Isolation and characterization of a novel strain of Stenotrophomonas maltophilia possessing various dioxygenases for monocyclic hydrocarbon degradation.
ABSTRACT: A Gram-negative bacterium, designated as strain KB2, was isolated from activated sludge and was found to utilize different aromatic substrates as sole carbon and energy source. On the basis of morphological and physiochemical characteristics and 16S rRNA gene sequence analysis, the isolated strain KB2 was identified as Stenotrophomonas maltophilia. Strain KB2 is from among different Stenotrophomonas maltophilia strains the first one described as exhibiting the activities of three types of dioxygenases depending on the structure of the inducer. The cells grown on benzoate and catechol showed mainly catechol 1,2-dioxygenase activity. The activity of 2,3-dioxygenase was detected after phenol induction. Protocatechuate 3,4-dioxygenase was found in crude cell extracts of this strain after incubation with 4-hydroxybenzoic acid, protocatechuic acid and vanillic acid. Because of broad spectrum of dioxygenases' types that Stenotrophomonas maltophilia KB2 can exhibit, this strain appears to be very powerful and useful tool in the biotreatment of wastewaters and in soil decontamination.
Project description:Stenotrophomonas maltophilia KB2 is known to produce different enzymes of dioxygenase family. The aim of our studies was to determine activity of these enzymes after induction by benzoic acids in cometabolic systems with nitrophenols. We have shown that under cometabolic conditions KB2 strain degraded 0.25-0.4 mM of nitrophenols after 14 days of incubation. Simultaneously degradation of 3 mM of growth substrate during 1-3 days was observed depending on substrate as well as cometabolite used. From cometabolic systems with nitrophenols as cometabolites and 3,4-dihydroxybenzoate as a growth substrate, dioxygenases with the highest activity of protocatechuate 3,4-dioxygenase were isolated. Activity of catechol 1,2- dioxygenase and protocatechuate 4,5-dioxygenase was not observed. Catechol 2,3-dioxygenase was active only in cultures with 4-nitrophenol. Ability of KB2 strain to induce and synthesize various dioxygenases depending on substrate present in medium makes this strain useful in bioremediation of sites contaminated with different aromatic compounds.
Project description:This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 °C and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K m and V max was 12.8 μM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 μM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.
Project description:A group of flavones, isoflavones, flavanones, and chalcones was subjected to small-scale biotransformation studies with the Gram-negative Stenotrophomonas maltophilia KB2 strain in order to evaluate the capability of this strain to transform flavonoid compounds and to investigate the relationship between compound structure and transformation type. The tested strain transformed flavanones and chalcones. The main type of transformation of compounds with a flavanone moiety was central heterocyclic C ring cleavage, leading to chalcone and dihydrochalcone structures, whereas chalcones underwent reduction to dihydrochalcones and cyclisation to a benzo-?-pyrone moiety. Substrates with a C-2-C-3 double bond (flavones and isoflavones) were not transformed by Stenotrophomonas maltophilia KB2.
Project description:Beijerinckia sp. strain B1 is able to grow on either biphenyl or m-xylene as the sole source of carbon and is capable of cooxidizing many polycyclic aromatic hydrocarbons. The catabolic pathways for biphenyl and m-xylene degradation are coinduced and share common downstream enzymatic reactions. The catabolic pathway for biphenyl degradation involves two meta-cleavage steps, one for 2,3-dihydroxybiphenyl and a second for catechol. The catabolic pathway for m-xylene involves one m-cleavage step for 3-methylcatechol. The genes for two meta-cleavage dioxygenases were cloned from Beijerinckia sp. strain B1 on a single fragment of genomic DNA. The two genes are located approximately 5.5 kb away from one another. Expression of each gene separately in Escherichia coli and analysis of the meta-cleavage dioxygenase produced showed that one enzyme was more specific for 2,3-dihydroxybiphenyl while the second was more specific for catechol. The genes for the two meta-cleavage enzymes were thus labeled bphC and xylE for 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase, respectively. Nondenaturing polyacrylamide gel electrophoresis followed by enzyme activity staining showed that the two meta-cleavage dioxygenases could be easily separated from each other. Similar analyses of Beijerinckia sp. strain B1 grown on succinate, biphenyl, or m-xylene indicate that both meta-cleavage enzymes are induced when cells are grown on either biphenyl or m-xylene. The nucleotide sequence was determined for both bphC and xylE. The two genes are transcribed in opposite directions, demonstrating that at least two operons must be involved in biphenyl degradation by Beijerinckia sp. strain B1. Analysis of the deduced amino acid sequence indicates that 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) falls into the class of meta-cleavage dioxygenases acting on dihydroxylated polycyclic aromatic hydrocarbons and is somewhat distinct from the main group of meta-cleavage dioxygenases acting on 2,3-dihydroxybiphenyl. Catechol 2,3-dioxygenase (XyIE) falls into the class of meta-cleavage enzymes acting on dihydroxylated monocyclic aromatic hydrocarbons but shows little similarity to the canonical TOL plasmid-encoded catechol 2,3-dioxygenase.
Project description:Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a meta-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930-5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2, 3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.
