Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida

Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source. Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism. We cloned from a HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of camA gene encoding NADH-putidaredoxin reductase and the entire camB gene encoding putidaredoxin. The 2.9 kbp CAM segment was adjacent to the 4.27 kbp HindIII CAM segment which has been previously cloned (Koga et al. (1986) J. Bacteriol. 166, 1089-1095). Thus, the total 7.17 kbp HindIII CAM directed all the genes responsible for early steps of D-camphor degradation, i.e. 5-exo-hydroxycamphor dehydrogenase (camD gene), cytochrome P-450cam (camC), NADH-putidaredoxin reductase (camA), and putidaredoxin (camB). These cam genes form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene. The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin.     

P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli

The gene camC, which encodes the cytochrome P450 monoxygenase protein, was cloned into the shuttle vector pKT240 and recovered as the recombinant pKG201 with a 2.3 kb insert from the CAM plasmid in the PstI site. The gene product is expressed constitutively in P. putida and in E. coli whereas the inverted insert clone lacks expression, indicating absence of an insert promoter.     

Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.

The regulatory gene camR on the CAM plasmid of Pseudomonas putida (ATCC 17453) negatively controls expression of the cytochrome P-450cam hydroxylase operon (camDCAB) for the camphor degradation pathway and is oriented in a direction opposite to that of the camDCAB operon. In this study, we examined expression of the camR gene by monitoring the beta-galactosidase activity of camR-lacZ translational fusions in P. putida camR and camR+ strains. We found that the camR gene was autogenously regulated by its own product, CamR. To search for an operator site of the camR gene, a cam repressor (CamR)-overproducing plasmid, pHAOV1, was constructed by placing the camR gene under the control of a pL promoter. The translational initiation codon of CamR was changed by site-directed mutagenesis from GTG to ATG to improve translation efficiency. Judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the CamR protein was expressed up to about 10% of the soluble protein of CamR-overproducing Escherichia coli JM83/pHAOV1 cells. Results of DNase I footprinting assays using the cell lysate indicated that the CamR repressor covered a single region between the camR gene and the camDCAB operon. Our findings also suggest that the camR gene autogenously regulates its own expression by binding of the gene product, CamR, to the operator, which also serves as an operator of the camDCAB operon.     

Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants.

The gln-gamma gene, which specifies the gamma subunit of glutamine synthetase in Phaseolus vulgaris L., has been isolated and the regulatory properties of its promoter region analyzed in transgenic Lotus corniculatus plants. A 2-kilobase fragment from the 5'-flanking region of gln-gamma conferred a strongly nodule-enhanced pattern of expression on the beta-glucuronidase reporter gene. Parallel studies on the promoter of another glutamine synthetase gene (gln-beta) showed that a 1.7-kilobase fragment directed 20-fold to 140-fold higher levels of beta-glucuronidase expression in roots than in shoots. Histochemical localization of beta-glucuronidase activity in nodules of the transgenic plants indicated that the chimeric gln-gamma gene was expressed specifically in the rhizobially infected cells; expression of the gln-beta construct was detected in both cortical and infected regions of young nodules, and became restricted to the vascular tissue as the nodule matured. We conclude that gln-beta and gln-gamma genes are differentially expressed both temporally and spatially in plant development and that the cis-acting regulatory elements responsible for conferring these contrasting expression patterns are located within a 2-kilobase region upstream of their coding sequences.     

Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein

Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12-kilobase insert. The malS containing portion was subcloned into pBR322 as a 4-kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose-dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase.     

Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli.

A 13.6-kilobase (kb) Sau3AI restriction endonuclease fragment of Clostridium acetobutylicum DNA cloned into pBR322 enabled Escherichia coli ato mutants to grow on butyrate as a sole carbon source (But+). Complementation of the ato defect by the recombinant plasmid pJC6 was due to expression of the genes for phosphotransbutyrylase (PTB) and butyrate kinase (BK). Both genes were efficiently expressed in E. coli, as their products were readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. PTB was found to have a polypeptide subunit molecular weight of approximately 31,000, while that of BK was approximately 39,000. Deletion analysis and Tn5 mutagenesis of plasmid pJC7 (a But+ subclone containing a 4.4-kb BamHI fragment from the insert of pJC6) localized the PTB and BK genes within a region spanning approximately 2.9 kb. Preliminary evidence suggests that the two genes may form an operon that is transcribed as a single unit from a promoter of clostridial origin within the 4.4-kb insert of pJC7.     

