentisate; and detection of key enzymes, viz, carbaryl hydrolase, 1-naphthol hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, and gentisate dioxygenase, in the cell extract suggest that carbaryl is metabolized via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. Here, we demonstrate 1-naphthol hydroxylase and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbaryl-grown cells. 1-Naphthol hydroxylase is present in the membrane-free cytosolic fraction, requires NAD(P)H and flavin adenine dinucleotide, and has optimum activity in the pH range 7.5 to 8.0. Carbaryl-degrading enzymes are inducible, and maximum induction was observed with carbaryl. Based on these results, the proposed metabolic pathway is carbaryl → 1-naphthol → 1,2-dihydroxynaphthalene → salicylaldehyde → salicylate → gentisate → maleylpyruvate.     

The metabolism of carbaryl by three bacterial isolates, Pseudomonas spp. (NCIB 12042 & 12043) and Rhodococcus sp. (NCIB 12038) from garden soil

At an alkaline pH and in aqueous solution, carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Two bacteria isolated from garden soil, Pseudomonas sp. (NCIB 12042) and Rhodococcus sp. (NCIB 12038), could grow on carbaryl as sole carbon and nitrogen source at pH 6.8 but failed to metabolize carbaryl rapidly. Both could use 1-naphthol as sole carbon source and NCIB 12042 metabolized 1-naphthol via salicylic acid which induced higher expression of enzymes in the pathway. Strain NCIB 12038 metabolized 1-naphthol via salicylic and gentisic acids. In contrast, Pseudomonas sp. (NCIB 12043) was selected in a soil perfusion column enrichment at pH 5.2 and metabolized carbaryl rapidly to 1-naphthol and methylamine. 1-Naphthol was metabolized via gentisic acid. Neither salicylate nor gentisate induced higher expression of enzymes for 1-naphthol catabolism in NCIB 12038 and NCIB 12043.     

The metabolism of carbaryl by three bacterial isolates, Pseudomonas spp. (NCIB 12042 & 12043) and Rhodococcus sp. (NCIB 12038) from garden soil

At an alkaline pH and in aqueous solution, carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Two bacterial isolated from garden soil, Pseudomonas sp. (NCIB 12042) and Rhodococcus sp. (NCIB 12038), could grow on carbaryl as sole carbon and nitrogen source at pH 6.8 but failed to metabolize carbaryl rapidly. Both could use 1-naphthol as sole carbon source and NCIB 12042 metabolized 1-naphthol via salicylic acid which induced higher expression of enzymes in the pathway. Strain NCIB 12038 metabolized 1-naphthol via salicylic and gentisic acids. In contrast, Pseudomonas sp. (NCIB 12043) was selected in a soil perfusion column enrichment at pH 5.2 and metabolized carbaryl rapidly to 1-naphthol and methylamine. 1-Naphthol was metabolized via gentisic acid. Neither salicylate nor gentisate induced higher expression of enzymes for 1-naphthol catabolism in NCIB 12038 and NCIB 12043.     

Hydrolysis of carbaryl by a Pseudomonas sp. and construction of a microbial consortium that completely metabolizes carbaryl

Two Pseudomonas spp. (isolates 50552 and 50581) isolated from soil degraded 1-naphthol and carbaryl, an N-methylcarbamate pesticide, respectively. They utilized these compounds as a sole source of carbon. 1-Naphthol was completely metabolized to CO2 by the isolate 50552, while the carbaryl was first hydrolyzed to 1-naphthol and then converted into a brown-colored compound by the isolate 50581. The colored metabolite was not degraded, but 1-naphthol produced by the isolate 50581 during the exponential phase of growth was metabolized by the isolate 50552. The two isolates were used to construct a bacterial consortium which completely catabolized carbaryl to CO2. No metabolite was detected in the cell cultures of the consortium. The isolate 50581 harbored a 50-kb plasmid pCD1, while no plasmid was detected in the isolate 50552. The isolated bacteria individually or as a consortium may be used for detoxification of certain industrial and agricultural wastes.     

