The role of esterases in the toxicity of organothiophosphorus insectoacaricides containing a fragment of mercaptoacetic acid

Interaction of insectoacaricide Me (EtO)P(S)SCH2SCH2COOMe (I), its activation metabolites (P = O (II), S = O, and P = O, S = O (III) analogues), and a detoxication product (-COOH analoque (IV) with rat liver carboxylesterase, acetylcholinesterase and butyrylcholinesterase of warm-blooded animals, as well as with cholinesterase and carboxylesterase of American cockroach has been studied. Low toxicity of (I) towards warm-blooded animals and American cockroach is shown to result from its rapid hydrolysis with corresponding carboxylesterases to form (IV). Monothiophosphonates (II) and (III) are not hydrolyzed by carboxylesterases but inhibit them irreversibly. High toxicity of (I) towards aphids can be ascribed to low activity of the carboxylesterase of that insect.     

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

Studies on the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) in vitro

1. The metabolism of the phosphorothionate parathion in vitro was examined by using [(32)P]parathion and microsomes isolated from the livers of various animal species. 2. The major metabolic products of parathion in this system in vitro were identified as diethyl 4-nitrophenyl phosphate (paraoxon), diethyl hydrogen phosphate, diethyl hydrogen phosphorothionate and p-nitrophenol. 3. The reaction leading to the formation of diethyl hydrogen phosphorothionate and p-nitrophenol requires the same cofactors (NADPH and oxygen) required for metabolism of parathion to its active anti-acetylcholinesterase paraoxon. 4. The enzyme activity towards parathion per unit weight of liver is increased some 65-130% by pretreatment of male rats with phenobarbital and 3,4-benzopyrene. 5. The metabolism of parathion is inhibited by incubation in a nitrogen atmosphere and in an atmosphere containing carbon monoxide. Pure oxygen is also inhibitory. These results are discussed in terms of a deficiency of oxygen for maximal activity as well as the lability of some component of the system to oxidation.     

Differential sensitivity of plasma carboxylesterase-null mice to parathion, chlorpyrifos and chlorpyrifos oxon, but not to diazinon, dichlorvos, diisopropylfluorophosphate, cresyl saligenin phosphate, cyclosarin thiocholine, tabun thiocholine, and carbofuran

Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase, butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the latter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition of acetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinesterase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has no known effect on an animal's well being. Paraoxonase hydrolyzes organophosphorus compounds and is not inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency on response to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygous plasma carboxylesterase deficient ES1(-/-) mice and wild-type littermates were observed for toxic signs and changes in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasma acetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found that wild-type mice were protected from the toxicity of 12.5mg/kg parathion applied subcutaneously. However, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophosphate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpected result was the finding that transdermal application of chlorpyrifos at 100mg/kg and chlorpyrifos oxon at 14mg/kg was lethal to wild-type but not to ES1(-/-) mice, showing that with this organochlorine, the presence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterase in mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is too low to protect from low toxicity compounds that require high doses to inhibit acetylcholinesterase.     

Studies of the enzymic mechanism of the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) by rat liver microsomes

1. The metabolism of parathion by rat liver microsomes is affected by various enzyme inhibitors in a manner quite typical of the ;mixed-function oxidase' enzyme systems. 2. With many of these inhibitors (p-chloromercuribenzoate, Cu(2+), 8-hydroxyquinoline) the conversion of parathion into diethyl hydrogen phosphorothionate is less inhibited than conversion into diethyl 4-nitrophenyl phosphate (paraoxon). 3. Compounds containing reduced sulphur stimulate the overall metabolism of parathion. However, the conversion of parathion into diethyl hydrogen phosphorothionate is stimulated more than its conversion into paraoxon. 4. The metabolism of parathion to diethyl hydrogen phosphorothionate is also stimulated by EDTA, Ca(2+) and Ba(2+), but these stimulatory effects are not additive. 5. The electron acceptors FAD, riboflavine, menadione and methylene blue exhibit a concentration-dependent differential inhibition of the metabolism of parathion to diethyl hydrogen phosphorothionate and to paraoxon. 6. The concentration of parathion required for the half-maximal rate of production of diethyl hydrogen phosphorothionate is significantly different from the concentration required for half-maximal rate of production of paraoxon. 7. The results are discussed in terms of either two separate enzyme systems metabolizing parathion to diethyl hydrogen phosphorothionate and to paraoxon or two different binding sites for parathion, which share a common electron-transport pathway.     

