Purification and characterization of lysophospholipase L2 of Escherichia coli K-12

Lysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene. Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and heparin-Sepharose affinity column chromatography. The final preparation showed a single protein band with a molecular weight of 38,500 daltons in SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined. It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB [Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1017-1025.] The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids. The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol. The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative. This enzyme was heat-labile and was inactivated at 55 degrees C within 5 min. The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E. coli K-12 homogenates [Doi, O. & Nojima, S. (1975) J. Biol. Chem. 250, 5208-5214].     

Nucleotide sequence of the asnA gene coding for asparagine synthetase of E. coli K-12.

We have subcloned the asnA gene of E. coli K-12, a gene coding for asparagine synthetase, from a previously cloned 6 mega-dalton segment of E. coli chromosome containing the DNA replication origin, ori, and asnA. The complete nucleotide sequence of the asnA gene was determined: the region of the structural gene extends 990 base-pairs at nucleotide positions 1434-2423 (see Fig. 3), which codes for a polypeptide of 330 amino-acid residues with a molecular weight of 36,688 daltons. The nucleotide sequences of the promoter and the ribosome-binding site of the gene are also assigned. We discuss the properties of its polypeptide.     

Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12.

The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon. The gene was cloned into the pUC13 vector. Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase. Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.     

Molecular cloning and DNA sequencing of the radC gene of Escherichia coli K-12.

The radC102 mutation that sensitizes E. coli K-12 cells to ultraviolet light, ionizing radiations and alkylating agents was localized between the fpg and pyrE genes at 81.7 min on the bacterial chromosome. E. coli strain BH20 (radC+, fpg-1::KnR) has a 10.5-kb EcoRI/KpnI DNA fragment spanning the region from pyrE to the insertion mutation fpg-1::KnR. The proximity of the radC gene to this insertion mutation provided a strategy to isolate the radC+ gene based on the cloning of radC+ and fpg-1::KnR on the same DNA fragment using the resistance to kanamycin as a selector. A library of EcoRI/KpnI DNA fragments of E. coli strain BH20 was inserted into pUC19. One recombinant plasmid conferring resistance to kanamycin was selected and named pRCV10. The pRCV10 plasmid partially restores the resistance to UV-radiation when transformed into SR1187 (radC102), but sensitizes the wild-type strain to the same treatment. The radC102 complementing region was localized on a 1.2-kb BglII/BglII DNA fragment which was sequenced. The DNA sequence complementing the radC102 mutation contained an ATG translation start codon with an open reading frame of 297 base pairs which encodes a polypeptide of Mr 11,500. The order of the genes in this region of the E. coli chromosome is: fpg--rpmBG--radC--pyrE.     

Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12.

Melibiose uptake and hydrolysis in E.coli is performed by the MelB and MelA proteins, respectively. We report the cloning and sequencing of the melA gene. The nucleotide sequence data showed that melA codes for a 450 amino acid long protein with a molecular weight of 50.6 kd. The sequence data also supported the assumption that the mel locus forms an operon with melA in proximal position. A comparison of MelA with alpha-galactosidase proteins from yeast and human origin showed that these proteins have only limited homology, the yeast and human proteins being more related. However, regions common to all three proteins were found indicating sequences that might comprise the active site of alpha-galactosidase.     

Molecular cloning of the N-acetylneuraminate lyase gene in Escherichia coli K-12

Summary The gene for N-acetylneuraminate lyase [N-acetylneuraminate pyruvate-lyase; NPL] of Escherichia coli C600 was cloned onto pBR322 as a 9.8 kilobase HindIII fragment of chromosomal DNA and the hybrid plasmid was designated pMK2. The gene in the hybrid plasmid was subcloned in pBR322 as a 1.2 kilobase HindIII-EcoRI fragment and the resultant hybrid plasmid was designated pMK6. NPL activity level was increased more than 5-fold in the pMK6-bearing strain compared with that of the wild type, when the cells were grown on a medium containing inducer (N-acetylneuraminate: NANA). The transformants harbouring pMK6 also showed higher activity even in the absence of inducer. The NPL produced by pMK6-bearing cells was structurally and immunologically the same as that purified from E. coli C600.     

Fertility-deficient E. coli K-12 mutants

Summary Fertility-deficient and f2-r mutants of E. coli K-12 were studied. The above mutants were isolated following the nitrogen-mustard treatment of the E. coli K-12 Hfr and E. coli F ¹ lac ⁺-strains. Isolation of these mutants from F ¹-strains showed that mutations occur in the F-factor no matter whether it was in autonomous state or integrated in a chromosome.The existence of mutants of two types, fertility-deficient and f2-r, was demonstrated. Type I mutants were characterized by the maintenance of a low level phage f2-adsorption activity and by a 10-fold decrease of their fertility as compared with the original strain when crossed in the liquid medium. In crosses in solid media the recombination frequency in the case of type I mutants used as donor was the same as with the original strain. Type II mutants were characterized by the entrie loss of their f2-phage adsorption ability, by a 1,000-fold decrease of fertility in liquid media, and by the inability to recombinate in solid media.     

Nucleotide sequence of the CytR regulatory gene of E. coli K-12.

We have determined the nucleotide sequence of the cytR gene, which codes for the Cyt repressor (CytR). The coding region consists of 1023 or 1029 bp. The subunits of CytR are thus predicted to consist of 341 or 343 residues. It is shown that the N-terminal segment of the polypeptide is structurally similar to the DNA-binding region of known DNA-binding proteins. In addition, there exists an exceptionally high amino acid sequence homology between CytR and the Gal repressor, indicating a common origin of evolution.     

Molecular cloning and expression in E. coli of a yeast gene coding for beta-galactosidase.

The yeast Kluyveromyces lactis synthesizes a beta-galactosidase (EC 3.2.1.32) which is inducible by lactose. We have isolated the gene that codes for this enzyme using recombinant DNA techniques. K. lactis DNA was partially digested with the restriction endonuclease Eco R1 and joined to Eco R1-digested pBR322 plasmid DNA using DNA ligase. ligase. A lac-mutant of Escherichia coli lacking the structural gene for beta-galactosidase was transformed with ligated DNA. Three lac+ transformants containing recombinant plasmids were selected. Two of the plasmids (pK15 and pK17) contain four Eco R1-K. lactis DNA fragments having molecular weights of 2.2, 1.4, 0.55 and 0.5 x 10(6) daltons. The other plasmid (pK16) lacks the smallest fragment. E. coli carrying any of these plasmids produce beta-galactosidase activity that has a sedimentation coefficient and immunological determinants that are nearly identical to K. lactis beta-galactosidase and distinctly different from E. coli beta-galactosidase. DNA-DNA hybridization studies show that the four Eco R1 fragments in pK15 hybridize to K. lactis but not to E. coli DNA.     

L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene.

A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by lambda plac Mu insertion and shown to be deficient in L-serine deaminase activity. The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced. The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase. However, other possibilities are also considered. No significant homology with previously reported DNA or protein sequences was detected.     

Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12.

A 1650 base pair (BP) fragment carrying the entire argF structural gene with its associated control regions was isolated from an EcoRI/BamHI digest of phi80argFilambda cI857 DNA. This segment was cloned using the EcoRI and BamHI cleavage sites in the plasmid pBR322. A preliminary restriction map of the argF region was prepared. RNA polymerase binding studies indicated that the argF promoter is located approx. 30 base pairs from the EcoRI terminus of the cloned DNA segment.     

Molecular cloning and physical mapping of the hyd gene of Escherichia coli K-12

Abstract Passive transfer between rates of protection against cholera toxin (CT) was studied. Extracts of various organs, obtained from CT-immunized rats, were injected intravenously into non-immunized recipient rats. The ability of the extracts to inhibit CT-induced secretion in ligated jejunal loop were tested. A significant inhibition of the response to CT was achieved by extracts from hypophysis, brain and jejunal mucosa. Extracts from pancreas, spleen or adrenal glands were without effect, as were all extracts obtained from control rats. The antisecretory effects of the hypophysis extracts became intensified with increasing numbers of immunizations, and the antisecretory effect was most pronounced when the extract was injected immediately before the CT challenge. The active component of the hypophysis extract was heat-labile and negatively charged, suggesting an acidic protein as the mediator of the protective effect against CT.     

Molecular cloning of pheR in Escherichia coli K-12.

The regulator gene pheR, which in Escherichia coli controls the expression of pheA, the structural gene for chorismate mutase P-prephenate dehydratase, was cloned on to multicopy plasmids directly from the E. coli chromosome; this was achieved with the aid of the tetracycline resistance transposon, Tn10, that had been inserted very close to the pheR gene. Subsequently, pheR was subcloned on a 1.1-kilobase-pair fragment on the plasmid vector pBR322; its position on the plasmid was localized by the method of gamma delta-mediated transpositional inactivation. The pheR gene product was identified in maxicells and found to be a protein of subunit molecular weight 19,000, suggesting that the coding segment of the gene is about 500 nucleotide pairs long.     

大肠杆菌苯丙氨酸合成途径关键酶基因pheA的克隆与表达

为了通过基因工程手段来增加苯丙氨酸的生物产量,利用ＰＣＲ方法从大肠杆菌中克隆了抗反馈抑制突变型及野生型的ｐｈｅＡ基因,进行了核苷酸序列分析,并利用高效的原核表达载体ＰＢＶ２２０对ｐｈｅＡ基因编码的突变型及野生型分支酸变位酶／预苯酸脱水酶（ＣＭ／ＰＤ）进行了表达。序列分析表明突变型基因碱基第５８０位由Ｔ变为Ｃ,相应氨基酸由Ｖａｌ变为Ａｌａ,ＳＤＳ－ＰＡＧＥ图谱扫描分析表明目的蛋白ＣＭ／ＰＤ的表达量占全菌体蛋白的４３％,占上清总蛋白的５７％。酶活性测定表明其ＣＭ和 ＰＤ活性分别提高了 １５．５和６．７倍,产酸量也有了一定的提高,为构建产苯丙氨酸的生物工程菌奠定了基础。