Showing papers on "Pseudomonas putida published in 1975"

Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.

Abstract: Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene.

Substrate inhibition kinetics: Phenol degradation by Pseudomonas putida

Abstract: A pure culture of Pseudoinonas putida was grown in both a batch and continuous culture using phenol as the limiting substrate. Of two substrate inhibition models examined, the Haldane function was found to statistically best describe the kinetics. The applicable kinetic constants were either measured (μM, KI) or estimated (KS) from the experimental data. Particularly in the continuous culture, wall growth was found to exert significant effects on the broth biomass concentration and phenol conversion, both of which decreased with increasing amounts of wall growth. These effects are opposite to those predicted by wall growth models and to experimental results of others using mixed culture (activated sludge) systems.

Initial reactions in the oxidation of naphthalene by Pseudomonas putida

Abstract: A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil. A mutant strain of this organism, P. putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium. The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry. Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P. Fluorescens (formerly ATCC, 17483), P. putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P. putida described in this report. Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor. A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate. Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P. putida. The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase. This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium.

Regulation of alkane oxidation in Pseudomonas putida.

Abstract: We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P putida chromosome Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain Some inducers are neither growth nor respiration substrates Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities

Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids.

Abstract: Pseudomonas putida PpG6 is able to utilize purified n-alkanes of six to ten carbon atoms for growth. It can also grow on the primary terminal oxidation products of these alkanes and on 1-dodecanol but not on the corresponding 2-ketones or 1,6-hexanediol, adipic acid, or pimelic acid. Revertible point mutants can be isolated which have simultaneously lost the ability to grow on all five n-alkane growth substrates but which can still grow on octanol or nonanol. An acetate-negative mutant defective in isocitrate lysase activity is unable to grow on even-numbered alkanes and fatty acids. Analysis of double mutants defective in acetate and propionate or in acetate and glutarate metabolism shows that alkane carbon is assimilated only via acetyl-coenzyme A and propionyl-coenzyme A. These results support the following conclusions: (i) The n-alkane growth specificity of P. putida PpG6 is due to the substrate specificity of whole-cell alkane hydroxylation; (ii) there is a single alkane hydroxylase enzyme complex; (iii) the physiological role of this complex is to initiate the monoterminal oxidation of alkane chains; and (iv) straight-chain fatty acids from butyric through nonanoic are degraded exclusively by beta-oxidation from the carboxyl end of the molecule.

A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system.

Abstract: A strain of Pseudomonas putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the O-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components: an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase. The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently. The absolute spectrum of the oxidized displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores. The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 415 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the NADH-dependent reductase. Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i.e., iron-containing and labile-sulfur-containing monooxygenases. From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed.

Transformation of Pseudomonas putida and Escherichia coli with plasmid-linked drug-resistance factor DNA

Abstract: Conditions optimal for the transformation of Pseudomonas putida and E. coli with a drug-resistance factor (RP 1) DNA, which specifies resistance to carbenicillin, tetracycline, kanamycin, and neomycin, are described. The transformants retain all the fertility, incompatibility, and drug-resistance characteristics present in the parent. Covalently-closed circular molecules of almost identical contour lengths have been isolated from the parent and the transformants. The frequency of transformation is drastically reduced by treatment of RP 1 DNA with DNase and by denaturation or sonication. Shearing of RP 1 DNA in vitro and their subsequent introduction in P. putida cells, by transformation, produces transformants that exhibit a wide range of drug-resistant phenotypes, including those which are resistant to neomycin but sensitive to kanamycin. Isolation of such neomycin-resistant but kanamycin-sensitive transformants indicates that there might be two separate mechanisms specified by RP 1 for resistance to the two antibiotics.

Pathways for the degradation of m-cresol and p-cresol by Pseudomonas putida.

