Showing papers on "Pseudomonas putida published in 1986"

The xylABC promoter from the Pseudomonas putida TOL plamid is activated by nitrogen regulatory genes in Escherichia coli

Abstract: The xylABC promoter (OP1), located on the TOL plasmid of Pseudomonas putida contains sequences homologous to the conserved regions found in nitrogen fixation (nif) promoters and in other promoters subject to nitrogen control. XylA-lac fusions were constructed in order to monitor expression from the OP1 promoter in Escherichia coli. Transcription was activated in the presence of the heterologous regulatory genes ntrC or nifA from Klebsiella pneumoniae as well as by the homologous P. putida regulatory gene xylR. In all cases activation was also dependent on the ntrA gene, whose product has been implicated as a specific sigma factor for ntr activatable operons. The 5′ ends of xylA mRNA, detected by S1 nuclease mapping of in vivo transcripts, were identical in strains containing xylR, ntrC or nifA as transcriptional activators. However, activation of the K. pneumoniae nifL or nifH promoters by xylR was not detected.

Iron, Siderophores, and Plant Diseases

Abstract: Siderophores and Biological Systems: An Overview.- Iron Uptake Systems in Fungi.- Absorption, Transport and Metabolic Significance of Iron in Plants.- Iron-Efficiency Reactions of Monocotyledonous and Dicotyledonous Plants.- Siderophore Involvement in Plant Iron Nutrition.- The Effect of Pseudomonas Siderophores on Iron Nutrition of Plants.- The Facilitation of Iron Uptake in Bacteria and Plants by Substituted Catechols.- Diverse Effects of some Bacterial Siderophores on the Uptake of Iron by Plants.- Characterization and Structural Analysis of the Siderophore Produced by the PGPR Pseudomonas putida strain WCS358.- Physical, Biological and Host Factors in Iron Competition in Soils.- Importance of Siderophores in Microbial Interactions in the Rhizosphere.- Suppression of Root Diseases of Wheat by Fluorescent Pseudomonads and Mechanisms of Action.- Biological Control of Fusarium Wilts by Pseudomonasputida and its Enhancement by EDDHA.- Pyoverdine-Facilitated Iron Uptake among Fluorescent Pseudomonads.- Bacterial Siderophores: Structure of Pyoverdins and Related Compounds.- Methods of Studying Plant Growth Stimulating Pseudomonads: Problems and Progress.- Emergence-Promoting Rhizobacteria: Description and Implications for Agriculture.- Nature of Intrageneric Competition between Pathogenic and Non-Pathogenic Fusarium in a Wilt-Suppressive Soil.- Antagonism and Siderophore Production by Biocontrol Agents, Plant Growth Promoting Organisms and the General Rhizosphere Population.- Herbicide-Induced Interactions between Cereal Roots and Fluorescent Pseudomonas spp.- Iron as a Factor in Disease Development in Animals.- Stimulation of Disease Development by Siderophores and Inhibition by Chelated Iron.- A New Siderophore in Aeromonas hudrophila: Possible Relationship to Virulence.- Ferrous Complexes and Chelating Compounds in Suppression of Fungal Diseases of Cereals.- The Effect of Chelating Agents on a Rust Fungus Developing on Induced Resistant Plants.- High Affinity Iron Transport in Ustilago maydis.- Production of Fusarinine and Iron Assimilation by Pathogenic and Non-pathogenic Fusarium.- Cell Surface Mutants of Erwinia chrysanthemi: Possible Involvement of Iron Acquisition in Phytopathogenicity.- Iron and Phytotoxins as Exemplified by Stemphyloxins and other Toxins.- Characterization of Cell Envelope Protein Patterns of Crop Yield Increasing Root-Colonizing Pseudomonas spp.- A Saga of Siderophores.- Genetic Analysis of the Iron-Uptake System of Two Plant Groups Promoting Pseudomonas Strains.- Genotypic Diversity of Fluorescent Pseudomonads as Revealed by Southern Hybridization Analysis with Siderophore-Related Gene Probes.- Biological Control of Phytopathogens by Pseudomonas spp.: Genetic Aspects of Siderophore Production and Root Colonization.- Participants.

