Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading Variovorax Strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading variovorax strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

Isolation and characterization of an isoproturon mineralizing <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. strain SH from a French agricultural soil

The phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized in an agricultural soil in France that had been periodically exposed to IPU. Enrichment cultures from samples of this soil isolated a bacterial strain able to mineralize IPU. 16S rRNA sequence analysis showed that this strain belonged to the phylogeny of the genus Sphingomonas (96% similarity with Sphingomonas sp. JEM-14, AB219361) and was designated Sphingomonas sp. strain SH. From this strain, a partial sequence of a 1,2-dioxygenase (catA) gene coding for an enzyme degrading catechol putatively formed during IPU mineralization was amplified. Phylogenetic analysis revealed that the catA sequence was related to Sphingomonas spp. and showed a lack of congruence between the catA and 16S rRNA based phylogenies, implying horizontal gene transfer of the catA gene cluster between soil microbiota. The IPU degrading ability of strain SH was strongly influenced by pH with maximum degradation taking place at pH 7.5. SH was only able to mineralize IPU and its known metabolites including 4-isopropylaniline and it could not degrade other structurally related phenylurea herbicides such as diuron, linuron, monolinuron and chlorotoluron or their aniline derivatives. These observations suggest that the catabolic abilities of the strain SH are highly specific to the metabolism of IPU.     

The quantity and quality of dissolved organic matter as supplementary carbon source impacts the pesticide-degrading activity of a triple-species bacterial biofilm

Effects of environmental dissolved organic matter (eDOM) that consists of various low concentration carbonic compounds on pollutant biodegradation by bacteria are poorly understood, especially when it concerns synergistic xenobiotic-degrading consortia where degradation depends on interspecies metabolic interactions. This study examines the impact of the quality and quantity of eDOM, supplied as secondary C-source, on the structure, composition and pesticide-degrading activity of a triple-species bacterial consortium in which the members synergistically degrade the phenylurea herbicide linuron, when grown as biofilms. Biofilms developing on 10 mg L^?1 linuron showed a steady-state linuron degradation efficiency of approximately 85 %. The three bacterial strains co-localized in the biofilms indicating syntrophic interactions. Subsequent feeding with eDOM or citrate in addition to linuron resulted into changes in linuron-degrading activity. A decrease in linuron-degrading activity was especially recorded in case of co-feeding with citrate and eDOM of high quality and was always associated with accumulation of the primary metabolite 3,4-dichloroaniline. Improvement of linuron degradation was especially observed with more recalcitrant eDOM. Addition of eDOM/citrate formulations altered biofilm architecture and species composition but without loss of any of the strains and of co-localization. Compositional shifts correlated with linuron degradation efficiencies. When the feed was restored to only linuron, the linuron-degrading activity rapidly changed to the level before the mixed-substrate feed. Meanwhile only minor changes in biofilm composition and structure were recorded, indicating that observed eDOM/citrate effects had been primarily due to repression/stimulation of linuron catabolic activity rather than to biofilm characteristics.     

A chlorophyll a fluorescence-based Lemna minor bioassay to monitor microbial degradation of nanomolar to micromolar concentrations of linuron

A plant-microbial bioassay, based on the aquatic macrophyte Lemna minor L. (duckweed), was used to monitor biodegradation of nano- and micromolar concentrations of the phenylurea herbicide linuron. After 7 days of exposure to linuron, log-logistic-based dose-response analysis revealed significant growth inhibition on the total frond area of L. minor when linuron concentrations > or = 80 nM were added to the bioassay. A plant-protective effect was obtained for all concentrations > 80 nM by inoculation with either a bacterial consortium or Variovorax paradoxus WDL1, which is probably the main actor in this consortium. The outcome of the plant-microbe-toxicant interaction was also assessed using pulse amplitude-modulated chlorophyll a fluorescence and chlorophyll a fluorescence imaging. Linuron toxicity to L. minor became apparent as a significant decrease in the effective quantum yield (Delta F/Fm') within 90 min after exposure of the plants to linuron concentrations > or = 160 nM. Inoculation of the bioassay with the linuron-degrading bacteria neutralized the effect on the effective quantum yield at concentrations > or = 160 nM, indicating microbial degradation of these concentrations. The chlorophyll a fluorescence-based Lemna bioassay described here offers a sensitive, fast and cost-effective approach to study the potential of biodegrading microorganisms to break down minute concentrations of photosynthesis-inhibiting xenobiotics.     

Environmental Dissolved Organic Matter Governs Biofilm Formation and Subsequent Linuron Degradation Activity of a Linuron-Degrading Bacterial Consortium

It was examined whether biofilm growth on dissolved organic matter (DOM) of a three-species consortium whose members synergistically degrade the phenylurea herbicide linuron affected the consortium''s integrity and subsequent linuron-degrading functionality. Citrate as a model DOM and three environmental DOM (eDOM) formulations of different quality were used. Biofilms developed with all DOM formulations, and the three species were retained in the biofilm. However, biofilm biomass, species composition, architecture, and colocalization of member strains depended on DOM and its biodegradability. To assess the linuron-degrading functionality, biofilms were subsequently irrigated with linuron at 10 mg liter⁻¹ or 100 μg liter⁻¹. Instant linuron degradation, the time needed to attain maximal linuron degradation, and hence the total amount of linuron removed depended on both the DOM used for growth and the linuron concentration. At 10 mg liter⁻¹, the final linuron degradation efficiency was as high as previously observed without DOM except for biofilms fed with humic acids which did not degrade linuron. At 100 μg liter⁻¹ linuron, DOM-grown biofilms degraded linuron less efficiently than biofilms receiving 10 mg liter⁻¹ linuron. The amount of linuron removed was more correlated with biofilm species composition than with biomass or structure. Based on visual observations, colocalization of consortium members in biofilms after the DOM feed appears essential for instant linuron-degrading activity and might explain the differences in overall linuron degradation. The data show that DOM quality determines biofilm structure and composition of the pesticide-degrading consortium in periods with DOM as the main carbon source and can affect subsequent pesticide-degrading activity, especially at micropollutant concentrations.     

Efficient biotransformation of herbicide diuron by bacterial strain <Emphasis Type="Italic">Micrococcus</Emphasis> sp. PS-1

A Gram-positive, Micrococcus sp. strain PS-1 capable of utilizing phenylurea herbicide diuron as a sole carbon source at a high concentration (up to 250 ppm) was isolated from diuron storage site by selective enrichment study. The taxonomic characterization with 16S rRNA gene sequencing (1,477 bp) identified PS-1 as a member of Micrococcus sp. It was studied for the degradation of diuron and a range of its analogues (monuron, linuron, monolinuron, chlortoluron and fenuron). The shake flasks experiments demonstrated fast degradation of diuron (up to 96% at 250 ppm within 30 h incubation) with the addition of small quantity (0.01%) of non-ionic detergent. The relative degradation profile by the isolate was in the order of fenuron > monuron > diuron > linuron > monolinuron > chlortoluron. Further, the biochemical characterization of catabolic pathway by spectroscopic and chromatographic techniques demonstrated that the degradation proceeded via formation of dealkylated metabolites to form 3,4-dichloroaniline (3,4-DCA). It was the major metabolite formed, associated with profound increase in degradation kinetics in presence of appropriate additive.     

Isolation from Agricultural Soil and Characterization of a Sphingomonas sp. Able To Mineralize the Phenylurea Herbicide Isoproturon

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

Effect of immobilization of a bacterial consortium on diuron dissipation and community dynamics

This work intended to study the relationship between diuron herbicide dissipation and the population dynamics of co-cultivated Delftia acidovorans WDL34 (WDL34) and Arthrobacter sp. N4 (N4) for different cell formulations: free cells or immobilization in Ca-alginate beads of one or both strains. GFP-tagged WDL34 and N4 Gram staining allowed analyzing the cell growth and distribution of each strain in both beads and culture medium in the course of the time. Compared to the free cell co-culture of WDL34 and N4, immobilization of WDL34 in Ca-alginate beads co-cultivated with free N4 increased the dissipation rate of diuron by 53% (0.141 mg ml⁻¹ h⁻¹). In that case, immobilization strongly modified the final equilibrium among both strains (highest total N4 to WDL34 ratio). Our results demonstrated that the inoculant formulation played a major role in the cell growth of each cultivated strain possibly increasing diuron dissipation. This optimized cell formulation may allow improving water and soil treatment.     

Genetic and Biochemical Analyses of the tec Operon Suggest a Route for Evolution of Chlorobenzene Degradation Genes

The TecA broad-spectrum chlorobenzene dioxygenase of Burkholderia sp. strain PS12 catalyzes the first step in the mineralization of 1,2,4,5-tetrachlorobenzene. The catabolic genes were localized on a small plasmid that belongs to the IncPβ incompatibility group. PCR analysis of the genetic environment of the tec genes indicated high similarity to the transposon-organized catabolic tcb chlorobenzene degradation genes of Pseudomonas sp. strain P51. Sequence analysis of the regions flanking the tecA genes revealed an upstream open reading frame (ORF) with high similarity to the todF 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase gene of Pseudomonas putida F1 and a discontinuous downstream ORF showing high similarity to the todE catechol 2,3-dioxygenase gene of strain F1. Both homologues in strain P51 exist only as deletion remnants. We suggest that different genetic events thus led to inactivation of the perturbing meta-cleavage enzymes in strains P51 and PS12 during the evolution of efficient chlorobenzene degradation pathways. Biochemical characterization of TodF-like protein TlpF and a genetically refunctionalized TodE-like protein, TlpE, produced in Escherichia coli provided data consistent with the proposed relationships.     

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.     

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.     

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).     

AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.     

Monooxygenase-Mediated 1,2-Dichloroethane Degradation by Pseudomonas sp. Strain DCA1

A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a K_m value below the detection limit of 0.5 μM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.     

Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture

A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities (H. Futamata, S. Harayama, and K. Watanabe, Appl. Environ. Microbiol. 67:4671-4677, 2001). To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter⁻¹ day⁻¹) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter⁻¹ day⁻¹ (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.     

Biodegradation of chlorpropham and its major products by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NKC-1

Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] (CIPC), an important phenyl carbamate herbicide, has been used as a plant growth regulator and potato sprout suppressant (Solanum tuberosum L) during long-term storage. A bacterium capable of utilizing the residual herbicide CIPC as a sole source of carbon and energy was isolated from herbicide-contaminated soil samples employing selective enrichment method. The isolated bacterial strain was identified as Bacillus licheniformis NKC-1 on the basis of its morphological, cultural, biochemical characteristics and also by phylogenetic analysis based on 16S rRNA gene sequences. The organism degraded CIPC through its initial hydrolysis by CIPC hydrolase enzyme to yield 3-chloroaniline (3-CA) as a major metabolic product. An inducible 3-CA dioxygenase not only catalyzes the incorporation of molecular oxygen but also removes the amino group by the deamination yielding a monochlorinated catechol. Further, degradation of 4-chlorocatechol proceeded via ortho- ring cleavage through the maleylacetate process. 3-Chloroaniline and 4-chlorocatechol are the intermediates in the CIPC degradation which suggested that dechlorination had occurred after the aromatic ring cleavage. The presence of these metabolites has been confirmed by using ultra-violet (UV), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Fourier transmission-infrared (FT-IR), proton nuclear magnetic resonance (¹H NMR) and gas chromatography-mass (GC-MS) spectral analysis. Enzyme activities of CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase were detected in the cell-free-extract of the CIPC culture and are induced by cells of NKC-1 strain. These results demonstrate the biodegradation pathways of herbicide CIPC and promote the potential use of NKC-1 strain to bioremediate CIPC-contaminated environment with subsequent release of ammonia, chloride ions and carbon dioxide.     

Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

A molecular toolbox to estimate the number and diversity of Variovorax in the environment: application in soils treated with the phenylurea herbicide linuron

Real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE) approaches that specifically target the Variovorax 16S rRNA gene were developed to estimate the number and diversity of Variovorax in environmental ecosystems. PCR primers suitable for both methods were selected as such that the enclosed sequence showed maximum polymorphism. PCR specificity was maximized by combining PCR with a targeted endonuclease treatment of template DNA to eliminate 16S rRNA genes of the closely related Acidovorax. DGGE allowed the grouping of PCR amplicons according to the phylogenetic grouping within the genus Variovorax. The toolbox was used to assess the Variovorax community dynamics in agricultural soil microcosms (SMs) exposed to the phenylurea herbicide linuron. Exposure to linuron resulted in an increased abundance within the Variovorax community of a subgroup previously linked to linuron degradation through cultivation-dependent isolation. SMs that were treated only once with linuron reverted to the initial community composition 70 days after linuron exposure. In contrast, SMs irrigated with linuron on a long-term base showed a significant increase in Variovorax number after 70 days. Our data support the hypothesis that the genus Variovorax is involved in linuron degradation in linuron-treated agricultural soils.