Cloning of genes for naphthalene metabolism in Pseudomonas putida.

Plasmid pIG7 DNA cloned in Pseudomonas putida with the broad-host-range vectors pRK290 and pKT240 expresses the genes encoding nephthalene oxidation in the presence of the intermediate substrate, salicylate, or the gratuitous inducer, anthranilate. Two operons, nahAF and nahGK, cloned from the EcoRI fragment A (25 kilobases) are under wild-type regulation by the nahR locus. Deletion plasmids provide a restriction map of both operons. Double transformants containing structural and regulatory cistron nahR in trans are used to demonstrate positive control of expression.     

Primary structure and properties of an inactive mutant aspartate transcarbamoylase.

A mutant form of Escherichia coli aspartate transcarbamoylase (ATCase) which lacks catalytic activity has been purified and characterized (Wall, K.A., Flatgaard, J.E., Gibbons, I., and Schachman, H.K. (1979) J. Biol. Chem 254, 11910-11916). Peptide mapping of the mutant and wild type catalytic chains followed by the determination of the amino acid sequence of the one altered peptide in the mutant indicated that a glycyl residue was replaced by aspartic acid. This substitution is located at position 125 in the tentative sequence kindly provided by W. Konigsberg (personal communication). The mutant protein has an overall secondary structure similar to that of the wild type as indicated by circular dichroism spectroscopy. However, marked changes in the reactivity of several amino acid residues were demonstrated. Lysyl residue 84 which in the wild type subunits reacts specifically with pyridoxal 5'-phosphate is only slightly reactive in the mutant even though the peptide containing that residue was not altered in amino acid composition. Another residue, cysteinyl 46, which is thought to be in the active site, is much more reactive toward p-hydroxymercuribenzoate in the mutant subunit than in the wild type protein. Finally, tyrosyl residue 213, which according to recent crystallographic studies is not near the active site and which exhibits an unusually low pK (9.1) in the wild type catalytic subunits, appears to have its pK shifted to 10.5 or higher as a result of the mutation. The evidence indicates that the substitution of an aspartyl for a glycyl residue at a region of the amino acid sequence remote from those residues in the active site causes sufficient modification of the tertiary structure to cause the loss of enzyme activity and to affect the reactivity of other residues in the protein. Moreover, the quaternary structure of the intact enzyme is altered as well since the subunit interactions are greatly weakened.     

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.     

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

Transfer of nitrogen fixation genes into Pseudomonas putida isolated from Finnish tundra soil

The plasmid pRD1 containing the nif genes from Klebsiella pneumoniae was transferred by conjugation from Escherichia coli to Pseudomonas putida isolated from the tundra soil. 6-Cyanopurine, acetylene reduction and immunological tests showed that the nif genes were not expressed in P. putida. Existence of the nif genes in P. putida transconjugants was detected by transferring them to E. coli C600, which does not fix nitrogen. Existence of the nif genes in E. coli C600 transconjugants was detected immunologically and by acetylene reduction tests.     

Supra-operonic clustering of genes specifying glucose dissimilation in Pseudomonas putida.

Summary The linkage arrangements of genes governing glucolysis in Pseudomonas putida have been determined by transductional analysis. Five genes (gdh, kgtA, kgtB, edd and eda), comprising at least three operons, are cotransducible with each other, but not with ggu (glucose and gluconate uptake) nor with genes of a known supra-operonic cluster of genes specifying enzymes of other dissimilatory pathways, nor with a biochemically uncharacterized his marker. It thus appears that P. putida may have more than one chromosomal region in which genes with dissimilatory function are clustered in a supra-operonic fashion.     

DNA sequence of the tryptophan synthase genes of Pseudomonas putida

Genes encoding the 2 subunits of tryptophan synthase in Pseudomonas putida have been identified and cloned by their similarity to the corresponding genes in Pseudomonas aeruginosa. The deduced amino acid sequences were confirmed by comparison with regions ascertained earlier by protein sequencing. The Pseudomonas amino acid sequences are 85% identical for the beta subunit and 70% identical for the alpha subunit. These sequences are compared to those of Salmonella typhimurium, where the structure is known from X-ray crystallography. Although amino acid conservation drops to 54% and 36% for the beta and alpha subunits, only 3 single residue gaps are required to maintain alignment throughout and most of the residues identified as important for catalysis or cofactor binding are conserved. The 23 residues surrounding the beta chain lysine that enters into a Schiff base linkage with the pyridoxal phosphate cofactor are compared in 13 species, including representatives from the eukaryotic and both prokaryotic kingdoms; appreciable conservation is apparent. The approximately 100 base pairs separating the trpB gene from its divergently transcribed activator gene are similar in the 2 pseudomonads, but do not resemble those of any other bacterium or fungus studied to date.     

