Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase.

A 2,598-base-pair (bp) SalI-HincII DNA fragment has been cloned which codes for vanillate demethylase, the enzyme responsible for the demethylation of vanillate (3-methoxy-4-hydroxybenzoate) to protocatechuate (3,4-dihydroxybenzoate). Complementation and insertional inactivation experiments have shown that this fragment carries two genes (vanA and vanB) which are predominantly cotranscribed from a promoter upstream of vanA. Nucleotide sequencing of the SalI-HincII fragment confirmed the genetic data: two open reading frames of 987 and 942 bp were present in the transcribed orientation. These had a very high G + C content in the third base of each codon, which is characteristic of Pseudomonas chromosomal genes. Expression of the genes in Escherichia coli with the T7 RNA polymerase-promoter system gave rise to two polypeptides of 36 and 33 kilodaltons which could be identified by deletion analysis as the products of vanA and vanB, respectively. A search of the protein sequence data bank indicated that the vanB gene product was related to the ferredoxin family.     

Cloning and sequencing of cDNAs encoding two SSS

A defective S-allele, S ₀, and a functional S-allele, S _x, have previously been found to be retained in an F₁ hybrid of a self-compatible commercial cultivar of Petunia hybrida. Pistil proteins associated with these two alleles have also been identified. Their amino-terminal sequences have been found to share a high degree of similarity with those of S-proteins characterized from self-incompatible solanaceous species. Here we report the isolation and sequencing of cDNAs encoding S ₀- and S _x-proteins. Their deduced amino acid sequences contain all the consensus primary structural features of S-proteins from self-incompatible solanaceous species. Both proteins also have ribonuclease activity. The implications of these findings are discussed in relation to the presumed function of the S-protein in the self-incompatibility interaction.     

Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida.

The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed.     

Cloning and sequencing of rpoH and identification of ftsE-ftsX in Pseudomonas putida PpG1.

The rpoH gene encoding the heat-shock sigma factor of Pseudomonas putida was cloned by using its ability to complement the temperature-sensitive growth of the Escherichia coli rpoH mutant. The cloned DNA contained an open reading frame for a 284 amino acid sequence exhibiting high homology to the sigmaH proteins of P. aeruginosa and E. coli. Moreover, homologs to the cell division genes ftsX and ftsE were found immediately upstream of the rpoH gene.     

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.     

Genes encoding bacterial RNA-polymerase. I. Cloning of rpoBC-operon of Pseudomonas putida and its physical map

The P. putida rpoBC operon, coding for beta and beta' subunits of RNA polymerase, was cloned and its physical map constructed.     

Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2.

The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grown on dihydrogen-fumarate medium and also produced very low levels of HYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3. The hyb operon was mapped at 65 min on the E. coli chromosome.     

Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis

The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced. The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively. Sequence comparisons at the amino acid level to the B. subtilis degS-degU genes showed 74% and 84% similarity, respectively. On a multicopy vector the B. brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B. subtilis rec^– strain as well as in a B. subtilis sacU(HY) strain.     

Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa

The cytochrome c-551 gene from Pseudomonas aeruginosa was cloned by using two oligonucleotide probes, which had been synthesized based on the known primary structure of the protein. The restriction map of the cloned DNA and sequence analysis showed that the cytochrome c-551 gene is located 50 bp downstream of the nitrite reductase gene, which has recently been cloned and sequenced. DNA sequence analysis also indicated that cytochrome c-551 is synthesized in vivo as a precursor having an amino-terminal signal sequence consisting of 22 amino acid residues.     

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.     

Cloning and sequencing of partial segment of cholesterol oxidase encoding gene fromStreptomyces luridus

Cloning, sequencing and analysis of partial segment of cholesterol oxidase(cho) encoding gene fromStreptomyces luridus using PCR technique is described. The primers used for PCR were designed on the basis of inter-species homology, and the pTZ57R/T vector was used for molecular cloning. The sequencing results lead to identification of the oxidoreductase domain ofcho enzyme with 818 nucleotide length encoding 272 amino acids, which was submitted in NCBI with DQ480365 accession number. The submitted sequence in cludes complete FAD-linked reductase C terminal domain with 170 amino acids size and N-terminal of GMC-oxidoreductase domain ofcho enzyme.     

Comparison of two dioxygenases from Pseudomonas putida.

Catechol 2,3-dioxygenase and homoprotocatechuate 2,3-dioxygenase were purified from the same strain of Pseudomonas putida. Molecular weights and subunit sizes were similar, but amino acid compositions showed some marked differences.     

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.     

