The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol.

Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde.     

P-cresol and 3,5-xylenol methylhydroxylases in Pseudomonas putida N.C.I.B. 9896.

Pseudomonas putida N.C.I.B. 9869, when grown on 3,5-xylenol, hydroxylates the methyl groups on 3,5-xylenol and on p-cresol by two different enzymes. 3,5-Xylenol methylhydroxylase, studied only in relatively crude extracts, requires NADH, is not active with p-cresol and is inhibited by cyanide, but not by CO. The p-cresol methylhydroxylase requires an electron acceptor and will act under anaerobic conditions. It was purified and is a flavocytochrome c of mol.wt. approx. 114,000 consisting of two subunits of equal size. The enzyme catalyses the hydroxylation of p-cresol (Km 16 micron) and the further oxidation of product, p-hydroxybenzyl alcohol (Km 27 micron) to p-hydroxybenzaldehyde. A different p-cresol methylhydroxylase of the flavocytochrome c type is induced by growth on p-cresol. It too was purified and has mol.wt. approx. 100,000, and again consisted of two equal-size subunits. The Km for p=cresol 3.6 micron and for p=hydroxybenzyl alcohol, 15 micron.     

Regulation of isofunctional enzymes in Pseudomonas alcaligenes mutants defective in the gentisate pathway

The regulation of the inducible set of gentisate pathway enzymes used by Pseudomonas alcaligenes (P25X1) has been studied in strains derived from mutant strains of P25X1 that had lost the constitutive enzymes that degrade m –cresol, 2,5–xylenol and 3,5–xylenol. The enzyme, 3-hydroxybenzoate 6-hydroxylase II, that catalyzes the oxidation of 3-hydroxybenzoate to gentisate is substrate- and product-induced while gentisate dioxygenase II is substrate induced. Neither 3-hydroxybenzoate nor gentisate could induce the synthesis of maleylpyruvate hydrolase II and fumarylpyruvate hydrolase II. The results suggest that the structural genes encoding these four inducible enzymes and maleylpyruvate hydrolase I (a constitutive enzyme) exist in at least four operons. There is strict induction specificity of expression of this inducible set of gentisate pathway enzymes. 3-Hydroxy-4-methyl-benzoate failed to induce whilst 3-hydroxybenzoate and 3-hydroxy-5-methylbenzoate served as inducers of 6-hydroxylase II. Degradation of 2,5-xylenol is mediated by constitutive enzymes whereas the inducible set of enzymes are responsible for the metabolism of m -cresol and 3,5-xylenol.     

Specific deletion of a large segment of pRA500: a 3,5-xylenol degradative plasmid

3,5-Xylenol degradative plasmid, pRA500 (approximately 500 kilobase pairs, kb) carried by Pseudomonas putida NCIB 9869, also encodes resistance to inorganic mercuric ions (Hg^r). Following growth of Ps. putida on benzoate, p -cresol, p -hydroxybenzoate or protocatechuate, the frequency of loss of the 3,5-xylenol phenotype was 75–90%. The deletion of a large segment of approximately 350 kb from pRA500, to give rise to archetypal plasmid designated pRA502, occurred in such 3,5-xylenol-negative derivatives. During the conjugational transfer of pRA500 following selection of transconjugants for Hg^ronly, frequency of loss of 3,5-xylenol phenotype was approximately 60%. A high number of these 3,5-xylenol-negative transconjugants carried pRA502. A site-specific deletion is suggested in the formation of archetypal plasmid pRA502 by growth of Ps. putida on the above compounds and during transfer of pRA500.     

Evidence for isofunctional enzymes used in m-cresol and 2,5-xylenol degradation via the gentisate pathway in Pseudomonas alcaligenes.

