Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

Organization and Regulation of meta Cleavage Pathway Genes for Toluene and o-Xylene Derivative Degradation in Pseudomonas stutzeri OX1

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.     

Bacterial Metabolism of Arylsulfonates: Role of meta Cleavage in Benzene Sulfonate Oxidation by Pseudomonas testosteroni

Pseudomonas testosteroni H-8 oxidizes certain lower alkylbenzene sulfonates at rates inversely related to the length of the alkyl group. Appreciable Q(O)2 values were observed for benzene sulfonate (BS), toluene sulfonate (TS), and ethylbenzene sulfonate (EBS), but not for propylbenzene sulfonate (PS) and higher homologues. Catechol oxidation was catalyzed by a constitutive catechol-2,3-oxygenase (EC 1.99.2.a). Yellow meta cleavage products accumulated when BS-grown cells were exposed to catechol, 4-methylcatechol, 3-methylcatechol, EBS and PS, but not BS or TS. Traces of a yellow metabolite (probably 2-hydroxymuconic semialdehyde) were detectable during growth on BS. PS completely inhibited growth on BS, but not on L-leucine or nutrient broth. Also, PS antagonized respiration on BS and catechol, but not glutamate, the extent of inhibition being directly related to PS concentration. Formation of a meta cleavage product from PS, and inhibition of catechol oxidation by PS, suggested that the actual inhibitor may not be PS itself, but a metabolite.     

Regiochemistry of Camphor Analog Oxidation by Pseudomonas putida

Pseudomonas putida cooxidized norcamphor and pericyclocamphanone to hydroxylated and lactonized products during growth on camphor. Norcamphor was hydroxylated at the 5 position, similar to the corresponding process in camphor, but pericyclocamphanone was oxidized at the 6 position. We conclude that the regiochemistry of the hydroxylation may be substrate controlled.     

Degradation of 4-Chlorophenol via the meta Cleavage Pathway by Comamonas testosteroni JH5

Comamonas testosteroni JH5 used 4-chlorophenol (4-CP) as its sole source of energy and carbon up to a concentration of 1.8 mM, accompanied by the stoichiometric release of chloride. The degradation of 4-CP mixed with the isomeric 2-CP by resting cells led to the accumulation of 3-chlorocatechol (3-CC), which inactivated the catechol 2,3-dioxygenase. As a result, further 4-CP breakdown was inhibited and 4-CC accumulated as a metabolite. In the crude extract of 4-CP-grown cells, catechol 1,2-dioxygenase and muconate cycloisomerase activities were not detected, whereas the activities of catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-oxopent-4-enoate hydratase were detected. These enzymes of the meta cleavage pathway showed activity with 4-CC and with 5-chloro-2-hydroxymuconic semialdehyde. The activities of the dioxygenase and semialdehyde dehydrogenase were constitutive. Two key metabolites of the meta cleavage pathway, the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde) and 5-chloro-2-hydroxymuconic acid, were detected. Thus, our previous postulation that C. testosteroni JH5 uses the meta cleavage pathway for the complete mineralization of 4-CP was confirmed.     

Propane and n-Butane Oxidation by Pseudomonas putida GPo1

Propane and n-butane inhibit methyl tertiary butyl ether oxidation by n-alkane-grown Pseudomonas putida GPo1. Here we demonstrate that these gases are oxidized by this strain and support cell growth. Both gases induced alkane hydroxylase activity and appear to be oxidized by the same enzyme system used for the oxidation of n-octane.     

Enzymes of the Tryptophan Synthetic Pathway in Pseudomonas putida

The first four enzymatic activities of the tryptophan synthetic pathway in Pseudomonas putida were found on separate molecules. Gel filtration and density gradient centrifugation experiments did not disclose any associations or aggregations among them. These findings contrast with the situation found in the enteric bacteria, where the first two activities are found in an aggregate and the third and fourth are catalyzed by a single enzyme. Tryptophan synthetase, the last enzyme of the pathway, consists of two dissociable components. The affinity of these components is less in P. putida than is the case in Escherichia coli.     