Structure of the Pseudomonas putida alkBAC operon. Identification of transcription and translation products

The structural genes of the Pseudomonas oleovorans alk (alkane utilization) system, which are localized on the alkBAC operon, were cloned as a 16.9-kilobase pair EcoRI fragment. We have measured the length and determined the position of the alkBAC operon on this fragment by electron microscopy of R-loops. Furthermore, the 7.3-kilobase pair long alkBAC operon was analyzed for translation products in Escherichia coli minicells. Using a spectrum of overlapping subclones, six different proteins were identified. Starting from the alkBAC promotor, these polypeptides had molecular masses of 41, 15, 49, 58, 59, and 20 kDa, respectively. The 41-kDa protein was identified as alkane hydroxylase by reaction with a specific antibody. The 15- and 49-kDa peptides are soluble components of the alkane hydroxylase complex. The 58-kDa protein is most likely involved in alkanol dehydrogenase activity.     

Characterization of copABCD operon from a copper-sensitive Pseudomonas putida strain

We describe an operon, copABCD, that encodes copper-binding and sequestering proteins for copper homeostasis in the copper-sensitive strain Pseudomonas putida PNL-MK25. This is the second operon characterized as being involved in copper homeostasis, in addition to a P1-type ATPase encoded by cueAR, which was previously shown to be active in the same strain. In this study, 3 copper-responsive mutants were obtained through mini-Tn5::gfp mutagenesis and were found to exhibit reduced tolerance to copper. Sequencing analysis of the transposon-tagged region in the 3 mutants revealed insertions in 2 genes of an operon homologous to the copABCD of P. syringae and pcoABCD of Escherichia coli. Gene expression studies demonstrated that the P. putida copABCD is inducible starting from 3 micromol/L copper levels. Copper-sensitivity studies revealed that the tolerance of the mutant strains was reduced only marginally (only 0.16-fold) in comparison to a 6-fold reduced tolerance of the cueAR mutant. Thus, the cop operon in this strain has a minimal role when compared with its role both in other copper-resistant strains, such as P. syringae pv. syringae, and in the cueAR operon of the same strain. We propose that the reduced function of the copABCD operon is likely to be due to the presence of fewer metal-binding domains in the encoded proteins.     

Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida.

The structural genes for the flavoprotein subunit and cytochrome c subunit of p-cresol (4-methylphenol) methylhydroxylase (PCMH) from Pseudomonas putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland) and P. putida NCIMB 9866 were cloned and sequenced. The genes from P.putida NCIMB 9869 were for the plasmid-encoded A form of PCMH, and the genes from P.putida NCIMB 9866 were also plasmid encoded. The nucleotide sequences of the two flavoprotein genes from P.putida NCIMB 9869 and P.putida NCIMB 9866 (pchF69A and pchF66, respectively) were the same except for 5 bases out of 1,584, and the translated amino acid sequences were identical. The nucleotide sequences of the genes for the cytochrome subunits of PCMH from the two bacteria (pchC69A and pchC66) varied by a single nucleotide in their 303-base sequences, and the translated amino acid sequences differed by a single residue at position 41 (Asp in PchC69A and Ala in PchC66). Both cytochromes had 21-residue signal sequences, as expected for periplasmic proteins, and these sequences were identical. On the other hand, no signal sequences were found for the flavoproteins.pchF69A and pchC69A were expressed, separately or together, in Escherichia coli JM109 and P.putida RA4007, with active PCMH produced in both bacteria. The E. coli-expressed flavocytochrome was purified. Our studies indicated that the E.coli-expressed subunits were identical to the subunits expressed in P.putida NCIMB 9869: molecular weights, isoelectric points, UV-visible spectra, and steady-state kinetic parameters were the same for the two sets of proteins. The subunits readily associated upon mixing two crude extracts of E.coli, one extract containing PchC69A and the other containing PchF69A. The courses of association of PchC69A and PchF69A were essentially identical for pure E. coli-expressed subunits and pure P. putida 9869-expressed subunits. E. coli-expressed PchC69A and PchF69A contained covalently bound heme and covalently bound flavin adenine dinucleotide, respectively, as the proteins expressed in nature.     

Cloning,nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816

We have cloned the naphthalene dioxygenase(ND)-coding genes from Pseudomonas putida strain NCIB9816 based on their ability to convert indole to indigo. The region coding for this enzyme activity was sequenced and three successive open reading frames were found. The corresponding gene products were identified using the T7 polymerase/promoter system. All of them are necessary for the ND activity. A comparison of the ND-coding genes with the ones coding for benzene dioxygenase revealed significant homology which was more pronounced at the nucleotide level than at the amino acid level.