Isolation and characterization of two types of cDNA for mitochondrial adenylate kinase and their expression in Escherichia coli

Two cDNA clones for mitochondrial adenylate kinase were isolated from a cDNA library of bovine liver poly(A)+ RNA by using synthetic oligodeoxynucleotides as probes. The clone containing a 0.9-kilobase insert had the reading frame for a 241-residue protein (AK2A), while the other clone containing a 1.6-kilobase insert had the frame for a 234-residue protein (AK2B). Nucleotide sequences of these two clones were the same in the 5' portion up to the coding sequence for the 233rd residue, but different in the remaining 3' portions. The reported amino acid sequence of mitochondrial adenylate kinase from bovine heart corresponded to AK2A. Neither AK2A nor AK2B had a cleavable NH2-terminal presequence as that found in other imported mitochondrial proteins. RNA blot analysis of poly(A)+ RNAs from bovine liver and heart revealed three species of mRNA with approximate sizes of 0.9, 1.4, and 1.7 kilobases. The 1.7- and 1.4-kilobase species were specific for AK2B, whereas the 0.9-kilobase species was specific for AK2A. In the liver, the 1.7-kilobase mRNA was more abundant, whereas in the heart the 0.9-kilobase mRNA was predominant. The 1.4-kilobase mRNA was present only in the heart. The AK2A- and AK2B-coding sequences were expressed in Escherichia coli cells under the control of trc promoter. Both the products reverted the temperature-sensitive phenotype of the adenylate kinase mutant of E. coli.     

Molecular cloning of nif DNA from Azotobacter vinelandii.

Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobacter vinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled 6.2-kilobase (kb) nif insert from pSA30 (which contains the Klebsiella pneumoniae nifK, nifD, and nifH genes) as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showed that the 2.6-kb insert from one of the plasmids (pLB1) contains nifK whereas the 1.4-kb insert from the other plasmid (pLB3) contains nifD. Marker rescue tests using genetic transformation indicated that the 2.6-kb A. vinelandii nif fragment contains the wild-type alleles for the nif-6 and nif-38 mutations carried by Nif- strains UW6 and UW38. The 1.4-kb insert contains the wild-type allele for the nif-10 mutation carried by Nif- strain UW10.     

Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma.

Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DR1 region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNAs, including the human autonomously replicating sequence 1 (ARS1).     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region.

Genes coding for three xylan-degrading activities, xylanase, xylosidase, and arabinosidase, were simultaneously cloned from the colonic anaerobic organism Bacteriodes ovatus. The genes for the three enzymes were located on a 3.8-kilobase EcoRI genomic insert and were cloned by using plasmid pUC18. All three activities were expressed in Escherichia coli JM83, and all were cell associated. Expression of the xylanase gene was independent from expression of the xylosidase and arabinosidase genes, whereas expression of the latter two genes appeared to be coordinated. Restriction endonuclease analysis of the arabinosidase and xylosidase genes and partial purification of these enzyme activities from E. coli suggested that these activities were catalyzed by a bifunctional protein or two proteins of very similar molecular weight. All three enzyme activities were regulated in B. ovatus in response to the carbon source used for growth. This is the first report of the cloning and expression of B. ovatus genes.     

Spontaneous deletion of a 20-kilobase DNA segment carrying genes specifying isopropylbenzene metabolism in Pseudomonas putida RE204.

The genes encoding isopropylbenzene metabolism in Pseudomonas putida RE204 are readily lost in two ways: by loss (curing) of plasmid pRE4 which specifies the catabolic pathway and by deletion from pRE4 of an approximately 20-kilobase segment of DNA carrying the catabolic genes. The presence of DNA sequences at the ends of the catabolic gene region sharing homology with one another suggests that the deletions result from recombination events between these homologous sequences.     

Control of Reproductive Floral Organ Identity Specification in Arabidopsis by the C Function Regulator AGAMOUS

The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities.     

Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS.

The xylDEGF operon and the regulatory gene xylS of the TOL plasmid found in Pseudomonas putida mt-2 were cloned onto Escherichia coli vector plasmids. A 9.5-kilobase fragment, derived from the TOL segment of pTN2 deoxyribonucleic acid, carried the xyl genes D, E, G, and F, which encode toluate oxygenase, catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase, respectively. The enzymes were noninducible unless a 3-kilobase PstI fragment, derived also from the TOL segment, was provided in either cis or trans. The PstI fragment appeared to contain the regulatory gene xylS, which produced a positive regulator. The regulator was activated by m-toluate or benzoate, but not by m-xylene or m-methylbenzyl alcohol. the map positions of xylG and xylF were also determined.     

Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.     

Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.