Regulation of histidine catabolism by succinate in Pseudomonas putida

The regulation of the histidine-degrading pathway is known to involve induction and repression. Our studies have shown that succinate may control the histidine-degrading pathway by sequential negative feedback inhibition. Succinate inhibited urocanase, and urocanate in turn inhibited histidase. Crude preparations of the two enzymes were made from Pseudomonas putida grown on l-histidine. Succinate was a competitive inhibitor of urocanase (K(i), 1.8 mm). Lactate, pyruvate, alpha-ketoglutarate, and glutamate did not inhibit urocanase. Urocanate inhibited histidase competitively (K(i), 0.13 mm). A multienzyme system (histidine to glutamate), when incubated with histidine and succinate, exhibited the combined effect. Succinate caused the level of accumulated urocanate to increase and indirectly blocked histidine disappearance. Growth of cells on urocanate as a nitrogen source was inhibited by 1% succinate. Succinate may play a physiological role in the biological regulation of histidine metabolism.     

Benzoate Metabolism in Pseudomonas putida(arvilla) mt-2: Demonstration of Two Benzoate Pathways

Benzoate-grown cells of Pseudomonas putida(arvilla) mt-2 contain both metapyrocatechase and pyrocatechase activities, although the former activity is much higher than that of the latter. A spontaneous mutant deficient in metapyrocatechase and 2-hydroxymuconic semialdehyde hydrolyase, the first two enzymes in the meta-cleavage pathway of the ring of catechol, has been isolated from this strain. This mutant grows well on a minimal medium containing benzoate as a sole carbon source and has the high activity of pyrocatechase. These findings indicate that the strain mt-2 possesses the genetic capacity for enzymes of both the meta- and ortho-cleavage pathways of benzoate degradation, but its phenotypic expression is the meta pathway.     

Identification of a Two-Component Regulatory Pathway Essential for Mn(II) Oxidation in Pseudomonas putida GB-1