New Route to Bacterial Production of Indigo

Abstract: Cells of Escherichia coli K–12 containing a cloned fragment of Pseudomonas putida TOL plasmid pWW0 produce the dye indigo. Analysis of the cloned fragment and Tn1000 transposon insertion mutagenesis has identified the xylA gene as being responsible for this phenotype. The xylA gene specifies xylene oxidase, a relaxed specificity enzyme that hydroxylates or monooxygenates toluene and xylenes and their corresponding alcohols. Indole, which is formed from tryptophan by tryptophanase in E. coli, was shown to be a precursor in the reaction sequence leading to indigo formation. These results suggest a novel route for bacterial production of indigo via hydroxylation of indole.

Oxygen uptake of microorganisms entrapped in Ca-alginate

Abstract: The oxygen uptake rates of Pseudomonas putida, Saccharomyces cerevisiae and Aspergillus niger, immobilized in Ca-alginate gel, were determined in comparison with the respiration of free cells. The specific oxygen uptake rate of immobilized microorganisms decreased with increasing cell content of the gel beads and increasing alginate concentration.

Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli.

Abstract: This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed.

Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

Abstract: A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.

Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida.

Abstract: Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida. A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon. In parallel, a series of N-methyl-N'-nitro-N-nitro-soguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon. Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined. Four cistrons for toluate 1,2-dioxygenase were thus identified. DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate. This analysis also identified four cistrons. Examination of the products of these cistrons, by means of E. coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xylY, encodes a 20 kilodalton protein.

Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2.

Abstract: Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.

Purification and Some Properties of the 2-Hydroxy-6-oxohepta-2,4-dienoate Hydrolase (2-Hydroxymuconic Semialdehyde Hydrolase) Encoded by the TOL Plasmid pWW0 from Pseudomonas putida mt-2

Abstract: SUMMARY: The 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolase encoded by the TOL plasmid pWW0 from Pseudomonas putida mt-2 (PaWl) was purified to homogeneity. It has an M r of 65000 and is dissociated by SDS into two subunits of equal size. Alanine was the only N-terminal residue detected, and each subunit contained one cysteine thiol group. The pH optimum for activity and for enzyme stability was around 7·5, whereas the isoelectric point was 4·7. Only the products of catechol 2,3-oxygenase action on linear chain alkylcatechols and 4-chlorocatechol served as substrates. Kinetic measurements showed that the higher activity against the ketone substrates (from 3-substituted catechols) over aldehyde substrates (from 4-substituted catechols) was the result of higher V max values rather than lower K m values. Antisera prepared against this purified HOD hydrolase were shown by Ouchterlony double diffusion and inhibition studies to be related to the HOD hydrolase from phenol-grown P. putida strain U (NCIB 10015) in which the enzyme is chromosomally encoded.

Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453.

Abstract: The oxygenating component of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453 was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatography on DEAE-cellulose and polyanion SI-17 columns. It had an Mr of 78,000, bound one molecule of nonautooxidizable flavin mononucleotide (FMN), consisted of two subunits of equal molecular weight, and existed in two electrophoretically distinguishable active forms. The oxygenating complex was constructed from equimolecular amounts of an NADH oxidase, which could be purified separately (Mr, 36,000), and the oxygenating component. Most of the NADH oxidase dissociated from the oxygenating component during purification, although traces remained, to give the final preparation of the oxygenating component significant oxygenase activity. FMN did not dissociate significantly from the oxygenating component during purification, but it was not covalently bound and could be removed under a variety of conditions. Binding between the two proteins that made up the active complex was fairly weak and freely reversible. It probably occurred through the FMN which was strongly bound to the oxygenating component and for which the NADH had a weak binding site. Iron was not present at a significant level in the oxygenating component, and in common with other characterized Baeyer Villiger monooxygenases, 2,5-diketocamphane 1,2-monooxygenase was found to be a simple flavoprotein.

Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage.

Abstract: SUMMARY: A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.

Formaldehyde dismutase, a novel NAD-binding oxidoreductase from Pseudomonas putida F61

