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W Reineke

Publications and source records attributed to W Reineke.

At least 37 records · Page 2Linked to original sources
PubMed1993

Microbial degradation of chlorinated acetophenones.

A defined mixed culture, consisting of an Arthrobacter sp. and a Micrococcus sp. and able to grow with 4-chloroacetophenone as a sole source of carbon and energy, was isolated. 4-Chlorophenyl acetate, 4-chlorophenol, and 4-chlorocatechol were identified as metabolites through comparison of retention times and UV spectra with those of standard substances. The proposed pathway was further confirmed by investigation of enzymes. The roles of the two collaborating strains were studied by growth experiments and on the level of enzymes. If transient accumulation of 4-chlorophenol was avoided either by the use of phenol-absorbing substances or by careful supplement of 4-chloroacetophenone, the Arthrobacter sp. was able to grow as a pure culture with 4-chloroacetophenone as a sole source of carbon and energy. Several mono-, di-, and trichlorinated acetophenones were mineralized by the Arthrobacter sp.

Acetophenones↗
PubMed1993

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.

Chromatography, DEAE-Cellulose↗
PubMed1992

Degradation of chlorotoluenes by in vivo constructed hybrid strains: problems of enzyme specificity, induction and prevention of meta-pathway.

The activities of the TOL plasmid-coded xylene oxygenase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase of Pseudomonas putida strain PaW1 were tested with substituted toluenes, benzylalcohols and benzaldehydes, respectively, as substrates. Several chlorinated toluenes were shown to induce enzymes of the xylene degradation sequence. Conjugative transfer of the TOL plasmid from Pseudomonas putida strain PaW1 to Pseudomonas sp. strain B13 and Pseudomonas cepacia strain JH230 allowed the isolation of hybrid strains capable of growing in the presence of 3-chloro-, 4-chloro- and 3,5-dichlorotoluene. Hybrid strains revealed new ways to prevent the dead-end meta-pathway for cholorocatechols.

Alcohol Oxidoreductases↗
PubMed1992

Maleylacetate reductase of Pseudomonas sp. strain B13: dechlorination of chloromaleylacetates, metabolites in the degradation of chloroaromatic compounds.

The maleylacetate reductase of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 has been purified 50-fold. The enzyme converted 2-chloromaleylacetate to 3-oxoadipate with temporary occurrence of maleylacetate; 1 mol of chloride was eliminated during the conversion of 1 mol of 2-chloro- and 2,3-dichloromaleylacetate; 2 mol of NADH were consumed per mol of 2-chloro- and 2,3-dichloromaleylacetate while only 1 mol was necessary to catalyze the conversion of maleylacetate or 2-methylmaleylacetate. The maleylacetate reductase failed to use fumarylacetate as a substrate. The role of the enzyme in the chloroaromatics degradation is discussed.

Adipates↗
PubMed1991

Total degradation of various chlorobiphenyls by cocultures and in vivo constructed hybrid pseudomonads.

Cocultures consisting of strains converting chlorobiphenyls to the respective benzoates or catechols and of chlorobenzoate degraders were investigated for the mineralization of chlorobiphenyls. Stable mixed cultures were obtained with 4-chlorobiphenyl, while those with 2-chloro- or 3-chlorobiphenyl were found to be unstable and released only low yields of chloride. When both sets of enzyme sequences were combined in one organism, Pseudomonas cepacia strain JH230, by conjugative transfer of genes of the biphenyl degradation sequence, the total degradation of 2-chloro-, 3-chloro-, 4-chloro-, 2,4-dichloro-, and 3,5-dichlorobiphenyl was achieved.

Aroclors↗
PubMed1990

Degradation of 3-chlorobiphenyl by in vivo constructed hybrid pseudomonads.

3-Chlorobiphenyl-degrading bacteria were obtained from the mating between Pseudomonas putida strain BN10 and Pseudomonas sp. strain B13. Strains such as BN210 resulted from the transfer of the genes coding the enzyme sequence for the degradation of chlorocatechols from B13 into BN10, whereas B13 derivatives such as B131 have acquired the biphenyl degradation sequence from BN10. During growth of the hybrid strains on 3-chlorobiphenyl 90% chloride was released. Activities of phenylcatechol 2,3-dioxygenase, benzoate dioxygenase, catechol 1,2-dioxygenase, chloromuconate cyloisomerase and 4-carboxymethylenebut-2-en-4-olide hydrolase were found in 3-chlorobiphenyl-grown cells. The hybrid strains were found to convert some congeners of the Aroclor 1221 mixture such as mono- and dichloro-substituted biphenyls.

Aroclors↗
PubMed1987

Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.

Alcaligenes↗
PubMed1986

Construction of bacterial strains with novel degradative capabilities for chloroaromatics.

Biodegradation of most naturally occurring compounds is relatively fast, since microorganisms have evolved appropriate enzyme systems. However, biodegradation is less likely in the case of man-made compounds like haloaromatics, which have structural features rarely or never encountered in natural products. One strategy to develop new metabolic activities for novel compounds by strains of microorganisms is by the alteration of their existing genetic information. The present paper summarizes results of studies where the degradation capabilities of bacteria were expanded by genetic material from external sources either by natural exchange or by cloning procedures.

Bacteria↗
PubMed1986

Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria.

Stimulation of transcription from positively regulated promoters involves regulatory proteins that have been activated, generally as a consequence of binding low molecular weight effector molecules. To define essential structural features of effectors for one positively acting gene regulator, the xylS-encoded protein, which activates the TOL plasmid meta-cleavage pathway operon promoters, effector activities of a wide range of benzoate derivatives have been systematically analyzed, and mutant xylS-encoded proteins exhibiting altered effector specificities have been generated and characterized. Cloned mutant xylS genes were trans dominant in partial diploids containing the wild-type xylS allele and could therefore be used to effect expansion of the range of aromatic compounds completely or partially degraded by Pseudomonas bacteria. The method developed to isolate mutant xylS-encoded proteins has general applicability and could in principle be used to isolate gene regulator specificity mutants of any inducible regulatory system.

Alleles↗
PubMed1986

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.

Bacterial Proteins↗
PubMed1984

Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters.

Expression of the meta-cleavage pathway operon of TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the xylS gene product. We have sequenced the promoter region of this operon and localized the transcription initiation sites. Two overlapping promoters, designated Pm1 and Pm2, are responsible for the positively regulated expression of the meta-pathway operon. Mutants of P. putida were isolated that expressed the meta-cleavage pathway operon constitutively. Several plasmid-located mutations that led to constitutivity were characterized by sequencing and the transcription initiation sites on mutant plasmids localized. This resulted in the identification of newly created promoters whose functioning did not require the xylS product. Comparison of the promoter sequences obtained suggests a tentative consensus sequence for promoters of P. putida which is significantly different from that of E. coli.

Base Sequence↗
PubMed1984

Microbial metabolism of haloaromatics: isolation and properties of a chlorobenzene-degrading bacterium.

A chlorobenzene-degrading bacterium was isolated by continuous enrichment from a mixture of soil and sewage samples. This organism, strain WR1306, was grown in a chemostat on a mineral medium with chlorobenzene being supplied through the vapor phase with a critical Dc value at a dilution rate of 0.55 h-1. Maximum growth rates in batch culture were accomplished at substrate concentrations of less than or equal to 0.5 mM in the culture medium. During growth on chlorobenzene, stoichiometric amounts of chloride were released. Respiration data and enzyme activities in cell extracts as well as the isolation of 3-chlorocatechol from the culture fluid are consistent with the degradation of chlorobenzene via 3-chloro-cis-1,2-dihydroxycyclohexa-3,5-diene, 3-chlorocatechol, 2-chloro-cis,cis-muconate, trans-4-carboxymethylenebut-2-en-4-olide, maleylacetate, and 3-oxoadipate.

Bacteria↗
PubMed1984

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols.

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K(2)) were 1.62 x 10 sec for 3-chlorocatechol and 2.38 x 10 sec for 3-fluorocatechol. The inhibitor constants (K(i)) were 23 muM for 3-chlorocatechol and 17 muM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.

Journal Article↗
PubMed1984

Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13.

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .

Base Sequence↗
PubMed1982

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: enzyme regulation and DNA structure.

WR211 and WR216 are derivatives of halobenzoate-degrading Pseudomonas sp. strain B13 into which the 117-kilobase TOL degradative plasmid pWW0 has been transferred from Pseudomonas putida mt-2. WR211 has lost the ability to grow on the TOL-specific substrate m-xylene but retains the ability to grow on its metabolite, m-toluate. An analysis of the induction of enzymes was consistent with WR211 carrying a nonfunctional regulatory gene, xy1R, WR216 is a spontaneous derivative of WR211 which grows on one of the TOL substrates and yet expresses the nonspecific toluate oxidase, which enables it to grow on the novel substrate 4-chlorobenzoate. In addition to the xy1R lesion inherited from WR211, WR216 appears to carry a mutation in the structural gene for catechol 2,3-oxygenase, xy1E. The plasmids in both strains were analyzed by restriction endonuclease digestion. pWW0-1211 in WR211 has a large deletion (39 kilobases) compared with pWW0 and appears to be identical to a previously described plasmid (pWW0-8) which encodes none of the TOL degradative functions. pWW0-1216 in WR216 has undergone a major structural reorganization relative to its parent, pWW0-1211. This plasmid has a smaller deletion (19 kilobases), which is staggered relative to the deletion in pWW0-1211, and in addition it has two 3-kilobase insertions of unknown origin, one of which appears to cause the xylE mutation.

Base Sequence↗
PubMed1982

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: prevention of meta pathway.

The hybrid pathway for chlorobenzoate metabolism was studied in WR211 and WR216, which were derived from Pseudomonas sp. B13 by acquisition of TOL plasmid pWW0 from Pseudomonas putida mt-2. Chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. When WR211 utilizes 3-chlorobenzoate (3CB), the expression of catechol 2,3-dioxygenase (C23O) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivation of the meta cleavage activity by 3-chlorocatechol. Utilization of 4-chlorobenzoate (4CB) by WR216 presupposes the suppression of C23O by a spontaneous mutation in the structural gene, so that 4-chlorocatechol is not misrouted into the meta pathway. Such C23O- mutants were also selected when WR211 was grown continuously on 3CB. Our data explain why the phenotypic characters 3CB+ and Mtol+ (m-toluate) are compatible, whereas 4CB+ and Mtol+ are incompatible.

Benzoates↗