Parathion and methyl parathion toxicity and metabolism in piperonyl butoxide and diethyl maleate pretreated mice

Pretreatment of male mice with piperonyl butoxide, 400 mg/kg 1 h before challenge with insecticides, resulted in a 40-fold antagonism of the acute i.p. toxicity of methyl parathion but potentiated the toxicity of parathion two-fold. Piperonyl butoxide had no effect on the toxicity of the oxygen analogs of these insecticides, methyl paraoxon and paraoxon. Diethyl maleate (1 ml/kg) depleted liver glutathione by 80% after one hour, potentiated the toxicity of both methyl parathion and methyl paraoxon, and partially counteracted the protective effect of piperonyl butoxide on methyl parathion toxicity. Piperonyl butoxide delayed the onset of brain cholinesterase inhibition by parathion. Studies of the metabolism of the insecticides by liver homogenates in vitro demonstrated that piperonyl butoxide inhibited both the oxidative formation of the oxygen analogs (activation) and oxidative cleavage to p-nitrophenol and dialkylphosphorothioic acid (detoxification). While parathion metabolism was mostly oxidative, methyl parathion metabolism appeared to be predominantly via glutathione-dependent enzymes. Studies of in vitro distribution of the insecticides demonstrated that piperonyl butoxide pretreatment resulted in elevated tissue concentrations of parathion and methyl parathion; however, the rate constant for elimination from plasma for both insecticides was unaffected by piperonyl butoxide. The overall rate of metabolism of methyl parathion in vivo was approximately twice that of parathion. These results suggest that during piperonyl butoxide inhibition of oxidative activation and cleavage, methyl parathion detoxification continues through uninhibited glutathione-dependent pathways of metabolism. The net result is a reduction in the acute toxicity of methyl parathion. Lack of an effective alternate pathway of detoxification may explain the delayed but greater toxicity of parathion in piperonyl butoxide pretreated mice.     

Activation of the phosphorothionate insecticide parathion by rat brain in situ

The ability of the rat brain to activate the phosphorothionate insecticide parathion to its potent anticholinesterase metabolite paraoxon in situ was observed by ligating the posterior portion of the circulatory system and thus removing the liver from the circulation. Under these conditions no acetylcholinesterase inhibition was observed in 15 min at a dosage of parathion (nominally 2.4 mg/kg) which yielded 95% inhibition when the liver was in the circulation. However, at a higher dose (nominally 48 mg/kg) there was substantial (about 70%) inhibition of brain acetylcholinesterase after 15 min, suggesting that the brain does have the ability to activate parathion in the intact situation.     

Purification and properties of a carboxylesterase from the liver of tiger shark (Galeocerdo cuvier).

A procedure is described for the purification of a carboxylesterase from shark liver, using a chloroform-acetone powder prepared from the liver as the starting material. The yield of purified enzyme is approximately 50 mg from 530 g of chloroform-acetone powder. The preparation is electrophoretically homogeneous. Active-site titrations with paraoxon gave an equivalent weight of approximately 83 000. The molecular weight, found from sedimentation equilibrium experiments, is approximately 80 000. There is no evidence of any association or dissociation of this species. The enzyme shows a marked preference for aryl esters over alkyl esters, in contrast to other carboxylesterases so far studied. The amino acid composition of the purified enzyme is reported.     