Abstract: A novel enzyme, formaldehyde dismutase, was purified and crystallized from the cell extract of an isolated bacterium, Pseudomonas putida F61. The enzyme catalyzes the dismutation of aldehydes and alcohol: aldehyde oxidoreduction in the absence of an exogenous electron acceptor. The enzyme is composed of four identical subunits with a Mr of 44000. Each subunit contains 1 mol NAD(H) and 2 mol zinc/mol. The ratio of NAD+ and NADH in a crystalline preparation of the enzyme was about 7:3. The enzyme-bound coenzyme was completely reduced and oxidized on the addition of a large amount of an alcohol and an aldehyde respectively. Both the oxidized and reduced enzymes catalyzed the dismutation reaction to the same extent. Steady-state kinetics of the enzyme were investigated using an oxidoreduction reaction between an alcohol and p-nitroso-N,N-dimethylaniline. The enzyme obeys a ping-pong mechanism and is competitively inhibited by an alcoholic substrate analogue, pyrazole, but not coenzyme analogues, such as AMP, N-methylnicotinamide. These results indicate that NAD(H) binds firmly (but not covalently) at each active site. The enzyme-bound NAD(H) was reduced and oxidized only by the added second substrates, alcohol and aldehyde respectively, and not by exogenous electron acceptors [including NAD(H)].

Naphthalene association and uptake in Pseudomonas putida.

Abstract: Two methods for bacterial membrane transport, filtration and flow dialysis, were used to study cellular association of Pseudomonas putida with naphthalene. It is not technically possible to determine the exact cellular or vesicular location of the naphthalene, and because it is hydrophobic, it could be at the membrane(s) rather than inside the cells. As an index of naphthalene having crossed the inner membrane we used the intracellular formation of its first catabolite. An energized membrane or ATP was not essential for association or movement into the cell. Evidence for a nonspecific association and a movement into cells by simple diffusion are the lack of saturation of association, an absence of inhibition of association by protein inhibitors and structural analogs, and the passage of naphthalene through cell membranes in the presence of iodoacetamide. Specific naphthalene metabolism gene expression was not required for association.

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

Abstract: 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.

Chromosomal location of TOL plasmid DNA in Pseudomonas putida.

Abstract: Thesoil isolate Pseudomonas putida MW1000can grow on toluene andother hydrocarbons; inthis respect itissimilar tostrains ofPseudomonas which carrytheTOLplasmid. Byconjugation experiments, thegenes conferring these growth abilities havebeenshowntobelocated onthebacterial chromosome, linked tovil and catB.A56-kilobase segment ofthebacterial chromosome ofMW strains carrying theTOLgenescantranspose totheIncP-1 plasmid R18-18. Physical analysis ofthese TOLR18-18 hybrids hasshownthat theTOLsegment isalmost identical tothesame region found intheTOLplasmid pWWO. A numberofdegradative plasmids havebeendescribed whichenable Pseudomonas putida toutilize avariety of hydrocarbons forgrowth. Inalmost allofthese cases, the plasmid involved isautonomous andself-replicating . Howeverthere hasbeenlimited evidence that suchplasmids can becomeintegrated into thebacterial chromosome. Williams andWorsey(36) isolated arange ofpseudomonads fromsoil whichhadtheability toutilize m-toluate forgrowth. Notall ofthese strains couldtransfer this ability atconjugation, indicating thepossibility thattheTOL genescouldbe located inthebacterial chromosome oronanontransmissible plasmid. Jeenes andWilliams (12)havedescribed a strain (WR211) whichbehaves asifithasachromosomal insert ofTOLDNA inaddition toaphenotypically TOLplasmid. Meulin andBroda(15) presented Southern hybridization evidence forthechromosomal location ofsegments oftheTOL plasmid pWWO insomephenotypically cured strains. Thus,evidence forthechromosomal location of TOLgenesislimited tonontransmissibility atconjugation,

Cadmium-Binding Proteins in Pseudomonas putida: Pseudothioneins

Abstract: Pseudomonas putida adapted to growth in 3 mM cadmium. The resistance mechanism involved complexation of cadmium in polyphosphate granules, changes in the structure of the cell membrane and induction of three cysteine-rich, low molecular weight proteins (3500-7000) containing 4 to 7 g-atoms per mole of cadmium, zinc, and copper. Each protein was produced during a different phase of growth, and the smallest protein (3500) was released into the environment when the cells lysed at the end of the exponential phase. The metal binding sites of the major protein were further characterized using a range of physical methods, including 113Cd NMR. The properties of the bacterial pseudothioneins are compared to those of metallothioneins.