Study of the reaction sequence by which Pseudomonas alcaligenes (P25X1) and derived mutants degrade m-cresol, 2,5-xylenol, and their catabolites has provided indirect evidence for the existence of two or more isofunctional enzymes at three different steps. Maleylpyruvate hydrolase activity appears to reside in two different proteins with different specificity ranges, one of which (MPH1) is expressed constitutively; the other (MPH11) is strictly inducible. Two gentisate 1,2-dioxygenase activities were found, one of which is constitutively expressed and possesses a broader specificity range than the other, which is inducible. From oxidation studies with intact cells, there appear to be two activities responsible for the 6-hydroxylation of 3-hydroxybenzoate, and again a broadly specific activity is present regardless of growth conditions; the other is inducible by 3-hydroxybenzoate. Three other enzyme activities are also detected in uninduced cells, viz., xylenol methylhydroxylase, benzylalcohol dehydrogenase, and benzaldehyde dehydrogenase. All apparently possess broad specificity. Fumarylpyruvate hydrolase was also detected but only in cells grown with m-cresol, 3-hydroxybenzoate, or gentisate. Mutants, derived either spontaneously or after treatment with mitomycin C, are described, certain of which have lost the ability to grow with m-cresol and 2,5-xylenol and some of which have also lost the ability to form the constitutive xylenol methylhydroxylase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase, 3-hydroxybenzoate 6-hydroxylase, and gentisate 1,2-dioxygenase. Such mutants, however, retain ability to synthesize inducibly a second 3-hydroxybenzoate 6-hydroxylase and gentisate 1,2-dioxygenase, as well as maleylpyruvate hydrolase (MPH11) and fumarylpyruvate hydrolase; MPH1 was still synthesized. These findings suggest the presence of a plasmid for 2,5-xylenol degradation which codes for synthesis of early degradative enzymes. Other enzymes, such as the second 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, maleylpyruvate hydrolase (MPH1 and MPH11), and fumarylpyruvate hydrolase, appear to be chromosomally encoded and, with the exception of MPH1, strictly inducible.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. II. Isolation and properties of blocked mutants

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. II. Isolation and properties of blocked mutants. J. Bacteriol. 91:1155-1160. 1966.-Mutants of Pseudomonas putida blocked in early reactions of the pathway for oxidation of d-mandelate were isolated and partially characterized. The specific genetic lesions in these mutants made normal inducer-metabolites of the pathway nonmetabolizable. Under the conditions of gratuitous enzyme synthesis so obtained, it could be shown that the d and l isomers of mandelate are equipotent inducers, and that the synthesis of the first five enzymes of the mandelate pathway is coordinate. Further experiments with the blocked mutants showed that benzoylformate, the third intermediate of the pathway, acts as an inducer without prior conversion to mandelate, and that there is no inducible, concentrating permease for mandelate.     

Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.     

Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867

The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols.

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID50's for prostaglandin (PG) E2 synthesis in these cells were 0.1 muM for 2,6-xylenol, 5 muM for tricresol, 6 muM for p-cresol, 7 muM for o-cresol, 15 muM for 3,5-xylenol, 30 muM for m-cresol and 100 muM for phenol. The corresponding values for aspirin and indomethacin were 4 muM and 0.02 muM, respectively. The substituted phenols also inhibited serotinin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG2.     

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.     

The molecular cloning of genes specifying some enzymes of the 3,5-xylenol degradative pathway

Enzymes for the degradation of 3,5-xylenol ofPseudomonas putida NCIB9869 are encoded on a transmissible plasmid, pRA500. Genes specifying the inducible synthesis of some enzymes encoded on pRA500, namely, citraconase (G), citramalate coenzyme-A-transferase (H), citramalyl-coenzyme-A lyase (I), maleyl-pyruvate isomerase (J) and fumarylpyruvate hydrolase (K) have been cloned on a 7.9-kbHindIII fragment into the vector pKT231 to give pRA507. Biochemical and restriction analysis of pRA507 and some of its deleted derivatives has enabled preliminary locations to be assigned to the genes encoding these enzymes. The structural genes for enzymes H and I, together with a regulatory gene controlling their expression, are located on a 1.6-kbHindIII/XhoI fragment of pRA507 and the genes for enzymes G, J and K are located within a 4.5-kbXhoI/ClaI fragment of pRA507, which lies immediately adjacent to the former fragment. Biochemical analysis of the strain carrying pRA507, of its plasmid-free derivative and of other plasmid-free derivatives of the wild-typeP. putida has provided indirect evidence for the presence of two isofunctional enzymes at three different steps of the degradative pathway. One set of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase and maleylpyruvate hydrolase is encoded on pRA500 and these enzymes are inducible only. A second set is encoded on the chromosome and these enzymes are constitutive and not further inducible. The chromosomally located genes appeared to be silent until 3,5-xylenol-negative strains were incubated for prolonged periods with compounds such asm-hydroxybenzoate orp-cresol.     