Identification of a carboxylesterase expressed in protoplasts using fluorescence-activated cell sorting

A method was developed to identify plant carboxylesterases using a homologous expression system with the capacity for high-throughput screening based on fluorescence-activated cell sorting (FACS). Protoplasts of Arabidopsis thaliana were prepared and transfected with a mutated (Cys59Ser) Arabidopsis S-formylglutathione hydrolase ( atsfghm ), which encoded a carboxylesterase highly active in the hydrolysis of the vital marker methylumbelliferyl acetate (MUA) to the fluorophore methylumbelliferone (MU). Unlike all other Arabidopsis carboxylesterases studied to date, At SFGH and its more stable mutant variant At SFGHm are insensitive to inhibition by organophosphate insecticides, such as paraoxon. By making use of the combined traits of a high carboxylesterase activity towards MUA and a lack of sensitivity to paraoxon, FACS was employed to selectively collect catalytically active atsfghm -transformed protoplasts. A population of 400 000 protoplasts containing 8000 sfghm transformants was treated with paraoxon to inhibit endogenous esterase activity and then fed with MUA. Fluorescent cells expressing the At SFGHm enzyme were then collected by FACS, and the presence of the respective transgene was confirmed by polymerase chain reaction, with 9.6% of the transformants recovered. We suggest that the use of FACS to identify other carboxylesterases which can be catalytically determined using plant cell fluorescence-based assays could be a powerful method for the high-throughput screening of new enzymes, especially those which do not express well in microbial hosts.     

BIOCHEMICAL ASPECTS OF INSECTICIDE RESISTANCE IN COTTON BOLLWORM FROM HANDAN OF HEBEI PROVINCE*

Abstract The cotton bollworms, Helicoverpa armigera Hübner, collected from Handan of Hebei Province, have evolved high resistance to pyrethroid, organophosphate and carbamate insecticides, The sensitivity of acetylcholinesterase (AChEs) to paraoxon and methomyl varied with the development stages of the cotton bollworm. After the treatments with LD₅ and LD₅₀ of parathion and methomyl to the cotton bollworms, the affinity of AChE to acetylthiocholine (ATCh) and acetyl-β-bmethyl-thio choline (MeTCh) increased significantly except the treatment of parathion using LD₅₀ dosage while the sensitivity of AChEs to paraoxon significantly decreased. The sensitivity of AChEs to methomyl strongly increased in the treatment of parathion using LD₅₀ dosages while strongly decreased in other treatments. The affinity of carboxylesterase to β-naphthyl acetate (β-NA) was higher in groups of treatment with insecticides than in group of control. The glutathione-s-transferase (GST) activity significantly decreased in the induced groups using LD₅ dosages, while increased in the selection groups using LD₅₀ dosages. The effects of parathion and methomyl on the phosphatases of cotton bollworm were related to the dosages of application and the time after treatment and the effect on the alkaline phosphatase was stronger than on acid phosphatase.     

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.     

Cloning and sequencing of a human liver carboxylesterase isoenzyme

A human liver lambda gt11 library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxylesterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.     

The effect of high and low dosages of paraoxon in β-naphthoflavone-treated rats

Aliesterases (carboxylesterases) are serine esterases that can serve a protective role for the target acetylcholinesterase (AChE) during organophosphorus insecticide intoxication because the former esterases are alternate phosphorylation sites. The levels of aliesterase activity in liver and plasma and AChE activity in brain regions were investigated after the intravenous administration of paraoxon (P = O) into female rats. The rats were pretreated intraperitoneally with β-naphthoflavone (BNF), which decreases hepatic aliesterase activity following a 3 day in vivo treatment, and/or tri-o-totyl phosphate (TOTP) to inhibit aliesterases. The liver aliesterases were inhibited less by P = O in BNF-treated rats than in control rats, which suggests that either BNF exposure may have resulted in aliesterases that are less sensitive to P = O inhibition or BNF may have altered P = O's availability. The BNF treatment did not seem to alter the degree of inhibition of the brain AChE activity following the low dosage of paraoxon (0.04 mg/kg). However, the brain AChE activity in the P = O/TOTP/BNF-treated rats was lower than that in the P = O/TOTP-treated rats, suggesting that BNF also caused changes in systems affecting the disposition of P = O in addition to the changes in the hepatic aliesterases. At the high dosage of paraoxon (0.12 mg/kg), the AChE and aliesterase activities showed a pattern similar to that of the low dosage. This suggests that the aliesterases, as altered by BNF exposure, even when nearly completely inhibited, did not alter the response of the target enzyme, AChE, and, therefore, the magnitude of the toxic response. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 263–268, 1997.     

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.     