Immunological comparison of microbial TPP-dependent non-oxidative α-keto acid decarboxylases

Abstract: Antigenic, and hence possible evolutionary, relationships amongst various TPP-dependent non-oxidative α-keto acid decarboxylases were determined by the Ouchterlony double diffusion method and by measuring the degree of antibody-induced enzyme inhibition. The results show that: (a) phenylglyoxylate decarboxylases of various wild-type strains of Acinetobacter calcoaceticus are antigenically indistinguishable; (b) there seems to be no antigenic cross-reactivity between the phenylglyoxylate decarboxylase of A. calcoaceticus and of Pseudomonas aeruginosa or Pseudomonas putida; and (c) no antigenic homology can be detected amongst phenylglyoxylate decarboxylase and phenylpyruvate decarboxylase of A. calcoaceticus and pyruvate decarboxylase of brewers' yeast.

Nucleotide sequence of a DNA segment promoting transcription in Pseudomonas putida.

Abstract: A DNA segment that promotes gene expression in Pseudomonas putida was identified in pTN8, a mutant plasmid of an RP4-TOL recombinant. A promoter on the segment was cloned with a promoter-probe vector containing the xylE gene of the TOL plasmid. The xylE gene was expressed under the control of the promoter, and the gene product catechol 2,3-dioxygenase was constitutively synthesized. As analyzed by an S1 nuclease protection assay, the amount of mRNA produced in P. putida was more than that in Escherichia coli. Fine S1 nuclease mapping and reverse transcriptase mapping revealed three tandem transcription start sites in both P. putida and E. coli. The nucleotide sequence preceding the transcription start sites was determined; a part of this sequence contained a sequence homologous to E. coli promoter sequences. A tentative consensus sequence for P. putida constitutive promoters is proposed.

Characterization and Structural Analysis of the Siderophore Produced by the PGPR Pseudomonas putida Strain WCS358

Abstract: Under iron limitation, the plant-growth-promoting Pseudomonas putida strain WCS358 produces a yellow-green fluorescent siderophore (Geels and Schippers, 1983; Geels and Schippers, 1983; Marugg et al., in press). The absorption spectra of WCS358 culture medium before and after addition of FeCl3 and at different pHs are shown in an accompanying paper by our group (weisbeek et al., in press). They strongly resemble those of the pseudobactin/pyoverdine class of siderophores (Hider, 1984).

Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.

Abstract: Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.

Plasmid-determined cadmium resistance in Pseudomonas putida GAM-1 isolated from soil.

Abstract: Cadmium-resistant pseudomonas putida GAM-1, which was able to grow in concentrations of CdCl/sub 2/ as high as 7 mM, was isolated from soil in a rice paddy. This bacterium harbored a DNA plasmid of about 52 kilobases. The plasmid (pGU100) transformed Escherichia coli C600 to cadmium resistance. A cadmium-resistant transformant of E. coli C600 contained a plasmid corresponding to that seen in P. putida GAM-1. The transformant did not take up cadmium as well as P. putida GAM-1 did. This is the first report on the mechanism of plasmid-determined Cd/sup 2 +/ resistance in gram-negative bacteria.

Adaptation of Pseudomonas putida mt-2 to growth on aromatic amines.

Abstract: Pseudomonas putida mt-2 (ATCC 33015) carrying the TOL plasmid pWW0 could adapt to growth on the aromatic amines aniline and m- and p-toluidine. In strain UCC2, a derivative adapted to rapid growth on these compounds, they were oxidatively deaminated to catechol or 4-methylcatechol, which in turn were dissimilated by a meta-cleavage pathway. The aniline/toluidine oxygenase and the meta-cleavage pathway enzymes were inducible by aromatic amines. Evidence is presented that in strain UCC2, plasmid pWW0 has undergone deletion of its catabolic genes, and that it is a novel plasmid, pTDN1, which is involved in the catabolism of aniline and m- and p-toluidine. The meta-cleavage pathway genes which are carried by pTDN1 were shown not to have originated in pWW0.

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

Abstract: Halogenated benzoates have been used as models for the study of the biodegradation of herbicides and PCBs. The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the ..beta..-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB/sup -/), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

Abstract: The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.

Comparative analysis of different Pseudomonas strains that degrade cinnamic acid.

Abstract: Strains of Pseudomonas stutzeri (CINNS) and Pseudomonas putida (CINNP and CINNW) isolated from soil with cinnamic acid as the sole carbon source were found to be simultaneously adapted to grow on phenylpropionic and p-hydroxybenzoic acids. In cinnamic acid-grown cultures, phenylpropionic acid was isolated. A catabolic plasmid of approximately equal to 75 kilobase pairs encoding the metabolism of cinnamic acid was found in strains CINNP and CINNS.