Properties of an inducible uptake system for beta-ketoadipate in Pseudomonas putida.

Wild-type strains of Pseudomonas putida form an inducible uptake system that appears to act on beta-ketoadipate under normal physiological conditions. The system is induced by beta-ketoadipate and is represented by catabolites derived from it. Adipate is metabolized very slowly by wild-type P. putida cultures; [14C]adipate was used as an analogue of beta-ketoadipate to measure the transport activity in wild-type cells and in cells that constitutively produced the uptake system. Constitutive cells that contained high levels of the uptake system concentrated adipate to a level up to 200-fold above the concentration in the external medium. The process was energy dependent. The activity of the system with radioactive adipate was inhibited by beta-ketoadipate, by beta-ketoadipate analogues, and by some compounds (e.g., acetate, glucose) that are structurally unrelated to beta-ketoadipate; it is not known if the inhibitory effects are exerted directly by the compounds themselves or indirectly by catabolites derived from the compounds. The discovery of the beta-ketoadipate uptake system is surprising in view of earlier studies that had indicated that beta-ketoadipate does not permeate the membrane of wild-type P. putida cells. Contradictions between the former investigations and the present analysis are due primarily to the relatively high concentrations of substrate used in the earlier experiments. The existence of the beta-ketoadipate uptake system indicates that beta-ketoadipate may exist as a selective nutrient in the natural niche of P. putida and may play a determinative role in the evolution of induction mechanisms that are characteristic of fluorescent pseudomonads.     

Characterization of an OprL null mutant of Pseudomonas putida.

A Pseudomonas putida oprL null mutant was generated with reverse genetics by using an in vitro-truncated oprL::xylE construct and in vivo allelic exchange. The nature of the mutation introduced in P. putida was confirmed by Southern blotting. Western blots (immunoblots) of peptidoglycan-associated proteins revealed that the OprL protein was not made in the mutant strain, whereas it was detectable as a 19-kDa band in protein preparations of the wild-type strain. The P. putida oprL, mutant exhibited altered cell morphology as revealed by electron microscopy and was more sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA than the wild-type strain. The oprL gene was conserved in a wide variety of the Pseudomonas strains belonging to rRNA group I, which suggests that this gene is important for the maintenance of the cell envelope and cell morphology in this group of microorganisms.     

Cloning, sequencing, and expression of the Pseudomonas putida protocatechuate 3,4-dioxygenase genes.

The genes that encode the alpha and beta subunits of protocatechuate 3,4-dioxygenase (3,4-PCD [EC 1.13.11.3]) were cloned from a Pseudomonas putida (formerly P. aeruginosa) (ATCC 23975) genomic library prepared in lambda phage. Plaques were screened by hybridization with degenerate oligonucleotides designed using known amino acid sequences. A 1.5-kb SmaI fragment from a 15-kb primary clone was subcloned, sequenced, and shown to contain two successive open reading frames, designated pcaH and pcaG, corresponding to the beta and alpha subunits, respectively, of 3,4-PCD. The amino acid sequences deduced from pcaHG matched the chemically determined sequence of 3,4-PCD in all except three positions. Cloning of pcaHG into broad-host-range expression vector pKMY319 allowed high levels of expression in P. putida strains, as well as in Proteus mirabilis after specific induction of the plasmid-encoded nahG promoter with salicylate. The recombinant enzyme was purified and crystallized from P. mirabilis, which lacks an endogenous 3,4-PCD. The physical, spectroscopic, and kinetic properties of the recombinant enzyme were indistinguishable from those of the wild-type enzyme. Moreover, the same transient enzyme intermediates were formed during the catalytic cycle. These studies establish the methodology which will allow mechanistic investigations to be pursued through site-directed mutagenesis of P. putida 3,4-PCD, the only aromatic ring-cleaving dioxygenase for which the three-dimensional structure is known.