Simultaneous purification and comparative characterization of six serine hydrolases from rat liver microsomes

Rat liver microsomes contain many serine hydrolases, which can be demonstrated in electropherograms with carboxylesterase stain and with an active-site-directed radioactive organophosphate. Five of the most prominent of these enzymes plus dipeptidyl aminopeptidase IV, a microsomal serine hydrolase without activity against simple esters, have been highly purified with a simultaneous procedure after solubilization with saponin. The five carboxylesterases belong to at least three groups of chemically different proteins. Terminal amino acids, amino acid composition, and substrate specificity are different, while the subunit molecular weight of all esterases is very similar (about 60,000). All purified carboxylesterases have monooleylglycerol-cleaving capacity. The subunit weight (84,000) and the N-terminal amino acid (serine) of the peptidase differ from those of all isolated carboxylesterases. The data are correlated to other reports on individual serine hydrolases from rat liver.     

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.     

The carboxylesterase family exhibits C-terminal sequence diversity reflecting the presence or absence of endoplasmic-reticulum-retention sequences.

Resident proteins of the endoplasmic reticulum lumen are continuously retrieved from an early Golgi compartment by a receptor-mediated mechanism. The sorting or retention sequence on the endoplasmic reticulum proteins is located at the C-terminus and was initially shown to be the tetrapeptide KDEL in mammalian cells and HDEL in Saccharomyces cerevisiae. The carboxylesterases are a large family of enzymes primarily localized to the lumen of the endoplasmic reticulum. Retention sequences in these proteins have been difficult to identify due to atypical and heterogeneous C-terminal sequences. Utilizing the polymerase chain reaction with degenerate primers, we have identified and characterized the C-termini of four members of the carboxylesterase family from rat liver. Three of the carboxylesterases sequences contained C-terminal sequences (HVEL, HNEL or HTEL) resembling the yeast sorting signal which were reported to be non-functional in mammalian cells. A fourth carboxylesterase contained a distinct C-terminal sequence, TEHT. A full-length esterase cDNA clone, terminating in the sequence HVEL, was isolated and was used to assess the retention capabilities of the various esterase C-terminal sequences. This esterase was retained in COS-1 cells, but was secreted when its C-terminal tetrapeptide, HVEL, was deleted. Addition of C-terminal sequences containing HNEL and HTEL resulted in efficient retention. However, the C-terminal sequence containing TEHT was not a functional retention signal. Both HDEL, the authentic yeast retention signal, and KDEL were efficient retention sequences for the esterase. These studies show that some members of the rat liver carboxylesterase family contain novel C-terminal retention sequences that resemble the yeast signal. At least one member of the family does not contain a C-terminal retention signal and probably represents a secretory form.     

Organophosphorus and carbamate resistance in the predacious miteTyphlodromus pyri due to insensitive acetylcholinesterase

The cause of parathion and propoxur resistance inTyphlodromus pyri was studied in a Dutch strain in which resistance was dependent on a semi-dominant gene. Activity of glutathione S-transferase and acetylcholinesterase and reaction rate of acetylcholinesterase with paraoxon and propoxur were measured in this resistant (R) and in a susceptible (S) strain. The R strain was 100-fold resistant to parathion and 2300-fold resistant to propoxur. A 36-fold reduction was found in rate of inhibition of acetylcholinesterase in the R strain for paraoxon, and a 14-fold reduction for propoxur. In combination with the monogenic nature of the resistance, this proves that the insensitivity of acetylcholinesterase is the cause of resistance. The rate constant of acetylcholinesterase inhibition at 25°C in the S and R strains was 1.5×10⁵ and 4.2×10³ M ^–1 min^–1 respectively for paraoxon, and 5.1×10⁴ and 3.6×10³ M ^–1 min^–1 for propoxur. There was no significant difference between the R and S strains in glutathione S-transferase activity. The R strain had a somewhat lower acetylcholinesterase activity than the S strain.     

A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion

A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 microM parathion (146 mg l(-1)) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis-Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 microM p-nitrophenol (83 mg l(-1)) and P. putida KT2440 pSB337 inhibited by 1,000 microM p-nitrophenol (139 mg l(-1)). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.