Biodegradation of nitrobenzene through a hybrid pathway in Pseudomonas putida

The biodegradation of nitrobenzene was attempted by using Pseudomonas putida TB 103 which possesses the hybrid pathway combining the tod and the tol pathways. Analysis of the metabolic flux of nitrobenzene through the hybrid pathway indicated that nitrobenzene was initially oxidized to cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene by toluene dioxygenase in the tod pathway and then channeled into the tol pathway, leading to the complete biodegradation of nitrobenzene. A crucial metabolic step redirecting the metabolic flux of nitrobenzene from the tod to the tol pathway was determined from the genetic and biochemical studies on the enzymes involved in the tol pathway. From these results, it was found that toluate-cis-glycol dehydrogenase could convert cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene to catechol in the presence of NAD(+) with liberation of nitrite and the reduced form of NAD(+) (NADH) into the medium. (c) 1995 John Wiley & Sons, Inc.     

XYL, a nonconjugative xylene-degradative plasmid in Pseudomonas Pxy.

Pseudomanas Pxy metabolizes p- or m-xylene through intermediate formation of the corresponding methylbenzyl alcohol and toluic acid via the meta pathway. The strain Pseudomonas Pxy spontaneously loses its ability to grow with xylene or toluate, and the rate of loss of this ability is greatly enhanced by treatment of the cells with mitomycin C. The assay of enzymes involved in xylene degradation in xylene-negative Pxy cells indicates the loss of the entire enzyme complement of the pathway. The genes specifying all the xylene-degradative enzymes, including those of the meta pathway, appear to be borne on a nonconjugative plasmid and can be transferred to xylene-negative Pxy or P. putida strain PpG1 cells only in the presence of a transfer plasmid termed factor K. When transferred to strain PpG1, the xylene-degradative plasmid, termed XYL, coexists stably with factor K, but transduction of XYL is not accompanied by a cotransfer of factor K. XYL appears to be compatible wit- all the other known degradative plasmids in P. putida. The xylene pathway is inducible in wild-type Pxy as well as in Pxy and PpG1 exconjugants, suggesting the cotransfer of regulatory genes along with the plasmid. The enzymes converting xylene to toluate are induced by xylene, methylbenzyl alcohol, or the aldehyde derivatives but not significantly by toluate, whereas catechol dioxygenase and other enzymes are induced by toluates and presumable by xylene as well.     

Intradiol pathway of para-cresol conversion by Rhodococcus opacus 1CP

The growth of Rhodococcus opacus 1CP in medium with different concentrations of p-cresol as the sole source of carbon and energy was studied. It was shown that the optimal concentration of p-cresol was 600 mg/L. The ability of this strain to transform practically all amounts of p-cresol to 4-methylcatechol followed by its utilization through ortho-pathway was shown. New enzymes (4-methylcatechol 1,2-dioxygenase, catechol 1,2-dioxygenase, and methylmuconate cycloisomerase) were purified to homogeneity and characterized. Based on the data obtained on p-cresol degradation, formation of intermediates, and the enzymes participating in this pathway, we suggest an ortho-pathway of p-cresol degradation by R. opacus 1CP through 4-methylcatechol and 3-methyl-cis, cis-muconate.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. 3. Isolation and properties of constitutive mutants