Bacterial manganese(II) oxidation has a profound impact on the biogeochemical cycling of Mn and the availability of the trace metals adsorbed to the surfaces of solid Mn(III, IV) oxides. The Mn(II) oxidase enzyme was tentatively identified in Pseudomonas putida GB-1 via transposon mutagenesis: the mutant strain GB-1-007, which fails to oxidize Mn(II), harbors a transposon insertion in the gene cumA. cumA encodes a putative multicopper oxidase (MCO), a class of enzymes implicated in Mn(II) oxidation in other bacterial species. However, we show here that an in-frame deletion of cumA did not affect Mn(II) oxidation. Through complementation analysis of the oxidation defect in GB-1-007 with a cosmid library and subsequent sequencing of candidate genes we show the causative mutation to be a frameshift within the mnxS1 gene that encodes a putative sensor histidine kinase. The frameshift mutation results in a truncated protein lacking the kinase domain. Multicopy expression of mnxS1 restored Mn(II) oxidation to GB-1-007 and in-frame deletion of mnxS1 resulted in a loss of oxidation in the wild-type strain. These results clearly demonstrated that the oxidation defect of GB-1-007 is due to disruption of mnxS1, not cumA::Tn5, and that CumA is not the Mn(II) oxidase. mnxS1 is located upstream of a second sensor histidine kinase gene, mnxS2, and a response regulator gene, mnxR. In-frame deletions of each of these genes also led to the loss of Mn(II) oxidation. Therefore, we conclude that the MnxS1/MnxS2/MnxR two-component regulatory pathway is essential for Mn(II) oxidation in P. putida GB-1.In living cells, manganese (Mn) is an essential trace element, required for enzymes such as superoxide dismutase and in photosystem II (7). In the environment, Mn cycles between a soluble reduced form [Mn(II)] and an insoluble oxidized form [Mn(III, IV)] that can adsorb other trace metals from the environment and serve as potent oxidizing agents. Thus, redox cycling of Mn has a profound effect on the bioavailability and geochemical cycling of many essential or toxic elements (40). Microorganisms, particularly bacteria, are capable of catalyzing the oxidation of Mn(II), thereby increasing the rate of formation of Mn(III, IV) by several orders of magnitude (39). Since Mn(III, IV) oxides are able to bind trace metals, the bacteria that catalyze their formation are good candidates for bioremediation of heavy metal contaminated sites (26, 39).Although bacterial Mn(II) oxidation is widespread, little is known about the physiological function of oxidation (40). The oxidation of Mn(II) to Mn(III) or Mn(IV) is thermodynamically favorable; thus, bacteria may derive energy from this reaction, although this has never been unequivocally proven (40). In addition, Mn(II) oxidation could protect cells from reactive oxygen species (4) or UV irradiation (11). Since oxidation occurs on the cell surface, the bacteria become coated with the solid Mn(IV) oxides, which may also provide protection from toxic heavy metals, predation, or phage infection (40). As a strong oxidant, Mn(IV) oxides could allow the bacteria to degrade refractory organic matter to low-molecular-weight compounds that could then be used to support bacterial growth (38). Conversely, Mn(II) oxidation may be a side reaction or the result of nonspecific interactions with cellular products (15). Identifying signals or conditions that regulate oxidation could provide some insight into the role of Mn(II) oxidation in the cell. Aside from a requirement for oxygen (28) and iron (27, 30), as well as the observation that oxidation occurs in stationary phase (23), very little is known about this regulation.The enzymes responsible for Mn(II) oxidation have been tentatively identified from some species of bacteria and in several cases the enzyme is a putative multicopper oxidase (MCO). MCOs are a family of enzymes that use four Cu ion cofactors to catalyze oxidation of diverse substrates such as metals and organic compounds (33). This family of enzymes is found in plants and fungi (laccase) and humans (ceruloplasmin), as well as in bacteria (35). Some fungi have been shown to use a laccase enzyme to oxidize Mn(II) (20). In both Leptothrix discophora SS-1 and Pedomicrobium sp. strain ACM 3067, the Mn(II)-oxidizing MCO was identified genetically (mofA [10] and moxA [31], respectively). A third MCO—MnxG—was identified both biochemically and genetically as the Mn(II) oxidase in Bacillus sp. strain SG-1 and related strains (14, 43). Recent work with the Mn(II)-oxidizing alphaproteobacterium Aurantimonas manganoxydans SI85-9A1 and Erythrobacter sp. strain SD21 has identified a second class of enzyme involved in Mn(II) oxidation: the heme-binding peroxidase named MopA (3). This class of enzyme had previously been shown to be used by fungi to oxidize Mn(II) (29), in some cases in concert with an MCO (34).Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium (9) whose genetic tractability and ease of growth under standard laboratory conditions make it an ideal model system for studying the physiology and mechanism of Mn(II) oxidation. Consequently, several random transposon mutagenesis screens have been undertaken with this organism to identify genes required for Mn(II) oxidation. These screens have identified several categories of genes as important for oxidation or the export of the oxidase to the cell surface: the ccm operon of c-type cytochrome synthesis genes (8, 13), genes encoding components of the trichloroacetic acid (TCA) cycle and the tryptophan biosynthesis pathway (8) and genes encoding a general secretory pathway (12). The Mn(II) oxidation-defective mutant GB-1-007 has a transposon insertion in the gene cumA that encodes a putative MCO (6). Therefore, P. putida GB-1 has been thought to use a similar mechanism as L. discophora SS-1, Pedomicrobium sp. strain ACM 3067, and Bacillus sp. to oxidize Mn(II).Because the available data suggested that CumA was an MCO essential for Mn(II) oxidation, we wanted to study its function in greater detail. We were hampered in this, however, by the fact that the transposon insertion in cumA resulted in a growth defect due to its polar effect on expression of the downstream cumB gene (6). In order to assess the role of CumA in Mn(II) oxidation without the complications arising from polarity, we generated an in-frame deletion of cumA and tested the ability of the resulting ΔcumA strain to form Mn(IV) oxides. Our results showed that cumA is dispensable for Mn(II) oxidation and have instead revealed a complex two-component regulatory pathway essential for Mn(II) oxidation in P. putida GB-1.     

Oxidation of n-alkanes: Growth of Pseudomonas putida

Summary The induction of alkane hydroxylase activity was investigated in two strains of Pseudomonas putida with a view to the production of primary alcohols. n-Nonanol production rates (16.0 mol/g dry wt/h) with an alcohol dehydrogenase negative mutant P. putida PpS173 were considerably lower than might be expected from the growth of a wild type on n-alkane. Production of cells by fed-batch culture on n-nonane, with a specific alkane hydroxylase activity of 3.9 mmol/g/h, was considered most suitable for isolation of the alkane hydroxylase.     

Regulation of valine catabolism in Pseudomonas putida

The activities of six enzymes which take part in the oxidation of valine by Pseudomonas putida were measured under various conditions of growth. The formation of four of the six enzymes was induced by growth on d- or l-valine: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydrogenase were synthesized constitutively. d-Amino acid dehydrogenase and branched-chain keto acid dehydrogenase were induced during growth on valine, leucine, and isoleucine, and these enzymes were assumed to be common to the metabolism of all three branched-chain amino acids. The segment of the pathway required for oxidation of isobutyrate was induced by growth on isobutyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. d-Amino acid dehydrogenase was induced by growth on l-alanine without formation of other enzymes required for the catabolism of valine. d-Valine was a more effective inducer of d-amino acid dehydrogenase than was l-valine. Therefore, the valine catabolic pathway was induced in three separate segments: (i) d-amino acid dehydrogenase, (ii) branched-chain keto acid dehydrogenase, and (iii) 3-hydroxyisobutyrate dehydrogenase plus methylmalonate semialdehyde dehydrogenase. In a study of the kinetics of formation of the inducible enzymes, it was found that 3-hydroxyisobutyrate and methylmalonate semialdehyde dehydrogenases were coordinately induced. Induction of enzymes of the valine catabolic pathway was studied in a mutant that had lost the ability to grow on all three branched-chain amino acids. Strain PpM2106 had lowered levels of branched-chain amino acid transaminase and completely lacked branched-chain keto acid dehydrogenase when grown in medium which contained valine. Addition of 2-ketoisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the growth medium of strain PpM2106 resulted in induction of normal levels of branched-chain keto acid dehydrogenase; therefore, the branched-chain keto acids were the actual inducers of branched-chain keto acid dehydrogenase.     

Regulation of leucine catabolism in Pseudomonas putida

The generation time of Pseudomonas putida with l-leucine was 20 h in synthetic media but only 3 h with d-leucine. Slow growth in the presence of l-leucine was partially overcome by addition of 0.1 mM amounts of either d-valine, l-valine, or 2-ketoisovalerate. The activities of five enzymes which take part in the oxidation of leucine by P. putida were measured under various conditions of growth. Four enzymes were induced by growth with dl-leucine as sole source of carbon: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-methylcrotonyl-coenzyme A carboxylase, and 3-hydroxy-3-methylglutaryl-coenzyme A lyase. The segment of the pathway required for oxidation of 3-methylcrotonate was induced by growth on isovalerate or 3-methylcrotonate without formation of the preceding enzymes. The synthesis of carboxylase and lyase appeared to have been repressed by the addition of l-glutamate or glucose to cells growing on dl-leucine as the sole carbon source. Mutants unable to grow at the expense of isovalerate had reduced levels of carboxylase and lyase, whereas the levels of three enzymes common to the catabolism of all three branched-chain amino acids and those of two isoleucine catabolic enzymes were normal.     

Regulation of alkane oxidation in Pseudomonas putida.

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.     

Impact of Growth in Benzoate and m-Toluate Liquid Media on Culturability of Pseudomonas putida on Benzoate and m-Toluate Plates

Pseudomonas putida grown in continuous culture on benzoate or m-toluate lost the ability to grow on benzoate or m-toluate plates. A similar effect was not seen with a glucose continuous culture. Cells carrying and expressing a TOL plasmid rapidly lost their ability to grow on benzoate solid medium.     

Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6

The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems. The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes. Pseudomonas sp. strain JS6 uses a similar system for growth on toluene or dichlorobenzenes. We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene. When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols. Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols. Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.     

Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches. Transposants carrying mini-Tn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3′-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.     

Evidence For Autogenous Regulation of Pseudomonas putida Tryptophan Synthase

Studies of a trpA mutant constitutive for tryptophan synthase production support the hypothesis of autogenous regulation (R. F. Goldberger, 1974; A. R. Proctor and I. P. Crawford, 1975) of the Pseudomonas putida trpAB loci.