Abstract: A comparison of the oxidation rates of various compounds by whole cells of Pseudomonas putida 3, 5 indicated that m-cresol is metabolized by oxidation to 3-hydroxybenzoate followed by hydroxylation to gentisate, the ring-fission substrate, when grown with 3, 5-xylenol. However, when m-cresol was the growth substrate, similar experiments suggested a different pathway involving a methyl-substituted catechol, and ring-fission by meta cleavage. Assays of ring-fission enzymes in cell-free extracts confirmed that different pathways are induced by the two growth substrates. 3, 5-Xylenol-grown cells contained high levels of gentisate oxygenase and only very small amounts of catechol oxygenase, whereas gentisate ocygenase could not be detected in m-cresol-grown cells, but levels of catechol oxygenase were greatly increased. Extracts of m-cresol-grown cells also contained 2-hydroxymuconic semialdehyde dehydrogenase and hydrolase, whose specificities enable them to metabolize the ring-fission products from catechol, 3-methylcatechol, and 4-methylcatechol. This catechol pathway is also used by m-cresol-grown cells for p-cresol metabolism. In contrast, the results for cells grown with p-cresol point to an alternative pathway involving oxidation to 4-hydroxybenzoate and hydrosylation to protocatechuate as ring-fission substrate. Extracts of these cells contained high levels of protocatechuate oxygenase and only small amounts of catechol oxygenase.

Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen.

Abstract: Pseudomonas putida, strain 40, originally isolated by enrichment on caffeine as the sole source of carbon and nitrogen, has been developed to grow on 0.5% caffeine. The organism will grow on any N-methyl derivative of xanthine containing one or more methyl groups at the 1, 3, or 7 positions. An investigation of the activities of resting cell suspensions and cell-free preparations together with the detection of metabolic intermediates suggest that caffeine is first metabolized by the action of an enzyme which is capable of hydrolytically removing all three methyl groups with the production of methanol and free xanthine. The methanol presumably is oxidized to the final product, CO2, through the sequential action of methanol, formaldehyde, and formate dehydrogenases, which are induced by growth on caffeine. Furthermore, the xanthine would seem to be channeled through conventional pathways of purine degradation through the action of xanthine dehydrogenase and uricase, both induced by growth on caffeine. However, a variety of data suggests that the metabolism of caffeine may be compartmentalized in the cell and metabolized separately from externally added xanthine. Additional studies indicated that the cell is permeable to the methylxanthines. The significance of these findings is discussed.

Metabolism ofToluene andXylenes byPseudomonas (putida (arvilla) mt-2: Evidence for aNewFunction ofthe

Abstract: Pseudomonas putida (arvilla) mt-2carries genes forthecatabolism oftoluene, m-xylene, andp-xylene on atransmissible plasmid, TOL.Thesecompounds are degraded byoxidation ofone ofthemethyl substituents viathecorresponding alcohols andaldehydes tobenzoate andm- andp-toluates, respectively, which arethenfurther metabolised bythemetapathway, alsocodedforbytheTOL plasmid. Thespecificities ofthebenzyl alcohol dehydrogenase andthebenzaldehydedehydrogenase fortheir three respective substrates areindependent ofthe carbon sourceusedforgrowth, suggesting that asingle setofnonspecific enzymes isresponsible forthedissimilation ofthebreakdown products oftoluene andmandp-xylene. Benzyl alcohol dehydrogenase andbenzaldehyde dehydrogenase arecoincidently andpossible coordinately induced bytoluene andthexylenes, andbythecorresponding alcohols andaldehydes. Theyarenotinduced incells grown on m-toluate butcatechol 2,3-oxygenase can beinduced bym-xylene.

Alternative routes of aromatic catabolism in Pseudomonas acidovorans and Pseudomonas putida: gallic acid as a substrate and inhibitor of dioxygenases.

Abstract: When 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) was added to Pseudomonase acidovorans growing at the expense of succinate, enzymes required for degrading homoprotocatechuate to pyruvate and succinate semialdehyde were strongly induced. These enzymes were effectively absent from cell extracts of the organism grown with 4-hydroxyphenylacetic acid, and this substrate was metabolized by the catabolic enzymes of the homogentisate pathway. Two separate ring-fission dioxygenases for 3,4,5-trihydroxybenzoic acid (gallic acid) were present in cell extracts of Pseudomonas putida when grown with syringic acid, and gallate was degraded by reactions associated with meta fission. One of the two gallate dioxygenases also attacked 3-O-methylgallic acid; the other, which did not, was induced when cells were exposed to gallate. This organism possessed ortho fission enzymes, including protocatechuate 3,4-dioxygenase (EC 1.13.11.3) and cis,cis-carboxymuconate-lactonizing enzyme (EC 5.5.1.2), after induction with 3,4-dihydroxybenzoic acid (protocatechuic acid). Gallate was a substrate for protocatechuate 3,4-dioxygenase, with a Vmax about 3% of that of protocatechuate and with an apparent Km slightly lower. Gallate was a powerful competitive inhibitor of protocatechuate oxidation.

New mechanisms for the biosynthesis and metabolism of 2-keto-L-gulonic acid in bacteria.

Abstract: L-Sorbose is oxidized to 2-keto-L-gulonic acid (KGA) via the following sequence of reactions which we call the “sorbosone pathway”: L-sorbose ⇌ L-sorbosone → KGA. The first step is reversible and is mediated by enzymes found in a soluble fraction obtained from Pseudomonas putida ATCC 21812. Although no cofactor requirements were found for the forward reaction, the reverse reaction clearly required NADH. Enzymes for this NADH-dependent synthesis of L-sorbose could be differentiated on the basis of molecular weights. The second step in the sorbosone pathway is catalyzed by a particulate enzyme found in extracts from P. putida and Gluconobacter melanogenus IFO 3293. The rate limiting reaction in the sorbosone pathway is the synthesis of L-sorbosone. In addition to P. putida, Klebsiella pneumoniae (ATCC 27858) and Serratia marcescens (ATCC 27857) also contain the enzymes which catalyze the reactions of the sorbosone pathway. Two of the bacteria studied, P. putida and G. melanogenus, also contain an enzyme involved in the further metabolism of KGA to L-idonic acid. This enzyme, referred to as KGA-reductase, is found in the soluble fraction of cell-free extracts and is dependent on NADH or NADPH.

Improved method of selection for mutants of Pseudomonas putida.

Abstract: Optimum conditions for enrichment of mutants of Pseudomonas putida in liquid culture were established using a procedure which combines N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis with an improved D-cycloserine selection.

Induction of alkane hydroxylase proteins by unoxidized alkane in Pseudomonas putida.

Abstract: In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase proteins in mutants lacking the ability to convert n-alkanes to their primary alcohols. Purified heptane is an effective inducer in a mutant lacking detectable hydroxylase activity.

Exchange reactions catalyzed by methioninase from Pseudomonas putida.

Abstract: Highly purified methioninase from Pseudomonas putida, which catalyzes alpha, gamma-elimination reactions of homocysteine and its S-substituted derivatives as well as alpha, beta-elimination reactions of cysteine and its derivatives, was found to catalyze exchange reactions between the substituent at the gamma-carbon of homocysteine substrates and exogenously added alkanethiols, forming the corresponding S-alkylhomocysteines. It also catalyzed similar beta-exchange reactions between cysteine and alkanethiols. Thus, all the substrates for the methioninase-catalyzed elimination reactions also appear to be available for the exchange reactions.

The uptake of glucose and gluconate by Pseudomonas putida.

Abstract: The uptake of glucose and gluconate is under inductive control inPseudomonas putida. Glucose, gluconate, and 2-ketogluconate were each good nutritional inducers of these transport abilities. Glucose and gluconate uptake obeyed saturation kinetics: the apparent Km for glucose was 6mm and that for gluconate was 0.5mm. Therefore, transport of both substrates appears to be mediated by enzyme-like carriers.

Physiological consequences of starvation in Pseudomonas putida: degradation of intracellular protein and loss of activity of the inducible enzymes of L-arginine catabolism.

Abstract: We investigated the degradation of radioisotopically labeled intracellular protein in starved, intact cells of Pseudomonas putida P2 (ATCC 25571) and the regulation of this process. Intracellular protein isotopically labeled with L-[4,5-3H]leucine during log-phase growth at 30 C is degraded at rates of 1 to 2%/h in log-phase cells and 7 to 9%/h in starved cells. Rifampin, chloramphenicol, and tosyllysine chloromethylketone lower the rate of protein degradation by starved cells. Addition to starved cells of a nutrient upon which the culture is induced for growth rapidly lowers the rate of protein degradation from 7 to 9%/h to less than 1.5%/h. A nutrient that is oxidized but that cannot immediately support growth also lowers the rate of starvation-induced protein degradation. Proteolytic activity of cell extracts requires a divalent metal ion and may be inhibited up to 60% by tosyllysine chloromethylketone or p-toluenesulfonyl fluoride. Rifampin and chloramphenicol have no effect. In contrast to intact cells, extracts of growing or starving cells degrade protein at equivalent rates. We also investigated the stabilities of the inducible transport system and of four inducible intracellular enzymes of L-arginine catabolism. These include: the membrane-associated, L-arginine-specific transport system; L-arginine oxidase (oxidase); alpha-ketoarginine decarboxylase (decarboxylase); gamma-guanidinobutyraldehyde dehydrogenase ( dehydrogenase); and gamma-guanidinobutyrate amidinohydrolase (hydrolase). In starved cells, the rates of loss of activities were: transport and dehydrogenase activities, stable; oxidase and decarboxylase activities, 20 to 30%/h; hydrolase activity, 5 to 8%/h. Chloramphenicol decreases the rate of loss of oxidase, decarboxylase, and hydrolase activity, whereas p-toluenesulfonyl fluoride lowers the rate of loss of decarboxylase but not of oxidase or hydrolase activity. Addition to starved cells of a nutrient for which they are already induced for growth (e.g., malate, a noninducer of arginine catabolic enzymes) decreases the rate of loss of oxidase and decarboxylase activity but not that of the hydrolase.

