Localization of a gene responsible for ozone sensitivity in Escherichia coli]

A gene, ozrC, responsible for sensitivity to ozone in Escherichia coli, was localized on the E. coli chromosome between argEH and metA by means of analysis of cotransduction frequencies of the gene ozrC with certain gene markers in the malB region of the chromosome.     

Disruption of the Escherichia coli cls gene responsible for cardiolipin synthesis

The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-1 mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally. The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cls mutants depended on the dosage of the pss gene, and attempts to transfer a null allele of the cls gene into a pss-1 mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.     

温度诱导Targetron系统用于大肠杆菌高效基因失活

构建基于Te I3c/4c嗜热二型内含子的温度诱导Targetron基因失活系统(Thermotargetron),并应用于中温微生物基因编辑。在大肠杆菌HMS174(DE3)基因组中,选择Subunitofflagellum基因(fliC)和C4dicarboxylate orotate:H+symporter基因(dctA)为靶基因。根据Te I3c/4c DNA识别规则,在fliC和dctA基因中选择fliC489a、fliC828s、fliC1038s和dctA2a位点为基因打靶位点。使用重叠延伸PCR方法,基于pHK-TT1A质粒构建打靶载体。打靶载体转化HMS174菌株,对数期转化子培养液48℃热激1h后涂布于氯霉素抗性LB平板上。使用菌落PCR和DNA测序检测突变株并计算基因失活效率。获得突变株后,通过琼脂穿刺和碳源代谢实验,鉴定ΔfliC、ΔdctA突变株表型变化。菌落PCR测序结果表明,Te I3c/4c插入到fliC和dctA基因设计位点,且打靶效率高达100%。突变株表型验证实验表明,ΔfliC突变株运动能力显著下降,ΔdctA突变株苹果酸代谢能力缺失。综上所述,...     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli.

The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region. The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.     

A new Escherichia coli cell division gene, ftsK.

A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer.     

Cloning of a gene responsible for the biosynthesis of glutathione in Escherichia coli B.

A gene (gshI) responsible for gamma-glutamylcysteine synthetase (GSH-I) activity was cloned to construct an Escherichia coli B strain having high glutathione synthesizing activity. For this purpose, two E. coli B mutants (strains C912 and RC912) were used. C912 was deficient in GSH-I activity. RC912, a revertant of C912, had a GSH-I activity that was desensitized to feedback inhibition of reduced glutathione. To clone gshI, chromosomal DNAs of RC912 and plasmid vector pBR322 were digested with various restriction endonucleases and then ligated with T4 DNA ligase. The whole ligation mixture was used to transform C912, and the transformants were selected as tetramethylthiuramdisulfide-resistant colonies. Of about 20 resistant colonies, 2 or 3 became red when treated with nitroprusside and showed appreciably high GSH-I activities. The chimeric plasmid DNA, designated pBR322-gshI, was isolated from the strain having the highest GSH-I activity and transformed into RC912. The structure and molecular size of pBR322-gshI in RC912 were determined. The molecular size of this plasmid was 6.2 megadaltons, and the plasmid contained a 3.4-megadalton segment derived from RC912 chromosomal DNA, which included gshI gene. The GSH-I activity of RC912 cells containing pBR322-gshI was fourfold higher than that of RC912 cells without pBR322-gshI.     

Identification of the dicyclohexylcarbodiimide-reactive protein component of the adenosine 5''-triphosphate energy-transducing system of Escherichia coli.

Membranes of Escherichia coli contain an adenosine 5'-triphosphate (ATP) energy-transducing system that is inhibited by treatment with dicyclohexylcarbodiimide (DCCD). The carbodiimide-reactive protein component of this system has been identified after treatment with [14C]DCCD. This protein has an apparent molecular weight of 9,000 as judged from acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is extracted from the membrane with chloroform-methanol (2:1). These properties are similar to the analogous protein previously identified in mitochondria (Cattell et al., 1971). A mutant strain, RF-7, has been isolated which derives energy from oxidative phosphorylation in the presence of 5 mM DCCD. The ATP hydrolase activity of the membraned system in the mutant was considerably less sensitive to inhibition by DCCD than that in the wild type. The carbodiimide-reactive protein, which was easily labeled by [14C]DCCD in the wild type, was labeled much less rapidly in the carbodiimide-resistant mutant. It is thus concluded that the reaction of DCCD with this specific protein leads to inhibition of the ATP energy-transducing reactions. The mutation causing carbodiimide resistance in strain RF-7 was mapped. It is cotransduced with the uncA gene at a frequency exceeding 90%. The mutationally altered protein causing the carbodiimide resistance was not conclusively identified. However, reconstitution experiments indicate that the altered protein is not one of the subunits of the soluble ATP hydrolase activity, which can be removed from the membrane by washing with 1 mM tris(hydroxymethyl)aminomethane buffer lacking Mg2+. The carbodiimide-reactive protein remains with the membrane residue after removal of the soluble ATP hydrolase and is thus distinct from these subunits as well.     

The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli

The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.     

A gene for DNA invertase and an invertible DNA in Escherichia coli K-12

An assay system for the pin gene function, which suppresses the vh2 mutation of Salmonella, was developed and used to show that most strains of Escherichia coli K-12 are Pin+, whereas all the strains of E. coli C examined are Pin-. An E. coli host strain was constructed and used for detection of DNA fragments carrying the E. coli K-12 pin gene cloned in the plasmid vector pBR322. Restriction analysis of the cloned fragments showed that the invertible DNA (designated P region) is adjacent to the pin gene and that its inversion is mediated by the pin gene product. The pin gene was found to be functionally homologous to the gin gene of Mu phage and the cin gene of P1 phage. The P region most probably resides within the cryptic prophage e14, and the Pin- phenotype is likely to be associated with the loss of e14.     

Refolding of recombinant Pasteurella haemolytica A1 glycoprotease expressed in an Escherichia coli thioredoxin gene fusion system

Pasteurella haemolytica A1 secretes an O-sialoglycoprotein endopeptidase (EC. 3.4.24.57) (glycoprotease: Gcp) which is specific for O-linked sialoglycoproteins. When the cloned gene is expressed in Escherichia coli, the recombinant glycoprotease (rGcp) is secreted to the peripalsm where it is present as a disulfide-linked aggregate which lacks enzymatic activity. In vitro refolding and activation of rGcp by mammalian protein disulfide isomerase (PDI) or by the E. Coli chaperones (DnaK, DnaJ and GrpE) indicate that the redox environment of rGcp is critical in restoring biological activity. A fusion protein, rTrx-Gcp, was constructed to investigate the role of thioredoxin (E. coli TrxA) in the production of enzymatically active rGcp. This 47 kDa protein was expressed at a high level, in a soluble, monomeric form, in the cytoplasm of E. coli. Cleavage of the fusion protein by enterokinase released the rGcp fragment (35 kDa) with glycoprotease activity. A higher recombinant glycoprotease activity was recoveref after anion exchange chromatography of lystates of E. coli expressing rTrx-Gcp. Thus when E. coli TrxA is combined in a recombinant fusion protein with P. haemolytica A1 Gcp, productive folding of the glycoprotease can occur as a result of the chaperone action of the protein disulfide reductase coupled with its ability to retain the fusion gene product in the E. coli cytopalsm.