Bacterial degradation of mixed-PAHs and expression of PAH-catabolic genes.

Polyaromatic hydrocarbons (PAHs) are hazardous organic compounds with established toxicity, carcinogenicity, and mutagenicity, ubiquitous distribution, and persistence in different environmental matrices. In the present study, degradation of the mixture of PAHs (phenanthrene, anthracene, fluorene, and pyrene) by Kocuria flava and Rhodococcus pyridinivorans was investigated. The individual strains and consortium of both degraded 55.6%, 59.5%, and 59.1% of 10 mg L-1 of mixed PAHs, respectively, within 15 days. The participation of catabolic enzymes [catechol 2,3-dioxygenase (C23O), dehydrogenase (DH), and peroxidase (POD)] was confirmed during catalytic oxidation through meta-cleavage of mixed PAHs in this study. The catabolic gene expression of naphthalene dioxygenase (NAH) and catechol 2,3-dioxygenase (C23O) during degradation was confirmed using RT-qPCR in the present study. This is the first study that shows significant gene expression of the catabolic genes during degradation of mixed PAHs by selected bacterial strains. The C23O gene showed a 6.02 log fold higher expression in Kocuria flava in comparison to Rhodococcus pyridinivorans whereas NAH gene exhibited a 7.9 log fold higher expression in Rhodococcus pyridinivorans in comparison to Kocuria flava. Hence it is likely to conclude that combination of Kocuria flava and Rhodococcus pyridinivorans can effectively remove hazardous mixture of PAHs from the contaminated environmental matrix.

Adaptation of phenol-degrading Pseudomonas putida KB3 to suboptimal growth condition: A focus on degradative rate, membrane properties and expression of xylE and cfaB genes.

Detailed characterization of new Pseudomonas strains that degrade toxic pollutants is required and utterly necessary before their potential use in environmental microbiology and biotechnology applications. Therefore, phenol degradation by Pseudomonas putida KB3 under suboptimal temperatures, pH, and salinity was examined in this study. Parallelly, adaptive mechanisms of bacteria to stressful growth conditions concerning changes in cell membrane properties during phenol exposure as well as the expression level of genes encoding catechol 2,3-dioxygenase (xylE) and cyclopropane fatty acid synthase (cfaB) were determined. It was found that high salinity and the low temperature had the most significant effect on the growth of bacteria and the rate of phenol utilization. Degradation of phenol (300 mg L-1) proceeded 12-fold and seven-fold longer at 10 °C and 5% NaCl compared to the optimal conditions. The ability of bacteria to degrade phenol was coupled with a relatively high activity of catechol 2,3-dioxygenase. The only factor that inhibited enzyme activity by approximately 80% compared to the control sample was salinity. Fatty acid methyl ester (FAMEs) profiling, membrane permeability measurements, and hydrophobicity tests indicated severe alterations in bacteria membrane properties during phenol degradation in suboptimal growth conditions. The highest values of pH, salinity, and temperature led to a decrease in membrane permeability. FAME analysis showed fatty acid saturation indices and cyclopropane fatty acid participation at high temperature and salinity. Genetic data showed that suboptimal growth conditions primarily resulted in down-regulation of xylE and cfaB gene expression.

Biodegradation of binary mixtures of octane with benzene, toluene, ethylbenzene or xylene (BTEX): insights on the potential of Burkholderia, Pseudomonas and Cupriavidus isolates.

The contamination of the environment by crude oil and its by-products, mainly composed of aliphatic and aromatic hydrocarbons, is a widespread problem. Biodegradation by bacteria is one of the processes responsible for the removal of these pollutants. This study was conducted to determine the abilities of Burkholderia sp. B5, Cupriavidus sp. B1, Pseudomonas sp. T1, and another Cupriavidus sp. X5 to degrade binary mixtures of octane (representing aliphatic hydrocarbons) with benzene, toluene, ethylbenzene, or xylene (BTEX as aromatic hydrocarbons) at a final concentration of 100 ppm under aerobic conditions. These strains were isolated from an enriched bacterial consortium (Yabase or Y consortium) that prefer to degrade aromatic hydrocarbon over aliphatic hydrocarbons. We found that B5 degraded all BTEX compounds more rapidly than octane. In contrast, B1, T1 and X5 utilized more of octane over BTX compounds. B5 also preferred to use benzene over octane with varying concentrations of up to 200 mg/l. B5 possesses alkane hydroxylase (alkB) and catechol 2,3-dioxygenase (C23D) genes, which are responsible for the degradation of alkanes and aromatic hydrocarbons, respectively. This study strongly supports our notion that Burkholderia played a key role in the preferential degradation of aromatic hydrocarbons over aliphatic hydrocarbons in the previously characterized Y consortium. The preferential degradation of more toxic aromatic hydrocarbons over aliphatics is crucial in risk-based bioremediation.

Impact of nanoparticles on transcriptional regulation of catabolic genes of petroleum hydrocarbon-degrading bacteria in contaminated soil microcosms.

This study was conducted to determine what effects nanoparticles (NPs) like TiO2 , ZnO, and Ag may pose on natural attenuation processes of petroleum hydrocarbons in contaminated soils. The solid NPs used were identified using x-ray diffraction technique and their average size was certified as 18.2, 16.9, and 18.3 nm for Ag-NPs, ZnO-NPs, and TiO2 -NPs, respectively. NPs in soil microcosms behave differently where it was dissolved as in case of Ag-NPs, partially dissolved as in ZnO-NPs or changed into other crystalline phase as in TiO2 -NPs. In this investigation, catabolic gene encoding catechol 2,3 dioxygenase (C23DO) was selected specifically as biomarker for monitoring hydrocarbon biodegradation potential by measuring its transcripts by RT-qPCR. TiO2 -NPs amended microcosms showed almost no change in C23DO expression profile or bacterial community which were dominated by Bacillus sp., Mycobacterium sp., Microbacterium sp., Clostridium sp., beside uncultured bacteria, including uncultured proteobacteria, Thauera sp. and Clostridia. XRD pattern suggested that TiO2 -NPs in microcosms were changed into other non-inhibitory crystalline phase, consequently, showing the maximum degradation profile for most low molecular weight oil fractions and partially for the high molecular weight ones. Increasing ZnO-NPs concentration in microcosms resulted in a reduction in the expression of C23DO with a concomitant slight deteriorative effect on bacterial populations ending up with elimination of Clostridium sp., Thauera sp., and uncultured proteobacteria. The oil-degradation efficiency was reduced compared to TiO2 -NPs amended microcosms. In microcosms, Ag-NPs were not detected in the crystalline form but were available in the ionic form that inhibited most bacterial populations and resulted in a limited degradation profile of oil, specifically the low molecular weight fractions. Ag-NPs amended microcosms showed a significant reduction (80%) in C23DO gene expression and a detrimental effect on bacterial populations including key players like Mycobacterium sp., Microbacterium sp., and Thauera sp. involved in the biodegradation of petroleum hydrocarbons.

Versatile catechol dioxygenases in Sphingobium scionense WP01T.

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413-416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.

Catechol 2,3-dioxygenase from a new phenolic compound degrader Thauera sp. K11: purification and biochemical characterization.

Catechol 2,3-dioxygenase (C23O) from a new phenolic compound degrader Thauera sp. K11 was purified and characterized. The native form of the enzyme was determined as a homotetramer with a molecular weight of 140 kDa, and its isoelectric point was close to 6.4. One iron per enzyme subunit was detected using atom absorption spectroscopy, and the effective size of C23O in its dilute solution (0.2 g L-1 , pH 8.0) was 14.5 nm. The optimal pH and temperature were 8.4 and 45 °C, respectively. The addition of Mg2+ , Cu2+ , Fe2+ , and Mn2+ could improve the enzyme activity, while Ag+ was found to be a strong inhibitor. C23O was stable in alkali conditions (pH 7.6-11.0) and thermostable below 50 °C. The final purified C23O had a sheet content of 53%, consistent with the theoretical value. This showed that the purified catechol 2,3-dioxygenase folded with a reasonable secondary structure.

Biotransformation of benzo[a]pyrene by the thermophilic bacterium Bacillus licheniformis M2-7.

Benzo[a]pyrene (BaP) is recognized as a potentially carcinogenic and mutagenic hydrocarbon, and thus, its removal from the environment is a priority. The use of thermophilic bacteria capable of biodegrading or biotransforming this compound to less toxic forms has been explored in recent decades, since it provides advantages compared to mesophilic organisms. This study assessed the biotransformation of BaP by the thermophilic bacterium Bacillus licheniformis M2-7. Our analysis of the biotransformation process mediated by strain M2-7 on BaP shows that it begins during the first 3 h of culture. The gas chromatogram of the compound produced shows a peak with a retention time of 17.38 min, and the mass spectra shows an approximate molecular ion of m/z 167, which coincides with the molecular weight of the chemical formula C6H4(COOH)2, confirming a chemical structure corresponding to phthalic acid. Catechol 2,3-dioxygenase (C23O) enzyme activity was detected in minimal saline medium supplemented with BaP (0.33 U mg-1 of protein). This finding suggests that B. licheniformis M2-7 uses the meta pathway for biodegrading BaP using the enzyme C23O, thereby generating phthalic acid as an intermediate.

Production of a Recombinant Catechol 2,3-Dioxygenase for the Degradation of Micropollutants.

Phenolic compounds such as catechol represent a particular type of micropollutant whose high stability prevents rapid decay and metabolization in the environment. We successfully cloned a catechol 2,3-dioxygenase (C2,3O) from Pseudomonas putida mt-2 and expressed it in Escherichia coli BER2566. The biomass isolated from shake-flask fermentations was used to partially purify the enzyme. The enzyme proved unstable in clarified liquid fractions (50 mM Tris buffer, pH 7.6) and lost more than 90% of its activity over 7 h at 25 °C. In the presence of 10% acetone, the process was slowed down and 30% residual activity was still present after 7 h incubation. Storage of the enzyme in clear liquid fractions also proved difficult and total inactivation was achieved after 2 weeks even when kept frozen at -20 °C. Lowering the storage temperature to -80 °C preserved 30% activity over the same period. Only minor reactivation of the affected enzyme could be achieved after incubation at 20 °C in the presence of FeSO4 and/or ascorbic acid. Activity loss seems to be due mostly to Fe2+ oxidation as well as to subunit dissociation in the tetrameric structure. However, complete degradation of 1.0 mM catechol could be achieved at 20 °C and pH 7.6 over a 3 h period when using a suspension of whole cells or alginate-encapsulated cells for the biotransformation. Contrary to the clear liquid fractions, these forms of biocatalyst showed no significant sign of inactivation under the working conditions.

Development of nitrilase promoter-derived inducible vectors for Streptomyces.

An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.

Bioinspired copper(I) complexes that exhibit monooxygenase and catechol dioxygenase activity.

New tripodal ligand L2 featuring three different pyridyl/imidazolyl-based N-donor units at a bridgehead C atom, from which one of the imidazolyl units is separated by a phenylene linker, was synthesized and investigated with regards to copper(I) complexation. The resulting complex [(L2)Cu]OTf (2(OTf)), the known complex [(L1)Cu]OTf (1(OTf); L1 differs from L2 in that it lacks the phenylene spacer) and [(L3)Cu]OTf (3(OTf)), prepared from a known chiral, tripodal, N-donor ligand featuring pyridyl, pyrazolyl, and imidazolyl donors, were tested as catalysts for the oxidation of sodium 2,4-di-tert-butylphenolate (NaDTBP) with O2. Indeed, they mediated NaDTBP oxidation to give mainly the corresponding catecholate and quinone (Q). None of the complexes 1(OTf), 2(OTf), and 3(OTf) is superior to the others, as yields were comparable and, if the presence of protons is guaranteed by concomitant addition of the phenol DTBP, the oxidation can also be performed catalytically. For all complexes stoichiometric oxidations under certain conditions (concentrated solutions, high NaDTBP content) were found to also generate products typical for metal-mediated intradiol cleavage of the catecholate with O2. As shown representatively for 1(OTf) this dioxygenation sets in at a later stage of the reaction. Initially a copper species responsible for the monooxygenation must form from 1(OTf)/NaDTBP/O2, and only thereafter is the copper species responsible for dioxygenation formed and consumes Q as substrate. Hence, under these circumstances complexes 1(OTf)-3(OTf) show both monooxygenase and catechol dioxygenase activity.

Isolation, characterization and community diversity of indigenous putative toluene-degrading bacterial populations with catechol-2,3-dioxygenase genes in contaminated soils.

Indigenous bacterial assemblages with putative hydrocarbon-degrading capabilities were isolated, characterized and screened for the presence of the catechol-2,3-dioxygenase (C23O) gene after exposure to toluene in two different (i.e., pristine and conditioned) soil communities. The indigenous bacterial populations were exposed to the hydrocarbon substrate by the addition of toluene concentrations, ranging from 0.5 % to 10 % V/W in 10 g of each soil and incubated at 30 °C for upwards of 12 days. In total, 25 isolates (11 in pristine soil and 14 in conditioned soil) were phenotypically characterized according to standard microbiological methods and also screened for the 238-bp C23O gene fragment. Additionally, 16S rRNA analysis of the isolates identified some of them as belonging to the genera Bacillus, Exiguobacterium, Enterobacter, Pseudomonas and Stenotrophomonas. Furthermore, the two clone libraries that were constructed from these toluene-contaminated soils also revealed somewhat disparate phylotypes (i.e., 70 % Actinobacteria and Firmicutes to 30 % Proteobacteria in conditioned soil, whereas in pristine soil: 66 % Actinobacteria and Firmicutes; 21 % Proteobacteria and 13 % Bacteroidetes). The differences observed in bacterial phylotypes between these two soil communities may probably be associated with previous exposure to hydrocarbon sources by indigenous populations in the conditioned soil as compared to the pristine soil.

Antioxidant enzymes are induced by phenol in the marine microalga Lingulodinium polyedrum.

Knowing the impacts of different anthropogenic activities on ecosystems promotes preservation of aquatic organisms. Aiming to facilitate the identification of polluted or contaminated areas, the study of microalga Lingulodinium polyedrum in phenol-containing medium comprises the determination of toxic and metabolic phenol effects, featuring a possible use of this microorganism as bioindicator for this pollutant. Marine microalga L. polyedrum exposure to phenol increases superoxide dismutase (SOD) and catalase (CAT) activities. The 20% and 50% inhibitory concentrations (IC20 and IC50) of cells exposed to phenol were 40 µmol L(-1) and 120 µmol L(-1), respectively. Phenol biodegradation by L. polyedrum was 0.02 µmol h(-1)cell(-1), and its biotransformation was catalyzed by glutathione S-transferase (GST), phenol hydroxylase and catechol 2,3-dihydroxygenase metabolic pathways. Phenol exposure produced the metabolites 2-hydroxymuconic semialdehyde acid, 1,2-dihydroxybenzene (catechol), and 2-oxo-4-pentenoic acid; also, it induced the activity of key antioxidant biomarker enzymes SOD and CAT by three folds compared to that in the controls. Further, phenol decreased the glutathione/oxidized glutathione ratio (GSH/GSSG), highlighting the effective glutathione oxidation in L. polyedrum. Overall, our results suggest that phenol alters microalga growth conditions and microalgae are sensitive bioindicators to pollution by phenol in marine environments.

Effects of LB broth, naphthalene concentration, and acetone on the naphthalene degradation activities by Pseudomonas putida G7.

Luria-Bertani broth and acetone were usually used in naphthalene degradation experiments as nutrient and solvent. However, their effect on the degradation was seldom mentioned. In this work, we investigated the effect of LB, naphthalene concentration, and acetone on the degradation of naphthalene by Pseudomonas putida G7, which is useful for the degradation of naphthalene on future field remediation. By adding LB, the naphthalene degradation efficiencies and naphthalene dioxygenase were both decreased by 98%, while the catechol dioxygenase was decreased by 90%. Degradation of naphthalene was also inhibited when naphthalene concentration was 56 ppm and higher, which was accompanied with the accumulation of orange-colored metabolism products. However, acetone can stimulate the degradation of naphthalene, and the stimulation was more obvious when naphthalene concentration was lower than 2000 ppm. By assaying the enzyme activities of naphthalene dioxygenase and catechol dioxygenase, it was thought that the degradation efficiency was depending on the more sensitive enzymes on the complicated conditions.

Catechol 2,3-dioxygenase and other meta-cleavage catabolic pathway genes in the 'anaerobic' termite gut spirochete Treponema primitia.

Microorganisms have evolved a spectacular diversity of metabolisms, some of which allow them to overcome environmental constraints, utilize abundant but inaccessible resources and drive nutrient cycling in various ecosystems. The termite hindgut microbial community is optimized to metabolize wood, and in recent years, the in situ physiological and ecological functions of community members have been researched. Spirochetes are abundant in the termite gut, and herein, putative aromatic meta-cleavage pathway genes typical of aerobic pseudomonads were located in genomes of homoacetogenic termite hindgut 'anaerobes', Treponema primitia str. ZAS-1 and ZAS-2. Phylogenetic analyses suggest the T. primitia catechol 2,3-dioxygenase and several other essential meta-pathway genes were acquired from an α-proteobacterium in the distant past to augment several genes T. primitia acquired from anaerobic firmicutes that do not directly catabolize aromatics but can contribute to the final pathway steps. Further, transcripts for each meta-pathway gene were expressed in strictly anaerobic cultures of T. primitia str. ZAS-2 indicative of constitutive pathway expression. Also, the addition of catechol + O(2) to T. primitia liquid cultures resulted in the transient accumulation of trace amounts of the yellow ring cleavage product, hydroxymuconic semialdehyde. This is the first evidence of aromatic ring cleavage in the phylum (division) Spirochetes. Results also support a possible role for T. primitia in termite hindgut O(2) /lignin aromatic monomer metabolism. Potential O(2) -dependent yet nonrespiratory microbial metabolisms have heretofore been overlooked and warrant further investigation. These metabolisms could describe the degradation of plant-derived and other aromatics in microoxic environments and contribute significantly to carbon turnover.

Degradation of dibenzofuran via multiple dioxygenation by a newly isolated Agrobacterium sp. PH-08.

AIMS: To demonstrate the biodegradation of dibenzofuran (DF) and its structural analogs by a newly isolated Agrobacterium sp. PH-08. METHODS AND RESULTS: To assess the biodegradation potential of newly isolated Agrobacterium sp. PH-08, various substrates were evaluated as sole carbon sources in growth and biotransformation experiments. ESI LC-MS/MS analysis revealed the presence of angular degrading by-products as well as lateral dioxygenation metabolites in the upper pathway. The metabolites in the lower pathway also were detected. In addition, the cometabolically degraded daughter compounds of DF-related compounds such as BP and dibenzothiophene (DBT) in dual substrate degradation were observed. Strain PH-08 exhibited the evidence of meta-cleavage pathway as confirmed by the activity and gene expression of catechol-2,3-dioxygenase. CONCLUSIONS: Newly isolated bacterial strain, Agrobacterium sp. PH-08, grew well with and degraded DF via both angular and lateral dioxygenation as demonstrated by metabolites identified through ESI LC-MS/MS and GC-MS analyses. The other heterocyclic pollutants were also cometabolically degraded. SIGNIFICANCE AND IMPACT OF THE STUDY: Few reports have described the complete degradation of DF by a cometabolic lateral pathway. Our study demonstrates the novel results that the newly isolated strain utilized the DF as a sole carbon source and mineralized it via multiple dioxygenation.

Isolation, identification and characterization of a novel Ralstonia sp. FD-1, capable of degrading 4-fluoroaniline.

A gram-negative strain, designated as FD-1, isolated from aerobic activated sludge was capable of metabolizing 4-fluoroaniline (4-FA) as its sole carbon and nitrogen source and energy supply. According to the Biolog GNIII detection method 17 of 71 carbon substrates were easily utilized, while 12 of 23 substrates did not inhibit strain FD-1. The 16S rDNA sequence from strain FD-1 was 99 % similar to Ralstonia sp., suggesting that it belonged to the genus Ralstonia. The optimal conditions for growth and 4-FA degradation were pH 7 and 30 °C. The tolerance to 4-FA were 1,250 mg/L, while the tolerance to salinity was 15 g/L. Catechol 2,3-dioxygenase activity was detected and degradation intermediates were analyzed by liquid chromatography mass spectrometry leading to a proposed degradation pathway and suggesting that extradiol cleavage was involved in 4-FA degradation. This is the first report on the degradation of 4-FA by a bacterium from the Ralstonia genus.

Activity of a carboxyl-terminal truncated form of catechol 2,3-dioxygenase from Planococcus sp. S5.

Catechol 2,3-dioxygenases (C23Os, E.C.1.13.12.2) are two domain enzymes that catalyze degradation of monoaromatic hydrocarbons. The catalytically active C-domain of all known C23Os comprises ferrous ion ligands as well as residues forming active site pocket. The aim of this work was to examine and discuss the effect of nonsense mutation at position 289 on the activity of catechol 2,3-dioxygenase from Planococcus strain. Although the mutant C23O showed the same optimal temperature for activity as the wild-type protein (35 °C), it exhibited activity slightly more tolerant to alkaline pH. Mutant enzyme exhibited also higher affinity to catechol as a substrate. Its K(m) (66.17 µM) was approximately 30% lower than that of wild-type enzyme. Interestingly, removal of the C-terminal residues resulted in 1.5- to 1.8-fold (P < 0.05) increase in the activity of C23OB61 against 4-methylcatechol and 4-chlorocatechol, respectively, while towards catechol the activity of the protein dropped to about 80% of that of the wild-type enzyme. The results obtained may facilitate the engineering of the C23O for application in the bioremediation of polluted areas.

Biodegradation characteristics of p-Chloroaniline and the mechanism of co-metabolism with aniline by Pseudomonas sp. CA-1.

Co-metabolism is a promising method to optimize the biodegradation of p-Chloroaniline (PCA). In this study, Pseudomonas sp. CA-1 could reduce 76.57 % of PCA (pH = 8, 70 mg/L), and 20 mg/L aniline as the co-substrate improved the degradation efficiency by 12.50 %. Further, the response and co-metabolism mechanism of CA-1 to PCA were elucidated. The results revealed that PCA caused deformation and damage on the surface of CA-1, and the -OH belonging to polysaccharides and proteins offered adsorption sites for the contact between CA-1 and PCA. Subsequently, PCA entered the cell through transporters and was degraded by various oxidoreductases accompanied by deamination, hydroxylation, and ring-cleavage reactions. Thus, the key metabolite 4-chlorocatechol was identified and two PCA degradation pathways were proposed. Besides, aniline further enhanced the antioxidant capacity of CA-1, stimulated the expression of catechol 2,3-dioxygenase and promoted meta-cleavage efficiency of PCA. The findings provide new insights into the treatment of PCA-aniline co-pollution.

Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT.

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.

Functionality of the TOL plasmid under varying environmental conditions following conjugal transfer.

Conjugation of catabolic plasmids in contaminated environments is a naturally occurring horizontal gene transfer phenomenon, which could be utilized in genetic bioaugmentation. The potentially important parameters for genetic bioaugmentation include gene regulation of transferred catabolic plasmids that may be controlled by the genetic characteristics of transconjugants as well as environmental conditions that may alter the expression of the contaminant-degrading phenotype. This study showed that both genomic guanine-cytosine contents and phylogenetic characteristics of transconjugants were important in controlling the phenotype functionality of the TOL plasmid. These genetic characteristics had no apparent impact on the stability of the TOL plasmid, which was observed to be highly variable among strains. Within the environmental conditions tested, the addition of glucose resulted in the largest enhancement of the activities of enzymes encoded by the TOL plasmid in all transconjugant strains. Glucose (1 g/L) enhanced the phenotype functionality by up to 16.4 (±2.22), 30.8 (±7.03), and 90.8 (±4.56)-fold in toluene degradation rates, catechol 2,3-dioxygenase enzymatic activities, and xylE gene expression, respectively. These results suggest that genetic limitations of the expression of horizontally acquired genes may be overcome by the presence of alternate carbon substrates. Such observations may be utilized in improving the effectiveness of genetic bioaugmentation.