Hydrolysis of carbaryl by a Pseudomonas sp. and construction of a microbial consortium that completely metabolizes carbaryl.

Two Pseudomonas spp. (isolates 50552 and 50581) isolated from soil degraded 1-naphthol and carbaryl, an N-methylcarbamate pesticide, respectively. They utilized these compounds as a sole source of carbon. 1-Naphthol was completely metabolized to CO2 by the isolate 50552, while the carbaryl was first hydrolyzed to 1-naphthol and then converted into a brown-colored compound by the isolate 50581. The colored metabolite was not degraded, but 1-naphthol produced by the isolate 50581 during the exponential phase of growth was metabolized by the isolate 50552. The two isolates were used to construct a bacterial consortium which completely catabolized carbaryl to CO2. No metabolite was detected in the cell cultures of the consortium. The isolate 50581 harbored a 50-kb plasmid pCD1, while no plasmid was detected in the isolate 50552. The isolated bacteria individually or as a consortium may be used for detoxification of certain industrial and agricultural wastes.     

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into ‘upper’ (carbaryl to salicylate), ‘middle’ (salicylate to gentisate) and ‘lower’ (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s).     

The specificity of 1-naphthol oxygenases from three bacterial isolates, Pseudomonas spp. (NCIB 12042 and 12043) and Rhodococcus sp. (NCIB 12038) isolated from garden oil

Abstract Three bacterial isolates which appeared to use the insecticide, carbaryl (1-naphthyl, N -methyl-carbamate) as their sole carbon and nitrogen sources were originally selected from garden soil. Only one isolate, Pseudomonas sp. (NCIB 12043) could metabolise carbaryl rapidly to 1-naphthol and methylamine. The other two isolates, Pseudomonas sp. (NCIB 12042) and Rhodococcus sp. (NCIB 12038) relied on slow chemical hydrolysis of carbaryl to 1-naphthol and methylamine. All three isolates used 1-naphthol as their sole carbon source; however, their ability to use naphthalene and a range of mono- and dihydroxy-substituted naphthalene compounds varied. NCIB 12038 and NCIB 12043 showed little or no growth on naphthalene, 2,3-dihydroxynaphthalene or 1,3-dihydroxynaphthalene as sole carbon sources and their 1-naphthol oxygenases had little activity with these substrates. In contrast, NCIB 12042 could use these compounds as sole carbon sources and its 1-naphthol oxygenase also showed activity with them. We conclude that 1-naphthol oxygenase from NCIB 12042 is a relatively non-specific dioxygenase, whereas the 1-naphthol oxygenases from NCIB 12038 and NCIB 12043 are relatively specific monooxygenases requiring hydroxylated naphthalene compounds as substrates.     

Relationship between the phagocytic inhibition of the rat reticuloendothelial system and the anticholinesterasic effect of an insecticide, Carbaryl (author's transl)]

Phagocytic activity of the reticuloendothelial system (RES) and blood cholinesterase activity were determined in male rats after veinous administrations of carbaryl and 1-naphthol, a carbaryl metabolite. The various parameters were measured 1, 24, 48 and 72 hours after administration of the following four doses per 100 g body weight : 1.875, 3.75, 7.5 and 15 mumol. 1. Results showed an inhibition of the RES phagocytic activity (clearance of colloidal carbon) after carbaryl administration; although 1.875 mumol/100 g had no effect, the other doses inhibited RES activity, blockade time being a function of the dose given. The phagocytic function had returned to normal 72 hr after carbaryl administration. 2. Reductions in spleen weight and protein content were observed together with the RES blockade. 3. At all four doses, the anticholinesterase effect was already apparent one hour after carbaryl administration. 4. 1-naphthol, one of carbaryl's chief metabolites, had no effect either on the RES or on the different parameters studied. These results show a relationship between the phagocytic inhibition of the reticuloendothelial system and the anticholinesterasic effect by carbaryl. They suggest an inhibition of some esterases of macrophages interfering with the phagocytosis.     