Project description:The release of synthetic chemical pollutants in the environment is posing serious health risks. Enzymes, including oxygenases, play a crucial role in xenobiotic degradation. In the present study, we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants. Fosmid clones possessing dioxygenase activity were further sequenced, and their genes were identified using bioinformatics tools. Two positive fosmid clones, SD3 and RW1, suggested the presence of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC-SD3) and catechol 2,3-dioxygenase (C23O-RW1), respectively. Recombinant versions of these enzymes were purified to examine their pollutant-degrading abilities. The crystal structure of BphC-SD3 was determined at 2.6-Å resolution, revealing a two-domain architecture, i.e., N-terminal and C-terminal domains, with the sequential arrangement of ????? in each domain, characteristic of Fe-dependent class II type I extradiol dioxygenases. The structure also reveals the presence of conserved amino acids lining the catalytic pocket and Fe3+ metal ion in the large funnel-shaped active site in the C-terminal domain. Further studies suggest that Fe3+ bound in the BphC-SD3 active site probably imparts aerobic stability. We further demonstrate the potential application of BphC-SD3 in biosensing of catecholic compounds. The halotolerant and oxygen-resistant properties of these enzymes reported in this study make them potential candidates for bioremediation and biosensing applications.IMPORTANCE The disposal and degradation of xenobiotic compounds have been serious issues due to their recalcitrant properties. Microbial oxygenases are the fundamental enzymes involved in biodegradation that oxidize the substrate by transferring oxygen from molecular oxygen. Among oxygenases, catechol dioxygenases are more versatile in biodegradation and are well studied among the bacterial world. The use of catechol dioxygenases in the field is currently not practical due to their aerobically unstable nature. The significance of our research lies in the discovery of aerobically stable and halotolerant catechol dioxygenases that are efficient in degrading the targeted environmental pollutants and, hence, could be used as cost-effective alternatives for the treatment of hypersaline industrial effluents. Moreover, the structural determination of novel catechol dioxygenases would greatly enhance our knowledge of the function of these enzymes and facilitate directed evolution to further enhance or engineer desired properties.
Project description:Isogenic L1 and L2 gene knockout mutants of Stenotrophomonas maltophilia KJ (KJDeltaL1 and KJDeltaL2, respectively) were constructed by xylE gene replacement. Induction kinetics of the L1 and L2 genes were evaluated by testing catechol 2,3-dioxygenase activity in the mutants. The results suggested that the induction of the L1 and L2 genes was differentially regulated.
Project description:The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K.-H. Engesser, V. Strubel, K. Christoglou, P. Fischer, and H. G. Rast, FEMS Microbiol. Lett. 65:205-210, 1989; V. Strubel, Ph.D. thesis, University of Stuttgart, Stuttgart, Germany, 1991; V. Strubel, H. G. Rast, W. Fietz, H.-J. Knackmuss, and K.-H. Engesser, FEMS Microbiol. Lett. 58:233-238, 1989). Two 2,3-dihydroxybiphenyl-1,2-dioxygenases (BphC1 and BphC2) and one catechol-2,3-dioxygenase (C23O) were shown to be expressed in Terrabacter sp. strain DPO360 growing with dibenzofuran as a sole source of carbon and energy. These enzymes exhibited strong sensitivity to oxygen. They were purified to apparent homogeneity as homodimers (BphC and BphC2) and as a homotetrameric catechol-2,3-dioxygenase (C23O). According to their specificity constants kcat/Km, both BphC1 and BphC2 were shown to be responsible for the cleavage of 2,2',3-trihydroxybiphenyl, the first metabolite in dibenzofuran mineralization along the angular dioxygenation pathway. With this substrate, BphC2 exhibited a considerably higher kcat/Km, value (183 microM/min) than BphC1 (29 microM/min). Catechol-2,3-dioxygenase was recognized to be not involved in the ring cleavage of 2,2',3-trihydroxybiphenyl (kcat/Km, 1 microM/min). Analysis of deduced amino acid sequence data of bphC1 revealed 36% sequence identity to nahC from Pseudomonas putida PpG7 (S. Harayama and M. Rekik, J. Biol. Chem. 264:15328-15333, 1989) and about 40% sequence identity to various bphC genes from different Pseudomonas and Rhodococcus strains. In addition, another 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (bphC3) was cloned from the genome of Terrabacter sp. strain DPO360. Expression of this gene, however, could not be detected in Terrabacter sp. strain DPO360 after growth with dibenzofuran.
Project description:Burkholderia sp. strain TH2, a 2-chlorobenzoate (2CB)-degrading bacterium, metabolizes benzoate (BA) and 2CB via catechol. Two different gene clusters for the catechol ortho-cleavage pathway (cat1 and cat2) were cloned from TH2 and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that while both catechol dioxygenases (CatA1 and CatA2) were produced in BA-grown cells, CatA1 was undetectable when strain TH2 was grown on 2CB or cis,cis-muconate (CCM), an intermediate of catechol degradation. However, production of CatA1 during growth on 2CB or CCM was observed when cat2 genes were disrupted. The difference in the production of CatA1 and CatA2 was apparently due to a difference in inducer recognition by the regulators of the gene clusters. The inducer of CatA1 was found to be BA, not 2CB, by using a 2-halobenzoate dioxygenase gene (cbd) disruptant, which is incapable of transforming (chloro)benzoate. It was also found that CCM or its metabolite acts as an inducer for CatA2. When cat2 genes were disrupted, the growth rate in 2CB culture was reduced while that in BA culture was not. These results suggest that although cat2 genes are not indispensable for growth of TH2 on 2CB, they are advantageous.
Project description:Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.