Tendency of using different aromatic compounds as substrates by 2,4-DNT dioxygenase expressed by pJS39 carrying the gene dntA from Burkholderia sp. strain DNT

The substrate range of 2,4-dinitrotoluene (DNT) dioxygenase was investigated by measuring substrate-dependent O₂ uptake and maximum growth (expressed in A₆₀₀) on substrate-containing minimal medium. The control for each strain had no added particular substrate. The following aromatic compounds: catechol, α-naphthalene acetic acid, β-dimethylaminobenzaldehyde, 3,4-dinitrosalicylic acid, p-nitrophenol, naphthanol, o-anisic acid, salicylic acid, toluene, and benzoic acid, were tried as possible substrates. Considering all substrates used, only p-nitrophenol showed zero oxygen uptake rate and zero growth. This indicates that it was rather unlikely that p-nitrophenol is a substrate analog for 2,4-DNT. Catechol was clearly used as a sole carbon source by both wild-type Escherichia. coli (JM103) and the dnt transformant (JS39). Using α-naphthalene acetic acid and β-dimethylaminobenzaldehyde as substrates resulted in DNT dioxygenase oxygen uptake rates of 11.8 and 14?μM/hr/mg protein, respectively. However, using both compounds as a carbon source, JS39 had twice the growth rate of E. coli JM103. For the remaining six substrates tested (3, 4-dinitrosalicylic acid, p-nitrophenol, o-anisic acid, salicylic acid, toluene, and benzoic acid), there appeared to be growth advantages for JS39 (even though the growth in the presence of substrate was less than the controls) suggesting a situation similar to that described for α-naphthalene and β-dimethylaminobenzaldehyde above. Combining results from our assay with respirometry and growth-based experiments will allow a better understanding of the biochemical consequences of these interactions. These results suggest that DNT dioxygenase gene, dntA carried by JS39, and those potential genes for substrates-degraded enzyme(s) system could have a common root.     

Oxidation of aminonitrotoluenes by 2,4-DNT dioxygenase of Burkholderia sp. strain DNT

Aminonitrotoluenes form rapidly from the reduction of dinitrotoluenes (DNTs) which are priority pollutants and animal carcinogens. For example, 4-amino-2-nitrotoluene (4A2NT) and 2A4NT accumulate from the reduction of 2,4-DNT during its aerobic biodegradation. Here, we show that 2,4-DNT dioxygenase (DDO) from Burkholderia sp. strain DNT oxidizes the aminonitrotoluenes 2A3NT, 2A6NT, 4A3NT, and 5A2NT to 2-amino-3-nitrobenzylalcohol, 2-amino-4-nitro-m-cresol and 3-amino-5-nitro-p-cresol, 4-amino-3-nitrobenzylalcohol and aminonitrocresol, and 2-amino-5-nitro-o-cresol, respectively. 2A5NT and 3A4NT are oxidized to aminonitrocresols and/or aminonitrobenzylalcohols, and 4A2NT is oxidized to aminonitrocresol. Only 2A4NT, a reduced compound derived from 2,4-DNT, was not oxidized by DDO or its three variants. The alpha subunit mutation I204Y resulted in two to fourfold faster oxidization of the aminonitrotoluenes. Though these enzymes are dioxygenases, they acted like monooxygenases by adding a single hydroxyl group, which did not result in the release of nitrite.     

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.     

Bioremediation of 2,4-dinitrotoluene (2,4-DNT) in immobilized micro-organism biological filter

Aims: To evaluate the biodegradability of 2,4‐DNT using an anaerobic filter (AF) combined with a biological aerated filter (BAF), and elucidate the degradation mechanism of 2,4‐DNT and analyze the bacterial community of the reactors over a long period of operation. Methods and Results: The pilot test experienced wide fluctuations influent concentrations and there was lower than 0.50 mg l^?1 of 2,4‐DNT in the effluent of the system. The removal efficiency was above 99%. GC‐MS analysis demonstrated that 2,4‐DNT was mainly reduced to 2‐amino‐4‐nitrotoluene (2‐A‐4‐NT), 4‐amino‐2‐nitrotoluene (4‐A‐2‐NT), and 2,4‐diaminotoluene (2,4‐DAT) during the anaerobic reaction. In addition, ethanol was added into the influent as the electron donor. Because of the use of part ethanol as an auxiliary carbon source, more than twice the theoretical requirement of ethanol was needed to achieve a high 2,4‐DNT removal efficiency (>93%). ESEM observations showed that the carrier could immobilize micro‐organisms, which flourished more in reactors operating over longer periods. Further research by PCR‐DGGE revealed that new 2,4‐DNT‐resistant bacterial had been generated during the stress of 2,4‐DNT for 150 days. The dominant species for 2,4‐DNT degradation were identified by a comparison with gene sequences in GenBank. Conclusions: 2,4‐DNT could be effectively degraded by the combined process and ethanol played an important role in the biotransformation. The proposed transformation pathway of 2,4‐DNT was concluded. During the 150‐day operation, some microbial taxa unaccustomed to 2,4‐DNT died out and some new 2,4‐DNT‐resistant microbial taxa appeared. Significance and Impact of the Study: The study provides a novel method for the bioremediation of 2,4‐DNT, which is difficult to degrade by traditional biological methods. The most 2,4‐DNT‐resistant microbial taxa have not been reported elsewhere and they may be helpful to the treatment of actual 2,4‐DNT wastewater.     

Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation

The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomonas) sp. strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT). Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1). When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A(600)) and reached slightly higher maximum viable cell numbers. YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain. YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days.     

Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation.

The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission. Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids. Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons.     

Protein engineering of the 4-methyl-5-nitrocatechol monooxygenase from Burkholderia sp. strain DNT for enhanced degradation of nitroaromatics

4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from Burkholderia sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 microM of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 +/- 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 +/- 2 nmol/min/mg protein). The values of kcat/Km of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s(-1) M(-1)), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 microM, the initial nitrite release rate of M22L/L380I enzyme was 17 +/- 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 +/- 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.     

Directed evolution of aniline dioxygenase for enhanced bioremediation of aromatic amines

The objective of this study was to enhance the activity of aniline dioxygenase (AtdA), a multi-component Rieske non-heme iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA, so as to create an enhanced biocatalyst for the bioremediation of aromatic amines. Previously, the mutation V205A was found to widen the substrate specificity of AtdA to accept 2-isopropylaniline (2IPA) for which the wild-type enzyme has no activity (Ang EL, Obbard JP, Zhao HM, FEBS J, 274:928–939, 2007). Using mutant V205A as the parent and applying one round of saturation mutagenesis followed by a round of random mutagenesis, the activity of the final mutant, 3-R21, was increased by 8.9-, 98.0-, and 2.0-fold for aniline, 2,4-dimethylaniline (24DMA), and 2-isopropylaniline (2IPA), respectively, over the mutant V205A. In particular, the activity of the mutant 3-R21 for 24DMA, which is a carcinogenic aromatic amine pollutant, was increased by 3.5-fold over the wild-type AtdA, while the AN activity was restored to the wild-type level, thus yielding a mutant aniline dioxygenase with enhanced activity and capable of hydroxylating a wider range of aromatic amines than the wild type. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of 2,4‐dinitrotoluene on the anaerobic bacterial community in marine sediment

Aims: To study the impact of added 2,4‐dinitrotoluene (DNT) on the anaerobic bacterial community in marine sediment collected from an unexploded ordnance dumping site in Halifax Harbour. Methods and Results: Marine sediment was spiked with 2,4‐DNT and incubated under anaerobic conditions in the presence and absence of lactate. Indigenous bacteria in the sediment removed 2,4‐DNT with subsequent formation of its mono‐ and diamino‐derivatives under both conditions. PCR–DGGE and nucleotide sequencing were used to monitor the change in the bacterial population in sediment caused by the presence of 2,4‐DNT. The results showed that denaturing gradient gel electrophoresis banding patterns of sediment microcosms treated with 2,4‐DNT were different from controls that did not receive 2,4‐DNT. Bacteroidetes, Firmicutes and δ‐Proteobacteria were present in sediment incubated in the absence of 2,4‐DNT. However, several γ‐Proteobacteria became dominant in sediment in the presence of 2,4‐DNT, two of which were 99% similar to Shewanella canadensis and Shewanella sediminis. In the presence of both 2,4‐DNT and lactate, two additional δ‐Proteobacteria were enriched, one closely related (98% similarity) to Desulfofrigus fragile and the other affiliated (96% similarity) to Desulfovibrio sp. In contrast, none of the above four Proteobacteria were enriched in sediment incubated with lactate alone. Conclusions: Presence of 2,4‐DNT led to a significant change in bacterial population of marine sediment with the enrichment of several γ‐ and δ‐Proteobacteria. Significance and Impact of the Study: Our results provided the first evidence on the impact of the pollutant 2,4‐DNT on the indigenous bacterial community in marine sediment, and provided an insight into the composition of bacterial community that degrade 2,4‐DNT.     

Effects of<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of<Emphasis Type="Italic"> Burkholderia</Emphasis> and on 2,4-DNT degradation in two-phase bioreactors

Expression of vgb, encoding Vitreoscilla hemoglobin (VHb), in Burkholderia strain YV1 was previously shown to improve cell growth and enhance 2,4-dinitrotoluene (2,4-DNT) degradation compared with control strain DNT, especially under hypoxic conditions. In the work reported here, the ratio of 2,4-DNT degraded to oxygen uptake was approximately 5-fold larger for strain YV1 than for strain DNT. The addition of purified VHb to cytosolic fractions of strain DNT increased 2,4-DNT degradation 1.5-fold, compared with 1.1-fold for control bovine Hb, but increased the 2,4-DNT degradation 2.7-fold when added to partially purified 2,4-DNT dioxygenase, compared with 1.3-fold for bovine Hb. This suggests a direct transfer of oxygen from VHb to the oxygenase. In a bioreactor at high 2,4-DNT concentration (using 100 ml oleyl alcohol containing 2 g 2,4-DNT as the second phase) with 1.5 l culture, both strains could remove 0.8 g 2,4-DNT by 120 h; and, under the same conditions in a fed-batch reactor, the degradation increased to 1 g for strain YV1 but not for strain DNT.     

Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase from Burkholderia sp. strain RASC.

The findings of previous studies indicate that the genes required for metabolism of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) are typically encoded on broad-host-range plasmids. However, characterization of plasmid-cured strains of Burkholderia sp. strain RASC, as well as mutants obtained by transposon mutagenesis, suggested that the 2,4-D catabolic genes were located on the chromosome of this strain. Mutants of Burkholderia strain RASC unable to degrade 2,4-D (2,4-D- strains) were obtained by insertional inactivation with Tn5. One such mutant (d1) was shown to have Tn5 inserted in tfdARASC, which encodes 2,4-D/alpha-ketoglutarate dioxygenase. This is the first reported example of a chromosomally encoded tfdA. The tfdARASC gene was cloned from a library of wild-type Burkholderia strain RASC DNA and shown to express 2,4-D/alpha-ketoglutarate dioxygenase activity in Escherichia coli. The DNA sequence of the gene was determined and shown to be similar, although not identical, to those of isofunctional genes from other bacteria. Moreover, the gene product (TfdARASC) was purified and shown to be similar in molecular weight, amino-terminal sequence, and reaction mechanism to the canonical TfdA of Alcaligenes eutrophus JMP134. The data presented here indicate that tfdA genes can be found on the chromosome of some bacterial species and suggest that these catabolic genes are rather mobile and may be transferred by means other than conjugation.     

Metabolism of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes sp. JS867 under oxygen limited conditions

Transformation of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes JS867 undervarying degrees of oxygen limitation was examined. Complete 2,4-DNT removalwas observed under oxygen excess with near stoichiometric release (83%) of nitrite.Average kinetic parameters were estimated based on a dual-Monod biokinetic modelwith 2,4-DNT and O₂ as growth limiting substrates. The negative impact of nitrite accumulation on the reaction rate was adequately described by inclusion of a noncompetitive inhibition term for NO₂ ^-. Under aerobic conditions, _max, K_sDNT, andK_iNO were 0.058(0.004) hr^-1, 3.3(±1.3) mg 2,4-DNT/L, and 1.2(±pm0.2) hr^-1, respectively. At increasing oxygen limitation, rates of 2,4-DNT disappearance and nitrite production decreased and incomplete removal of 2,4-DNT commenced. JS867 was able to use NO₂ ^- as a terminal electron acceptor whengrown on glucose or succinate under anaerobic conditions. However, during growthon 2,4-DNT and under O₂-limited conditions, JS867 did not use released nitrite as electron acceptor. The nearly constant molar ratios of DNT removed over NO₂ ^- released under various degrees of oxygen limitation suggested that oxygenolytic denitration pathways continued. No evidence of nitroreduction was obtained under the examined oligotrophic conditions. JS867 displayed a high affinity for oxygen consumption with K_SO2 value of 0.285(±0.198) mg O₂/L. Our results indicate thatunder oligotrophic conditions with 2,4-DNT as dominant carbon source, oxygen availability and nitrite accumulation may limit 2,4-DNT biomineralization, but the accumulation of reduced 2,4-DNT transformation products will be small.     

TNT and nitroaromatic compounds are chemoattractants for Burkholderia cepacia R34 and Burkholderia sp. strain DNT

Nitroaromatic compounds are toxic and potential carcinogens. In this study, a drop assay was used to detect chemotaxis toward nitroaromatic compounds for wild-type Burkholderia cepacia R34, wild-type Burkholderia sp. strain DNT, and a 2,4-dinitrotoluene (2,4-DNT) dioxygenase mutant strain (S5). The three strains are chemotactic toward 2,4,6-trinitrotoluene (TNT), 2,3-DNT, 2,4-DNT, 2,5-DNT, 2-nitrotoluene (NT), 4NT, and 4-methyl-5-nitrocatechol (4M5NC), but not toward 2,6-DNT. Of these, only 2,4-DNT is a carbon and energy source for B. cepacia R34 and Burkholderia sp. strain DNT, and 4M5NC is an intermediate in the 2,4-DNT degradation pathway. It was determined that the 2,4-DNT dioxygenase genes are not required for the chemotaxis for these nitroaromatic compounds because the DNT DDO mutant S5 has a chemotactic response toward 2,4-DNT although 2,4-DNT is not metabolized by S5; hence, 2,4-DNT itself is the chemoattractant. This is the first report of chemotaxis toward TNT, 2,3-DNT, 2,4-DNT, 2,5-DNT, 2NT, 4NT, and 4M5NC.     

Increase in the thermostability of Bacillus sp. strain TAR-1 xylanase using a site saturation mutagenesis library

Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43–Lys115 and Ala300–Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80°C for 15 min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase.

Abbreviations: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan     

Saturation mutagenesis of Burkholderia cepacia R34 2,4-dinitrotoluene dioxygenase at DntAc valine 350 for synthesizing nitrohydroquinone, methylhydroquinone, and methoxyhydroquinone

Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc alpha-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 +/- 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 +/- 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 +/- 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 +/- 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 +/- 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 +/- 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 +/- 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 +/- 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 +/- 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene. Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol).     

Molecular and catalytic properties of 2,4'-dihydroxyacetophenone dioxygenase from Burkholderia sp. AZ11

The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 μM and 6.28 μmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.     

Effect of propanil,linuron, and dicamba on the degradation kinetics of 2,4‐dichlorophenoxyacetic acid by Burkholderia sp. A study by differential analysis of 2,4‐dichlorophenoxyacetic acid degradation data

The successive application of distinct pesticides, or mixtures of them, is a frequent practice that could adversely affect the microbial species inhabiting soil and aquatic ecosystems. The ability of soil or aquatic microbiota to degrade a pesticide could be affected by the presence of another. If the degradation rate of the first compound is inhibited, its dissipation half‐life in the environment could be hazardously enlarged. Few studies have been made to quantify the impact on the biodegradation rate of pesticides in soils or water by the presence of other pesticides. In this work, a method for assessing the effect of a pesticide on the biodegradation rate of another, measuring its effect on the biodegradation kinetics of a single bacterial strain is presented. The mathematical analysis is a powerful tool to study the stoichiometry and kinetics of microbial processes, which was used to evaluate independently, in detail, the effect of three pesticides (propanil, linuron, and dicamba) on the biodegradation kinetics of 2,4‐dichlorophenoxyacetic acid by a strain of Burkholderia sp. It was evidenced that linuron and dicamba caused a decay of more than 40% in the top instantaneous degradation rate of 2,4‐dichlorophenoxyacetic acid, while propanil showed a minimal effect.     