Interactions of carbaryl with estuarine bacterial communities

The addition of carbaryl (100g/ml) to a model estuarine ecosystem did not affect the number of bacteria in the sediment, but reduced the diversity (as measured by the rarefaction technique) of the microbial community as compared with a control model ecosystem. Two carbaryltolerant strains of bacteria were isolated from the carbaryl-treated system, but none were isolated from the control system. Bacterial growth and filter paper decomposition in mixed cultures was prevented by 100g/ml carbaryl, but this amount had no effect on the extracellular cellulase of an estuarine isolate. Increasing the amount of organic matter in the medium attenuated the toxicity of carbaryl to pure cultures of an estuarine isolate. The addition of 1, 10, or 100g/ml carbaryl to field plots had no effect on bacterial numbers, diversity, or filter paper decomposition. The amount of carbaryl in sediments exposed to 100g/ml fell below the limit of detection by thin-layer chromatography within 12 hours. In sterile and nonsterile model systems, carbaryl rapidly adsorbed to sediment, and hydrolyzed to 1-naphthol in both sediment and water. Although carbaryl may be toxic to bacteria under some conditions, the amounts that might enter and persist in an estuary are insufficient to have a significant impact on the sediment microbial community.     

Degradation of sevin (1-naphthyl-N-methyl carbamate by Achrombacter sp.

Summary A strain of Achromobacter utilized 1-Naphthyl-N-methyl carbamate (Sevin, Carbaryl) as the sole source of carbon in salt medium. Four degradation products of sevin were identified to be 1-naphthol, hydroquinone, catechol and pyruvate. The organism grew on 1-naphthol, hydroquinone and catechol.     

Biodegradation of Phenanthrene by a Bacillus Species

A bacterial strain capable of utilizing phenanthrene as sole source of carbon was isolated from soil and identified as a Bacillus sp. The organism also utilized naphthalene, biphenyl, anthracene, and other aromatic compounds as growth substrates. The organism degraded phenanthrene through the intermediate formation of 1-hydroxy-2-naphthoic acid, which was further metabolized via o-phthalate by a protocatechuate pathway, as evidenced by oxygen uptake and enzymatic studies. Received: 1 December 1999 / Accepted: 5 January 2000     

Isolation of a constitutively expressed enzyme for hydrolysis of carbaryl in Pseudomonas aeruginosa.

A hydrolase constitutively expressed in Pseudomonas aeruginosa which converts carbaryl to 1-naphthol was purified 1,767-fold by using a combination of anion-exchange, hydroxylapatite, gel filtration, and hydrophobic interaction chromatography techniques. The presence of Triton X-100 in buffers was necessary for deaggregation and purification of the hydrolase. This is the first membrane-bound hydrolase involved in the hydrolysis of any methylcarbamate pesticide purified from a bacterial source to date. The enzyme exhibited a unique specificity of hydrolyzing only carbaryl (1-naphthyl N-methylcarbamate) but not any other methylcarbamates. The purified enzyme was a monomer with a molecular mass of 65,000 Da. The pH and temperature optima for the enzyme activity were 8.5 and 45 degrees C, respectively. No cofactor requirement for the hydrolase activity could be demonstrated, and none of the divalent cations studied affected the activity of the enzyme. Also, the enzyme activity was not affected by the thiols: dithioerythritol, dithiothreitol, and 2-mercaptoethanol. The Km and Vmax values for carbaryl were 9 microM and 7.9 mumol/min/mg of protein, respectively.     

Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene (cehA) from Rhizobium sp. Strain AC100

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.     

Involvement of two plasmids in the degradation of carbaryl by Arthrobacter sp. strain RC100

A bacterium capable of utilizing carbaryl (1-naphthyl N-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as Arthrobacter and was designated strain RC100. RC100 hydrolyzes the N-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah-) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat-) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah- mutants or Nat- mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes.