Evaluation of diuron tolerance and biotransformation by the white-rot fungus Ganoderma lucidum

The white rot basidiomycete Ganoderma lucidum was evaluated for its capability to tolerate and to degrade the herbicide diuron. Diuron at a subtoxic concentration was added at the start of the cultivation in glucose liquid stationary cultures. Under this condition diuron was a laccase inducer. Almost 50% of the initially present diuron was removed after 15 d of cultivation. Two diuron metabolites were found N′-(3,4-dichlorophenyl)-N-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The addition of the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide reduced significantly the capability of the fungus in degrading diuron. The activities of superoxide dismutase and catalase were significantly increased in the mycelial extracts by the presence of diuron. On the other hand, diuron did not cause any significant alteration in the levels of reactive oxygen species. Additionally, laccase could also degrade diuron in vitro and this degradation was increased by the addition of synthetic mediators, 3-ethylbenzthiazoline-6-sulphonic acid and acetylacetone. Significant reduction in the toxicity, as evaluated by the Lactuca sativa bioassay, was observed after G. lucidum treatment. In conclusion, G. lucidum is able to metabolize diuron by intra- and extracellular mechanisms, without the accumulation of toxic products.     

Rapid mineralization of the phenylurea herbicide diuron by Variovorax sp. strain SRS16 in pure culture and within a two-member consortium

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-(14)C]diuron to (14)CO(2), depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted phenylurea herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-(14)C]diuron was mineralized to (14)CO(2) when 15.5 to 38.9 mug liter(-1) diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.     

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).     

Isolation and characterization of three <Emphasis Type="Italic">Sphingobium</Emphasis> sp. strains capable of degrading isoproturon and cloning of the catechol 1,2-dioxygenase gene from these strains

Three strains of bacteria (designated as YBL1, YBL2, YBL3 respectively) capable of degrading isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea, were isolated from the soils of two herbicide plants. Based on the comparative analysis of the 16S rRNA gene, and phenotypic and biochemical characterization, these strains were identified as Sphingobium sp. The optimum conditions for isoproturon degradation by these strains were pH 7.0, and temperature 30°C. Mg²⁺ (1 mM) enhanced the isoproturon degradation rate, while Ni²⁺ and Cu²⁺ (1 mmol l⁻¹) inhibited isoproturon degradation significantly. These three strains also showed the ability to remove the residues of other phenylurea herbicides such as chlorotoluron, diuron and fluometuron in mineral salt culture medium. The N-demethylation was the first step of degradation of dimethylurea-substituted herbicides. Strain YBL1 was found capable of degrading both dimethylurea-substituted herbicides and methoxymethylphenyl-urea herbicides i.e. linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea). Using the PCR method, partial sequences of the catechol 1,2-dioxygenase gene were obtained from these strains.     

Biodegradation of the herbicide diuron by streptomycetes isolated from soil

The diuron degrading activity of 17 streptomycete strains, obtained from agricultural and non-agricultural soils, was determined in the laboratory. All strains were identified as Streptomyces sp. by phenotypic characteristics and PCR-based assays. The strains were cultivated in liquid medium with diuron (4 mg L⁻¹) at 25 °C for 15 days. Biodegradation activity was determined by high-performance liquid chromatography. The results indicated that all strains were able to degrade diuron, but to different amounts. Twelve strains degraded the herbicide by up to 50% and four of them by up to 70%. Strain A7-9, belonging to S. albidoflavus cluster, was the most efficient organism in the degradation of diuron, achieving 95% degradation after five days of incubation and no herbicide remained after 10 days. Overall, the strains isolated from agricultural soils exhibited higher degradation percentages and rates than those isolated from non-agricultural soils. Given the high degradation activity observed here, the streptomycete strains show a good potential for bioremediation of soils contaminated with diuron.     

Atrazine and diuron resistant plants from photoautotrophic protoplast-derived cultures of Nicotiana plumbaginifolia

Atrazine and diuron resistant clones were isolated from diploid photoautotrophic protoplastderived colonies of Nicotiana plumbaginifolia. Protoplasts were mutagenised with 0.1 mM N-ethyl-N-nitrosourea and colonies were screened for resistance after plating. Selection of calli was carried out on their ability to grow and green on a selective medium containing either atrazine or diuron. Plants were regenerated from most tolerant calli. Herbicide spray showed that plants of 6 and 4 clones were resistant to atrazine and diuron, respectively.Abbreviations Atrazine 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine - diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - NEU N-ethyl-N-nitrosourea - PSII photosystem II     

Modeling Based Structural Insights into Biodegradation of the Herbicide Diuron by Laccase-1 from Ceriporiopsis subvermispora

The herbicide diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is used in many agricultural crops and non-crop areas worldwide, leading to the pollution of the aquatic environment by soil leaching. White rot fungi and its lignin modifying enzymes, peroxidases and laccases, are responsible for its degradation. Therefore, it is of interest to explore the potential use of Ceriporiopsis subvermispora laccase (CersuLac1) in the biotransformation of this herbicide by using its enzyme laccase. However, the structure of laccase from Ceriporiopsis subvermispora is still unknown. Hence, a model of laccase was constructed using homology modeling. The model was further used to dock p-methylbenzoate in the presence of four copper ions to analyze molecular basis of its binding and interaction. The ligand-protein interaction is stereo-chemically favorable in nature. The presence of the single protonated Lys457 was necessary for catalysis, being coordinated by a cupper ion. The best pose of diuron on CersuLac1 has a theoretical Ki of 2.91 mM. This is comparable to the KM values for laccases from other organisms with similar compounds. Thus, we document the insights for the potential use of laccase from Ceriporiopsis subvermispora in the biotransfrormation of diuron.     

Biomineralization of 3-nitrotoluene by Diaphorobacter species

Three bacterial strains utilizing 3-nitrotoluene (3-NT) as a sole source of carbon, nitrogen and energy were isolated from an industrial wastewater treatment plant. Biochemical tests and 16S rDNA sequence analysis revealed that the isolated strains belonged to Diaphorobacter sp. Detailed studies were carried out with Diaphorobacter sp. strain DS2. Degradation of 3-NT by Diaphorobacter sp. strain DS2 was accompanied by the release of nitrite in the culture broth with increase in biomass. Total organic carbon analysis confirmed the extensive mineralization of 3-NT. The strain could degrade 3-methylcatechol, 4-methylcatechol and catechol easily suggesting that the degradation pathway could involve these as possible intermediates. Successful PCR amplification of the oxygenase large subunit and the presence of high activity for catechol 2,3-dioxygenase in the crude cell lysate further confirmed that the degradation of 3-NT occurred through (methyl)catechol intermediates in strain DS2. The strain DS2 was found to degrade other isomers of mononitrotoluene (2-NT and 4-NT) and nitrobenzene as well.     

Biodegradation of the herbicide diuron by streptomycetes isolated from soil

The diuron degrading activity of 17 streptomycete strains, obtained from agricultural and non-agricultural soils, was determined in the laboratory. All strains were identified as Streptomyces sp. by phenotypic characteristics and PCR-based assays. The strains were cultivated in liquid medium with diuron (4 mg L⁻¹) at 25 °C for 15 days. Biodegradation activity was determined by high-performance liquid chromatography. The results indicated that all strains were able to degrade diuron, but to different amounts. Twelve strains degraded the herbicide by up to 50% and four of them by up to 70%. Strain A7-9, belonging to S. albidoflavus cluster, was the most efficient organism in the degradation of diuron, achieving 95% degradation after five days of incubation and no herbicide remained after 10 days. Overall, the strains isolated from agricultural soils exhibited higher degradation percentages and rates than those isolated from non-agricultural soils. Given the high degradation activity observed here, the streptomycete strains show a good potential for bioremediation of soils contaminated with diuron.     

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L^?1 h^?1. Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L^?1 h^?1. At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.     

Nanocomplexes on the basis of Taunit associated with biocides as effective anti-cyanobacterial agents

Cyanobacteria are photoautotrophic bacteria that are known also as blue-green algae. They accumulate on different surfaces and objects and contribute to their biodegradation. Moreover, cyanobacteria produce toxins, which lead to harmful environmental and human health impacts. Hence, cyanobacterial growth control problem is very vital. The goal of the study was to obtain new nanocomplexes on the basis of a modern nanomaterial Taunit associated with antibiotic chloramphenicol and herbicide diuron and to test their antimicrobial effect against a model organism such as the unicellular cyanobacterium Synechocystis sp. PCC 6803. A nanomaterial made of multiwalled carbon nanotubes (MWCNTs) called Taunit was used for the first time to obtain nanocomplexes coupled either with herbicide diuron (DCMU (3-(3,4-dichlorophenyl)- 1,1-dimethylurea) or with antibiotic chloramphenicol. A small amount of Taunit (~1 mg) was needed to adsorb micrograms of diuron or chloramphenicol. The new formed nanocomplexes differentiate in their antimicrobial activity, which could be explained by the difference in their chemical mechanism of action. Taunit ? diuron complex showed a higher biocide action against cyanobacterium than the Taunit ? chloramphenicol complex. The results allow to discuss the prospects of research on the use of Taunit ? diuron complex as a coating for various surfaces exposed to cyanobacteria fouling.     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Studies on Diuron Uptake in a Blue-Green Alga Nostoc muscorum

A mutant of Nostoc muscorum that is resistant to 3-(3.4-dichlorophenyl)-I.1-dimethylurea (diuron) has been selected. This mutant maylack the step in photosynthesis that is affected by diuron (DCMU).but it can also use DCMU as a source of carbon and nitrogen.Another mutant of this organism resistant to L-methionine-DL-sulphoximine(MSO), that was isolated previously, also shows some cross-resistanceto DCMU. Key words: Nostoc muscorum, Diuron resistant mutants, MSO resistant mutants     

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H₂O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (¹⁴C-radiolabel assays) with binding constants (K) of 1.4 × 10⁻⁸ molar and 4 × 10⁻⁸ molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10⁻⁸ molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts.     

Isolation and Characterization of Diuron-degrading Bacteria from Lotic Surface Water

The bacterial community structure of a diuron-degrading enrichment culture from lotic surface water samples was analyzed and the diuron-degrading strains were selected using a series of techniques combining temporal temperature gradient gel electrophoresis (TTGE) of 16 S rDNA gene V1–V3 variable regions, isolation of strains on agar plates, colony hybridization methods, and biodegradation assays. The TTGE fingerprints revealed that diuron had a strong impact on bacterial community structure and highlighted both diuron-sensitive and diuron-adapted bacterial strains. Two bacterial strains, designated IB78 and IB93 and identified as belonging to Pseudomonas sp. and Stenotrophomonas sp., were isolated and shown to degrade diuron in pure resting cells in a first-order kinetic reaction during the first 24 h of incubation with no 3,4-DCA detected. The percentages of degradation varied from 25% to 60% for IB78 and 20% to 65% for IB93 and for a diuron concentration range from 20 mg/L to 2 mg/L, respectively. It is interesting to note that diuron was less degraded by single isolates than by mixed resting cells, thereby underlining a cumulative effect between these two strains. To the best of our knowledge, this is the first report of diuron-degrading strains isolated from lotic surface water.     

Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii

Background

Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost.

Results

21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources.

Conclusion

These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1467-7) contains supplementary material, which is available to authorized users.     

Oxidation and reduction of sulfite by chloroplasts and formation of sulfite addition compounds

After exposing intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) and capable of photoreducing CO₂ at high rates to different concentrations of radioactive sulfite in the light or in the dark, ³⁵SO₂ and H₂³⁵S were removed from the acidified suspensions in a stream of nitrogen. Remaining activity could be fractionated into sulfate, organic sulfides, and sulfite addition compounds. When chloroplast suspensions contained catalase, superoxide dismutase and O-acetylserine, the oxidation of sulfite to sulfate was slower in the light than the reductive formation of sulfides that exhibited a maximum rate of about 2 micromoles per milligram chlorophyll per hour, equivalent to about 1% of maximum carbon assimilation. Botht the oxidative and the reductive detoxification of sulfite were very slow in the dark. Oxidation was somewhat, but not much, accelerated in the light in the absence of O-acetylserine, which caused a dramatic decrease in the formation of organic sulfides and an equally dramatic increase in the concentration of sulfite addition compounds whose formation was light-dependent. The sulfite addition compounds were not identified. Addition compounds did not accumulate in the dark. In the light, the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, decreased not only the reduction, but also the oxidation of sulfite and the formation of addition compounds.     

Characteristics of Nitrate Reduction in a Mutant of the Blue-Green Alga Agmenellum quadruplicatum

Characteristics of nitrate reduction in terms of nitrite production in an N-methyl-N′-nitro-N-nitrosoguanidine-induced mutant of the blue-green alga Agmenellum quadruplicatum are described. Following induction of nitrate reduction a linear rate of nitrite production proportional to cell concentration was observed. Rate of nitrite production and growth rate showed similar responses to pH, temperature, and light intensity. If required, only trace amounts of carbon dioxide were necessary for nitrite production. Atmospheres of oxygen or nitrogen inhibited production of nitrite. In addition, a low but constant rate of nitrite production was observed in the dark. Nitrite production by mutant AQ-6 was studied in terms of photosynthesis. As nitrite production proceeded, rate of photosynthesis declined. Ultraviolet irradiation and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea poisoning did not prevent nitrite production. The action spectrum of nitrite production was chlorophyll a-like.     

Degradation of substituted phenylurea herbicides by Arthrobacter globiformis strain D47 and characterization of a plasmid-associated hydrolase gene, puhA

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).