Enhancement of 2,4-dinitrotoluene biodegradation by Burkholderia sp. in sand bioreactors using bacterial hemoglobin technology

Continuous flow sand column bioreactor experiments were conducted to investigate the effect of 2,4-dinitrotoluene (DNT) concentration (i.e. DNT loading rate) and influent dissolved oxygen (DO) concentration on aerobic biodegradation of DNT by wild type (DNT) and recombinant (YV1) Burkholderia sp., the latter containing plasmid pSC160 which carries the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla. The experiments were conducted in two continuous flow packed bed sand column bioreactors, one growing the wild type strain and the other growing YV1. Under oxygen-rich feed conditions (6.8 mg DO/L in the feed) with an influent DNT concentration of 99.6 mg/L (DNT loading rate approximately = 9.2 mg/m2/day), the effluent DNT concentration from the wild type bioreactor reached 0.7 mg DNT/L in 40 days whereas it was less than 0.2 mg DNT/L for the YV1 bioreactor in about 25 days. When influent DNT concentration was increased to 214 mg/L (DNT loading rate approximately = 20.3 mg/m2/day) while maintaining the same influent DO level of 6.8 mg/L, the effluent DNT concentration increased to about 5 mg/L for the wild type bioreactor whereas it was maintained at less than 0.2 mg/L for the YV1 bioreactor. Additionally, when influent DO was reduced from 6.8 mg/L to 3.1 mg/L while the influent DNT concentration remained at 214 mg/L, the effluent DNT concentration increased to more than 20 mg/L for the wild type bioreactor but up to only 1.7 mg/L for the YV1 bioreactor. A subsequent increase of influent DO back to 6.6 mg/L reduced the effluent DNT concentration to about 5 mg/L for the wild type bioreactor and to 0.10-0.19 mg/L for the YV1 bioreactor. These results confirm the utility of vgb technology to enhance biodegradation of aromatic compounds under hypoxic conditions and also that this enhancement can be maintained over extended periods of time as evidenced by plasmid stability in Burkholderia YV1.     

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.     

Multiple pathways for toluene degradation in Burkholderia sp. strain JS150.

Burkholderia (Pseudomonas) sp. strain JS150 uses multiple pathways for the metabolism of catechols that result from degradation of aromatic compounds. This suggests that the strain also uses multiple upstream pathways for the initial hydroxylation of aromatic substrates. Two distinct DNA fragments that allowed Pseudomonas aeruginosa PAO1c to grow with benzene as a sole carbon source were cloned from strain JS150. One of the recombinant plasmids containing the initial steps for the degradative pathway contained a 14-kb DNA insert and was designated pRO2016. We have previously shown that the DNA insert originated from a plasmid carried by strain JS150 and contained genes encoding a multicomponent toluene-2-monooxygenase (tbmABCDEF) as well as the cognate regulatory protein (tbmR) that controls expression of the 2-monooxygenase (G. R. Johnson and R. H. Olsen, Appl. Environ. Microbiol. 61:3336-3346, 1995). Subsequently, we have identified an additional region on this DNA fragment that encodes toluene-4-monooxygenase activity. The toluene-4-monooxygenase activity was also regulated by the tbmR gene product. A second DNA fragment that allowed P. aeruginosa to grow with benzene was obtained as a 20-kb insert on a recombinant plasmid designated pRO2015. The DNA insert contained genes encoding toluene-4-monooxygenase activity but no toluene-2-monooxygenase activity. The pRO2015 insert originated from the chromosome of strain JS150, unlike the region cloned in pRO2016. Southern blots and restriction map comparisons showed that the genes for the individual 4-monooxygenases were distinct from one another. Thus, strain JS150 has been shown to have at least three toluene/benzene monooxygenases to initiate toluene metabolism in addition to the toluene dioxygenase reported previously by others.