Characterization of the growth of Pseudomonas putida LP on lipoate and its analogues: transport, oxidation, sulphur source, and enzyme induction.

Abstract: SUMMARY: Pseudomonas putida LP, which grows on lipoate, NH4NO3 and mineral salts, converts most of the organic substrate to bisnor-lipoate (1,2-dithiolane-3-propanoic acid) and acetyl-CoA. D-, L-, or DL-lipoate serve equally well as carbon and sulphur sources. There was no growth on or bacterial oxidation of the chemically synthesized bisnor- or tetranor-(1,2-dithiolane-3-carboxylic acid) chain-shortened analogues, but these, as well as lipoate, could supply the sulphur needed for growth when acetate was provided as the sole source of carbon. The uptake of lipoate by the bacterium is very slow and non-inducible, while the uptake of acetate is faster than octanoate. The oxidation of octanoate is more rapid and extensive than that of lipoate. Levels of acyl-CoA synthetase are not affected by the source of carbon, but activities of isocitrate lyase and malate synthase are higher when the cells are grown in acetate, octanoate or lipoate and lower when glucose is the carbon source. The glyoxylate cycle is induced to facilitate utilization of acetyl-CoA derived from lipoate, which is also degraded to water-soluble catabolites that yield the much smaller amount of sulphur required for growth.

Host factor for coliphage Qβ RNA replication is present in Pseudomonas putida

Abstract: Host Factor (HF)1, is a 12000 molecular weight polypeptide that is found in uninfected Escherichia coli and is required as a hexamer along with Qβ replicase for in vitro replication of Qβ phage RNA. It has recently been found to be associated with ribosomes and to bind tightly to poly(A).

Physiological role for the membrane bound ascorbate-TMPD oxidase in pseudomonas putida.

Abstract: The activity of the membrane-bound ascorbate-TMPD oxidase in Pseudomonas putida varies with growth conditions and age of the culture. A comparison of the effects of cyanide and azide on the oxidation of various substrates suggests that ascorbate-tMPD oxidase is not the terminal oxidase for NADH or succinate oxidation. Nowever, it does have a role in the oxidation of nicotinate, and may act as an additional terminal oxidase under certain other growth conditions.

Microbial Degradation of DDT.

Abstract: : Pseudomonas putida, an organism capable of utilizing diphenlmethane as sole source of carbon and energy, converted bis(p-chlorophenyl)acetic acid to bis(p-chlorophenyl)methane, 4,4'-dichlorobenzhydrol, and 4,4'-dichlorobenzophenone by cometabolism. The organism also dehalogenated 4,4'-dichlorobenzhydrol and 4,4'-dichlorobenzophenone. This is the first report of such dehalogenations of ring chlorines derived from DDT. Pseudomonas putida was also shown to convert diphenylmethane to benzhydrol and benzophenone. The organism was also found to be capable of ring cleavage of diphenylmethane and benzhydrol, producing phenylacetic and phenylglycolic acids, respectively. Studies were conducted to assess the effect of salinity, temperature, oxygen tension and presence of sediment organic nutrients and algal cells on the decomposition of DDT in model marine ecosystems. In the model ecosystems receiving the alga Cylindrospermum sp. or diphenylmethane, DDD, DDE, and DBP were formed from DDT. DDT and its breakdown products had no significant effect on respiration of microbial communities or algal productivity.