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. III. Isolation and properties of constitutive mutants. J. Bacteriol. 91:1161-1167. 1966.-Mutants of Pseudomonas putida constitutive for the synthesis of l(+)-mandelate dehydrogenase were obtained after mandelate- or benzoylformate-limited growth in a chemostat. When grown in media noninducing for the wild type, the mutants are capable of coordinate, constitutive synthesis of the first five enzymes of the mandelate pathway. Later enzymes of the pathway that were examined are normally repressed. The constitutive mutants have two other noteworthy properties: they are superinducible by some compounds which induce the mandelate group enzymes in the wild type, or as a result of exhaustion of the carbon and energy source of the medium in which they are grown; and they exhibit a decreased specificity of induction, being inducible by a wide range of compounds devoid of inductive function for the wild type. These results, together with other evidence indicating that the five mandelate group enzymes comprise a regulatory unit, are discussed and evaluated in the context of the general problem of the regulation of complex dissimilatory pathways.     

Identification of cis-diols as intermediates in the oxidation of aromatic acids by a strain of Pseudomonas putida that contains a TOL plasmid.

Pseudomonas putida BG1 was isolated from soil by enrichment with p-toluate and selection for growth with p-xylene. Other hydrocarbons that served as growth substrates were toluene, m-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene. The enzymes responsible for growth on these substrates are encoded by a large plasmid with properties similar to those of TOL plasmids isolated from other strains of Pseudomonas. Treatment of P. putida BG1 with nitrosoguanidine led to the isolation of a mutant strain which, when grown with fructose, oxidized both p-xylene and p-toluate to (-)-cis-1,2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylic acid (cis-p-toluate diol). The structure of the diol was determined by conventional chemical techniques including identification of the products formed by acid-catalyzed dehydration and characterization of a methyl ester derivative. The cis-relative stereochemistry of the hydroxyl groups was determined by the isolation and characterization of an isopropylidene derivative. p-Xylene-grown cells contained an inducible NAD+-dependent dehydrogenase which formed catechols from cis-p-toluate diol and the analogous acid diols formed from the other hydrocarbon substrates listed above. The catechols were converted to meta ring fission products by an inducible catechol-2,3-dioxygenase which was partially purified from p-xylene-grown cells of P. putida BG1.     

Genetic Basis of the Biodegradation of Salicylate in Pseudomonas

The genetic basis of the biodegradation of salicylate in Pseudomonas putida R1 has been studied. This strain utilizes the meta pathway for oxidizing salicylate through formation of catechol and 2-hydroxymuconic semialdehyde. The enzymes of the meta pathway are induced by salicylate but not by catechol, and the genes specifying these enzymes are clustered. The gene cluster can be eliminated from some salicylate-positive cells by treatment with mitomycin C and appears to exist inside the cell as an extrachromosomal element. This extrachromosomal gene cluster, termed the SAL plasmid, can be transferred by conjugation from P. putida R1 to a variety of other Pseudomonas species.     

Rapid characterization of microbial biodegradation pathways by FT-IR spectroscopy

Fourier transform-infrared (FT-IR) spectroscopy has become an important tool for rapid analysis of complex biological samples. The infrared absorbance spectrum could be regarded as a "fingerprint" which is characteristic of biochemical substances. In this study, Pseudomonas putida NCIMB 9869 was grown with either 3,5-xylenol or m-cresol as the sole carbon source, each inducing different metabolic pathways for m-cresol biotransformation. FT-IR spectroscopy was capable of differentiating both induced cultures of P. putida NCIMB 9869 as well as the resulting biotransformation product mixtures. FT-IR spectral analysis indicated that carboxylic acids were key chemicals responsible for distinguishing the products of the two catabolic pathways. Gas chromatography-mass spectrometry (GC-MS) was performed to validate the FT-IR analysis, indicating that two carboxylic acids, 3-hydroxybenzoic acid and 2,5-dihydroxybenzoic acid, were present as m-cresol biotransformation products from 3,5-xylenol-grown cells, but were absent in m-cresol-grown cells. The ability to use FT-IR to rapidly distinguish between biotransformation product mixtures as well as differentially induced bacterial strains suggests this approach might be a valuable tool for screening large biotransformation assays for novel products and metabolic mutants.     

Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene.

Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway.