Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.     

Hydrogen production by Escherichia coli containing a cloned hydrogenase gene from Citrobacter freundii

Two hydrogenase genes of Citrobacter freundii complementing different Escherichia coli hyd mutations were cloned on the multicopy-plasmid pBR322. Recombinant plasmids pCBH2 and pCFH1 were obtained. Since hydrogenase activities of E. coli transformant HK-8 (pCBH2) and HK-7 (pCFH1) were much the same as E. coli C600 (wild type cells), the reduction in DNA size of recombinant plasmid pCBH2 (10.7 kb) was investigated. Reduced recombinant plasmids pCBH4 (6.2 kb) and pCBH6 (5.7 kb) were obtained, and a hydrogenase gene was found to be located on the 2.35 kb fragment between AvaI and EcoRI sites. Hydrogenase activity and hydrogen-evolving activity of E. coli HK-8 (pCBH4 or pCBH6) from sodium formate, sodium pyruvate or glucose were approximately 2-fold higher than those of E. coli C600 (wild type cells).On the other hand, a reduced recombinant plasmid pCBH10 (6.0 kb), which contained the adjacent DNA fragment (2.15 kb) to a hydrogenase gene, was obtained. Hydrogenase activity of E. coli C600 harboring pCBH10 was half that of E. coli C600. From these results we estimate that in plasmid pCBH2, the repressor gene suppressing the synthesis of hydrogenase might have been cloned together with a hydrogenase gene.     

Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis

Summary Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNA originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.Abbreviations kop kilobase pairs - OcTase ornithine carbamoyl transferase - CPSase carbamoyl phosphate synthetase     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Cloning and expression of R-factor mediated arsenate resistance in Escherichia coli

Summary The resistance transfer factor R773 confers inducible arsenate, arsenite and antimony resistance on Escherichia coli. The genes for these resistances were cloned into the EcoRi site of plasmid pBR322 to produce a 33 kilobase plasmid, pUM1. Bacterial strains transformed with pUM1 synthesized a polypeptide of the apparent molecular weight 64,000 daltons when induced with arsenite. This polypeptide could be visualized on sodium dodecyl sulfate polyacrylamide gels stained with Coomassie blue. It was observed both in the membrane and cytosol fractions but not among the periplasmic proteins present in osmotic shock fluid. Minicells isolated from strain JR410(pUM1) incorporated [³⁵S]methionine into an inducible 64,000 dalton polypeptide, as demonstrated on autoradiographs of electrophoresed [³⁵S]-labeled minicell lysates, confirming that this polypeptide is a plasmid gene product. A 4.3 kilobase HindIII fragment of pUM1 was subcloned into the HindIII site of pBR322, producing recombinant plasmid pUM3. This plasmid conferred constitutive resistance to arsenite and arsenate. Extensive synthesis of two polypeptides of 64,000 and 16,000 daltons was observed both in Coomassie stained gels of whole cells and autoradiographs of gels of [³⁵S]methionine-labeled minicells. Synthesis of both polypeptides was constitutive.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

The cloning of Aspergillus nidulans mitochondrial DNA in Escherichia coli on plasmid pBR322

Summary We report the cloning of almost the entire mitochondrial DNA of Aspergillus nidulans on plasmid pBR322 in Escherichia coli. Only one fragment containing about 11% of the mitochondrial genome has not been cloned. We believe that this fragment cannot be maintained in E. coli on the pBR322 plasmid.     

DNA-DNA hybridization analysis of nylon oligomer-degradative plasmid pOAD2: identification of the DNA region analogous to the nylon oligomer degradation gene.

Fine structure of the gene of 6-aminohexanoic acid cyclic dimer hydrolase, one of the enzymes responsible for the degradation of the nylon oligomer (6-aminohexanoic acid cyclic dimer), on the plasmid pOAD2 harbored in Flavobacterium sp. KI72 was determined by constructing miniplasmids from plasmid pNDH5 (a hybrid plasmid consisting of pBR322 and a 9.1-kilobase-pair HindIII fragment of pOAD2 ). The 6-aminohexanoic acid cyclic dimer hydrolase produced by cells of Escherichia coli C600 harboring pNDH5 or its miniplasmid was examined immunologically and electrophoretically and was found to be identical to that of Flavobacterium sp. KI72 . A fragment of pOAD2 (17.2- to 19.1-kilobase-pair region on pOAD2 ) was detected as hybridized fragment by Southern blotting experiments, indicating the presence of the DNA region analogous to the 6-aminohexanoic acid cyclic dimer hydrolase gene on the plasmid.     

Cloning of a gene for maltohexaose producing amylase of an alkalophilic Bacillus and hyper-production of the enzyme in Bacillus subtilis cells

Summary The gene for maltohexaose producing amylase from an alkalophilic bacterium, Bacillus sp. # 707, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a Bacillus subtilis plasmid pUB110, designated the resulting plasmids as pTUE306 and pTUB812, respectively. A common DNA region of approximately 2.5 kb was defined among the inserted DNAs. The enzymatic activity was lost when a part of the common region was deleted. The plasmids were stably maintained and the gene was well expressed in the bacterium, B. subtilis[pTUB812] which produced more than 70 times higher activity in the culture medium than did Bacillus sp. # 707. The major product of hydrolysis of starch by the enzymes of B. subtilis[pTUB812] and E. coli[pTUE306] was maltohexaose. The cloned gene corresponded to one of the genes for five components of malto-oligosaccharide-producing amylases of Bacillus sp. # 707.Abbreviations G1, G2, G3, G4, G5, and G6 glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose, respectively - kb kilobase pairs - kdal kilodalton - [] designated plasmid-carrier state     

Stability of the hybrid plasmid pIM138 and its curing by some eliminating agents

Hybrid plasmid pIM138 was constructed by insertion of a chromosomal fragment with the threonme operon fromEscherichia coli into the pBR322 vector. Molar mass of pIM138 was 2.8 Mg/mol. Heteroduplexes between pBR322 vector and pIM138 hybrid DNA molecules were prepared. The hybrid plasmid shows a high stability against the curing effect of rifampicin and clorobiocm inE. coli SK1590thr host.     

Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli

The closely linked structural genes of phosphofructokinase (pfkA) and triosephosphate isomerase (tpi) of Escherichia coli were separately cloned onto plasmid pBR322. By gene dosage effects, transformed cells of E. coli C600 with these pBR322 hybrid plasmids showed 7- and 16-fold increases in the specific activities of phosphofructokinase and triosephosphate isomerase, respectively, over the specific activities in C600. Dried preparations of E. coli cells dosed with these genes showed appreciably high ATP-regenerating activity.     

Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans

Summary A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli. The fungal fragment on pBR322, designated pHK29, complements a corresponding E. coli dehydroquinase structural gene (aroD) mutation. pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself. The biosynthetic dehydroquinase activity extracted from E. coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus. The protein specified by pHK29 appears to be 80 Kd. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E. coli.Standard Abbreviations Used SSC Standard saline citrate (3 M Sodium Chloride, 0.15 M Sodium citrate) - EDTA Ethylenediaminetetra acetic acid - DTT Dithiothreitol - PMSF Phenyl methyl sulphonyl fluoride - TEMED N N N N, Tetramethylethylenediamine - Md Megadaltons - Kd Kilodaltons     

Glutathione production by Escherichia coli cells with hybrid plasmid containing tandemly polymerized genes for glutathione synthetase

Summary A DNA fragment containing the structural and promoter regions of glutathione synthetase (GSH II) gene (gsh II) from Escherichia coli B were polymerized. The dimeric and trimeric DNA fragments obtained were inserted into Bam HI site of vector plasmid pBR325 and the resulting hybrid plasmids were designated pGS401-02 and pGS401-03, respectively. The GSH II activity of E. coli cells with these hybrid plasmids increased depending on the number of the genes (gsh II) contained. To construct hybrid plasmids useful for glutathione production, another DNA fragment with a gene (gsh I) for -glutamylcysteine synthetase (GSH I) from E. coli B was inserted into Pst I sites of pGS401-02 and pGS401-03 and the hybrid plasmids obtained (pGS501-12 and pGS501-13, respectively) were introduced into E. coli B cells. Although the glutathione-producing activities of the cells with these plasmids were little improved as compared with that of cells with the hybrid plasmid (pGS501-11) containing both gsh I and gsh II because of the low activity of GSH I, our method has brought to light a new type of gene amplification.     

Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli

A pBR322-T.leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA. In the Escherichia coli host, this plasmid coded for the β-IPM dehy drogenase (product of leuB) activity, the optimal temperature of which was 80°C, suggesting that information on the thermostability of the enzyme lies in its structural gene. 10-day propagation of E. coli [pBR322-T.leu] at 37°C decreased the temperature optimum from 80°C to 75°C. This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37°C. Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E. coli.     

Cloning of an endoglucanase gene fromPseudomonas fluorescens var.cellulosa intoEscherichia coli andPseudomonas fluorescens

Summary An endoglucanase chromosomal gene from the cellulolyticPseudomonas fluorescens var.cellulosa (NCIB 10462) was cloned inEscherichia coli. Chromosomal DNA was partially digested with the restriction enzymeEcoRI and ligated into the broad host-range, mobilizable plasmid pSUP104 that had been linearized with the same enzyme. After transformation ofEscherichia coli, and endoglucanase-positive clone was detected in situ by use of the Congo-red assay procedure. The endoglucanase gene on the recombinant plasmid pRUCL 100 was expressed in the non-cellulolyticPseudomonas fluorescens PF41. The DNA fragment carrying the gene was transferred to the plasmid pBR322, generating plasmids pRUCL150 and pRUCL151, and its restriction map was derived.Abbreviations CMC carboxymethylcellulose - EG endoglucanase - kb kilobase pairs - Mops 4-morpholinepropanesulfonic acid - Ap^r-s resistance-sensitivity to the antibiotic ampicillin - Cm^r-s resistance-sensitivity to the antibiotic chloramphenicol - Tc^r-s resistance-sensitivity to the antibiotic tetracycline - Sm^r-s resistance-sensitivity to the antibiotic streptomycin - Tp^r-s resistance-sensitivity to the antibiotic trimethoprim     

Cloning and expression of Citrobacter freundii β-isopropylmalate dehydrogenase gene in both Escherichia coli and Bacillus subtilis

Summary -Isopropylmalate (IPM) dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis. Plasmid pCBL 1 containing C. freundii -IPM dehydrogenase gene was isolated using E. coli (leuB) as a host, pBR 322 as a vector and Hind III as an enzyme. The molecular weight (mol.wt.) of pCBL 1 was 7.7 megadalton (Md) and the plasmid was restricted at two sites by Hind III or Sal I, at three sites by BamH I and at four sites by Pst I. The second hybrid plasmid pCBL 2 containing -IPM dehydrogenase gene was reconstructed from 2.1 Md Pst I fragment of pCBL 1 and pBR 322. -IPM dehydrogenase activities of E. coli transformants with pCBL 1 or pCBL 2 were 2–7-fold higher than those of the present strains. The -IPM dehydrogenase gene was transferred from pBR 322 to pLS 353, a shuttle vector between E. coli and B. subtilis. The third plasmid, pCBL 3 (mol.wt. 5.6Md), was cloned in B. subtilis (leuC) and expressed the enzyme activity which complemented the Leucharacter. The enzyme activities of B. subtilis transformants with pCBL 3 were about 5-fold higher than those of present strains. Thus, the C. freundii gene was effectively expressed in both E. coli and B. subtilis.     

Replication of plasmid chimera pBR-mtB-A containing rat liver mtDNA fragment in Escherichia coli polA cells

Escherichia coli K-12 strains p108 (polA6), p3478 (polA1), and KS55 (polA12, ts) deficient in DNA polymerase I were transformed by recombinant pBR-mtB-A plasmid containing BamHI-A fragment of rat liver mtDNA and pBR322 plasmid. The physical map of the pBR-mtB-A, containing the recognition sites for SalI, EcoRI and HinIII endonucleases, was constructed and the orientation of mtDNA fragment joined to pBR322 plasmid was studied. The phenotypic selection using ampicillin containing medium at permissive and nonpermissive temperature (KS55 strain), or at 37 °C (polA6 and polA1 strains) revealed that only the cells transformed with the hybrid plasmid are able to grow under these conditions. The presence of mtDNA insertions in chimeric DNA molecules of pBR-mtB-A in polA strains was proved by electrophoretic and hybridization analysis. Thus the results obtained demonstrate the replication of the vehicle containing both plasmid replicon and mitochondrial origin in the conditions nonpermissive for the stable reproduction of the plasmid DNA alone.