Degradation of chlorosubstituted aromatic compounds by Pseudomonas sp. strain B13: fate of 3,5-dichlorocatechol

The degradation of 3,5-dichlorocatechol by enzymes of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. The following compounds were formed from 3,5-dichlorocatechol: trans-2-chloro-4-carboxymethylenebut-2-en-4-olide, cis-2-chloro-4-carboxymethylenebut-2-en-4-olide, and chloroacetylacrylate as the decarboxylation product of 2-chloromaleylacetate. They were identified by chromatographic and spectroscopic methods (UV, MS, PMR). An enzyme activity converting trans-2-chloro-4-carboxymethylenebut-2-en-4-olide into the cis-isomer was observed.Abbreviations 3CB 3-chlorobenzoate - 4CB 4-chlorobenzoate - 3,5DCB 3,5-dichlorobenzoate - 2,4D 2,4-dichlorophenoxyacetate - NOE Nuclear-Overhauser-Effect     

Dienelactone hydrolase from Pseudomonas sp. strain B13.

Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13.     

Degradation of bromacil by a Pseudomonas sp.

A gram-negative rod, identified as a Pseudomonas sp., was isolated from soil by using bromacil as the sole source of carbon and energy. During growth on bromacil or 5-bromouracil, almost stoichiometric amounts of bromide were released. The bacterium was shown to harbor two plasmids approximately 60 and 100 kilobases in size. They appeared to be associated with the ability to utilize bromacil as a sole source of carbon and also with resistance to ampicillin. This microorganism also showed the potential to decontaminate soil samples fortified with bromacil under laboratory conditions.     

Degradation of chloroaromatics: purification and characterization of maleylacetate reductase from Pseudomonas sp. strain B13.

Maleylacetate reductase of Pseudomonas sp. strain B13 was purified to homogeneity by chromatography on DEAE-cellulose, Butyl-Sepharose, Blue-Sepharose, and Sephacryl S100. The final preparation gave a single band by polyacrylamide gel electrophoresis under denaturing conditions and a single symmetrical peak by gel filtration under nondenaturing conditions. The subunit M(r) value was 37,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Estimation of the native M(r) value by gel filtration gave a value of 74,000 with a Superose 6 column, indicating that the enzyme is dimeric. The pH and temperature optima were 5.4 and 50 degrees C, respectively. The pI of the enzyme was estimated to be 7.0. The apparent Km values for maleylacetate and NADH were 58 and 30 microM, respectively, and the maximum velocity was 832 U/mg of protein for maleylacetate. Maleylacetate and various substituted maleylacetates, such as 2-chloro- and 2-methyl-maleylacetate, were reduced at significant rates. NADPH was also used as a cofactor instead of NADH with nearly the same Vmax value, but its Km value was estimated to be 77 microM. Reductase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group in the enzyme.     

Degradation of 1,2-dichlorobenzene by a Pseudomonas sp.

A Pseudomonas sp. that was capable of growth on 1,2-dichlorobenzene (o-DCB) or chlorobenzene as a sole source of carbon and energy was isolated by selective enrichment from activated sludge. The initial steps involved in the degradation of o-DCB were investigated by isolation of metabolites, respirometry, and assay of enzymes in cell extracts. Extracts of o-DCB-grown cells converted radiolabeled o-DCB to 3,4-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene (o-DCB dihydrodiol). 3,4-Dichlorocatechol and o-DCB dihydrodiol accumulated in culture fluids of cells exposed to o-DCB. The results suggest that o-DCB is initially converted by a dioxygenase to a dihydrodiol, which is converted to 3,4-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,4-dichlorocatechol is by a catechol 1,2-oxygenase to form 2,3-dichloro-cis,cis-muconate. Preliminary results indicate that chloride is eliminated during subsequent lactonization of the 2,3-dichloro-cis,cis-muconate, followed by hydrolysis to form 5-chloromaleylacetic acid.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Degradation of 1,4-dichlorobenzene by a Pseudomonas sp.

A Pseudomonas species able to degrade p-dichlorobenzene as the sole source of carbon and energy was isolated by selective enrichment from activated sludge. The organism also grew well on chlorobenzene and benzene. Washed cells released chloride in stoichiometric amounts from o-, m-, and p-dichlorobenzene, 2,5-dichlorophenol, 4-chlorophenol, 3-chlorocatechol, 4-chlorocatechol, and 3,6-dichlorocatechol. Initial steps in the pathway for p-dichlorobenzene degradation were determined by isolation of metabolites, simultaneous adaptation studies, and assay of enzymes in cell extracts. Results indicate that p-dichlorobenzene was initially converted by a dioxygenase to 3,6-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene, which was converted to 3,6-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,6-dichlorocatechol was by a 1,2-oxygenase to form 2,5-dichloro-cis, cis-muconate. Enzymes for degradation of haloaromatic compounds were induced in cells grown on chlorobenzene or p-dichlorobenzene, but not in cells grown on benzene, succinate, or yeast extract. Enzymes of the ortho pathway induced in cells grown on benzene did not attack chlorobenzenes or chlorocatechols.     

Degradation of 2-chloroallylalcohol by a Pseudomonas sp.

Three Pseudomonas strains capable of utilizing 2-chloroallylalcohol (2-chloropropenol) as the sole carbon source for growth were isolated from soil. The fastest growth was observed with strain JD2, with a generation time of 3.6 h. Degradation of 2-chloroallylalcohol was accompanied by complete dehalogenation. Chloroallylalcohols that did not support growth were dechlorinated by resting cells; the dechlorination level was highest if an alpha-chlorine substituent was present. Crude extracts of strain JD2 contained inducible alcohol dehydrogenase activity that oxidized mono- and dichloroallylalcohols but not trichloroallylalcohol. The enzyme used phenazine methosulfate as an artificial electron acceptor. Further oxidation yielded 2-chloroacrylic acid. The organism also produced hydrolytic dehalogenases converting 2-chloroacetic acid and 2-chloropropionic acid.     

Hybrid pathway for chlorobenzoate metabolism in Pseudomonas sp. B13 derivatives.

Derivatives of Pseudomonas sp. B13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the TOL plasmid of Pseudomonas putida mt-2 were studied. The utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. During growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deoxygenase and both dihydrodihydroxybenzoate dehydrogenases of the parent strains were induced. On the other hand, no catechol 2,3-dioxygenase from P. putida mt-2 was detectable, so that degradation of chlorocatechols by the nonproductive meta-cleavage pathway was avoided. Instead of that, chlorocatechols were subject to ortho cleavage and totally degraded by the preexisting enzymes of Pseudomonas sp. B13.     

Degradation of bromacil by a Pseudomonas sp

A gram-negative rod, identified as a Pseudomonas sp., was isolated from soil by using bromacil as the sole source of carbon and energy. During growth on bromacil or 5-bromouracil, almost stoichiometric amounts of bromide were released. The bacterium was shown to harbor two plasmids approximately 60 and 100 kilobases in size. They appeared to be associated with the ability to utilize bromacil as a sole source of carbon and also with resistance to ampicillin. This microorganism also showed the potential to decontaminate soil samples fortified with bromacil under laboratory conditions.     

The Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas sp.

To clarify biodegradation pathways of isoalkyl substituted aromatic hydrocarbons, oxidation products of isopropylbenzene and isobutylbenzene by Ps. desmolytica S449B1 and Ps. convexa S107B1 were examined.

Oxidation products from isopropylbenzene were determined to be 3-isopropylcatechol and (+)-2-hydroxy-7-methyI-6-oxooctanoic acid. Isobutylbenzene was also oxidized to 3-isobutylcatechol and (+)-2-hydroxy-8-methyl-6-oxononanoic acid by the same strains.

From these results, the existence of an unknown reductive step in the degradation of these isoalkyl substituted aromatic hydrocarbons and the initial oxidation of these aromatic hydrocarbons by the strains were made clear. The degradation pathways of isopropylbenzene and isobutylbenzene by these strains were discussed.     

Degradation of o-toluate by Pseudomonas sp. strain WR401

Abstract A Pseudomonas sp. strain WR401 was isolated for growth on 3-, 4-, and 5-methylsalicylate. The organism was capable of growth on o -toluate. The data on enzyme activities in cell-free extracts, DHB dehydrogenase and catechol 2,3-dioxygenase, as well as the cooxidation of the substrate analog 2-chlorobenzoate yielding 3-chlorocatechol indicated a pathway for o -toluate degradation through 6-methyldihydrodihydroxybenzoate, 3-methylcatechol and further through the meta -pathway. In contrast to other toluate dioxygenating enzymes found in m - and p -toluate degrading organisms, strain WR401 was able to dioxygenate a wider range of chlorobenzoates including 2-chlorobenzoate.     

Degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9

Abstract The aerobic degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9 was studied. Intact cells of Pseudomonas sp. BN9 grown with 2,4-dihydroxybenzoate oxidized 2,4-dihydroxybenzoate but not salicylate. Cell-free extracts of Pseudomonas sp. BN9 converted 2,4-dihydroxybenzoate after the addition of NAD(P)H. A partially purified protein fraction converted 2,4-dihydroxybenzoate with NADH to 1,2,4-trihydroxybenzene. 1,2,4-Trihydroxybenzene was converted by a 1,2-dioxygenase to maleylpyruvate, which was reduced by a NADH-dependent enzyme to 3-oxoadipate. 2,4-Dihydroxybenzoate 1-monooxygenase, 1,2,4-trihydroxybenzene 1,2-dioxygenase and maleylpyruvate reductase were induced in Pseudomonas sp. BN9 after growth with 2,4-dihydroxybenzoate.     

Biotin-vitamer Formation from Salicylic Acid by Pseudomonas sp.

Biotin-vitamer formation from salicylic acid was investigated. Strains of Pseudomonas sp., No. 102 and No. 362, isolated from soil samples utilized well salicylic acid as a sole source of carbon, and formed biotin-vitamers in culture broth. The metabolites were partially purified by the methods of active carbon adsorption and anion-exchange column chromatography, and clarified as desthiobiotin, bisnordesthiobiotin and 7-keto-8-aminopelargonic acid.     

Degradation of alkylphenol ethoxylates by Pseudomonas sp. strain TR01.

An alkylphenol ethoxylate-degrading bacterium was isolated from activated sludge of a municipal sewage treatment plant by enrichment culture. This organism was found to belong to the genus Pseudomonas; since no corresponding species was identified, we designated it as Pseudomonas sp. strain TR01. This strain had an optimal temperature and pH of 30 degrees C and 7, respectively, for both growth and the degradation of Triton N-101 (a nonylphenol ethoxylate in which the average number of ethylene oxide [EO] units is 9.5). The strain was unable to mineralize Triton N-101 but was able to degrade its EO chain exclusively. The resulting dominant intermediate was identified by normal-phase high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry as a nonylphenol ethoxylate with 2 mol of EO units. A carboxylated metabolite, [(nonylphenoxy)ethoxy]acetic acid, was detected by gas chromatography-mass spectrometry. This bacterium also metabolized alcohol ethoxylates with various numbers of EO units but not polyethylene glycols whatever their degree of polymerization. By oxygen consumption assay, the alkyl group or arene corresponding to the hydrophobic part of alcohol ethoxylates or alkylphenol ethoxylates was shown to contribute to the induction of the metabolic system of the EO chain of Triton N-101, instead of the EO chain itself, which corresponds to its hydrophilic part. Thus, the isolated pseudomonad bacterium has unique substrate assimilability: it metabolizes the EO chain only when the chain linked to bulky hydrophobic groups.     

Degradation of 1,4-dichlorobenzene by a Pseudomonas sp

A Pseudomonas species able to degrade p-dichlorobenzene as the sole source of carbon and energy was isolated by selective enrichment from activated sludge. The organism also grew well on chlorobenzene and benzene. Washed cells released chloride in stoichiometric amounts from o-, m-, and p-dichlorobenzene, 2,5-dichlorophenol, 4-chlorophenol, 3-chlorocatechol, 4-chlorocatechol, and 3,6-dichlorocatechol. Initial steps in the pathway for p-dichlorobenzene degradation were determined by isolation of metabolites, simultaneous adaptation studies, and assay of enzymes in cell extracts. Results indicate that p-dichlorobenzene was initially converted by a dioxygenase to 3,6-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene, which was converted to 3,6-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,6-dichlorocatechol was by a 1,2-oxygenase to form 2,5-dichloro-cis, cis-muconate. Enzymes for degradation of haloaromatic compounds were induced in cells grown on chlorobenzene or p-dichlorobenzene, but not in cells grown on benzene, succinate, or yeast extract. Enzymes of the ortho pathway induced in cells grown on benzene did not attack chlorobenzenes or chlorocatechols.     

Degradation of 2-Methylnaphthalene by Pseudomonas sp. Strain NGK1

Pseudomonas sp. strain NGK1, a soil bacterium isolated by naphthalene enrichment from biological waste effluent treatment, capable of utilizing 2-methylnaphthalene as sole source of carbon and energy. To deduce the pathway for biodegradation of 2-methylnaphthalene, metabolites were isolated from the spent medium and identified by thin-layer chromatography and high-performance liquid chromatography. The characterization of purified metabolites, oxygen uptake studies, and enzyme activities revealed that the strain degrades 2-methylnaphthalene through more than one pathway. The growth of the bacterium, utilization of 2-methylnaphthalene, and 4-methylsalicylate accumulation by Pseudomonas sp. strain NGK1 were studied at various incubation periods. Received: 20 March 2001 / Accepted: 25 April 2001     

Degradation of 1,2-dichlorobenzene by a Pseudomonas sp

A Pseudomonas sp. that was capable of growth on 1,2-dichlorobenzene (o-DCB) or chlorobenzene as a sole source of carbon and energy was isolated by selective enrichment from activated sludge. The initial steps involved in the degradation of o-DCB were investigated by isolation of metabolites, respirometry, and assay of enzymes in cell extracts. Extracts of o-DCB-grown cells converted radiolabeled o-DCB to 3,4-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene (o-DCB dihydrodiol). 3,4-Dichlorocatechol and o-DCB dihydrodiol accumulated in culture fluids of cells exposed to o-DCB. The results suggest that o-DCB is initially converted by a dioxygenase to a dihydrodiol, which is converted to 3,4-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,4-dichlorocatechol is by a catechol 1,2-oxygenase to form 2,3-dichloro-cis,cis-muconate. Preliminary results indicate that chloride is eliminated during subsequent lactonization of the 2,3-dichloro-cis,cis-muconate, followed by hydrolysis to form 5-chloromaleylacetic acid.     

假单胞菌S-2降解甲胺磷性能的研究

从甲胺磷生产车间分离到一株假单胞菌编号为S-2。S-2可利用甲胺磷为唯一氮源,但不能利用甲胺磷为唯一磷源。该文对S-2体内具有的降解甲胺磷的酶类进行了研究,初步断定：S-2可代谢产生酸性磷酸酶,主要在胞外降解甲胺磷。S-2在甲胺磷诱导的情况下,这些降解酶类可大量聚积。用诱导过的菌液降解甲胺磷比未经诱导的快了2d左右。