Summary
An 8 kb BamHI fragment of the Escherichia coli K12 chromosome has been cloned which complemented the pheotype of CRM+ pckA mutants with inactive phosphoenolpyruvate (PEP) carboxykinase. The pckA + clones expressed levels of enzyme activity elevated up to 30-fold and produced a Mr 55000 product in maxicells, which coelectrophoresed with purified PEP carboxykinase. The cloned fragment expressed the pckA, ompR and envZ gene products in maxicells. The order of genes on the chromosome inferred from restriction mapping, was (74 min)...pckA, envZ ompR...(75 min). Transcription of the pckA, gene cloned on multicopy plasmids increased in stationary phase and was also regulated by catabolite repression. The transcriptional control region has been located by genetic fusions to the chloramphenicol acetyltransferase (cat) gene and pckA, was transcribed in the direction of envZ (clockwise direction on the chromosome).
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References
Atlung T, Nielsen A, Hansen FG (1989) Isolation, characterization and nucleotide sequence of appY, a regulatory gene for growthphase dependent gene expression in Escherichia coli. J Bacteriol 171:1683–1691
Bachmann BJ (1983) Linkage map of Escherichia coli K12, Edition 7. Microbiol Rev 47:180–230
Backman K, Boyer H (1983) Tetracycline resistance determined by pBR322 is mediated by one polypeptide. Gene 26:197–203
Bolivar F, Backman K (1976) Vehicles and hosts for the cloning of recombinant DNA. Methods Enzymol 68:245–267
Chan YH (1987) Genetic fusions in loci affecting the synthesis of phosphoenolpyruvate carboxykinase in Escherichia coli K12. Ph.D. Thesis, University of Saskatchewan
Chin AM, Feught BU, Saier MH Jr (1987) Evidence for the regulation of gluconeogenesis by the fructose phosphotransferase system in Salmonella typhimurium. J Bacteriol 169:897–899
Chin AM, Feldheim DA, Saier MH Jr (1989) Altered transcriptional patterns affecting several metabolic pathways in strains of Salmonella typhimurium which overexpress the fructose regulon. J Bacteriol 171:2424–2434
Clewell DB, Helsinki DR (1970) Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry 9:4428–4448
Close TJ, Rodriguez RL (1982) Construction and characterization of the chloramphenicol resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene 20:305–316
Comeau DE, Ikenaka K, Tsung K, Inouye M (1985) Primary characterization of the protein products of the Escherichia coli ompB locus: Structure and regulation of synthesis of the ompR and envZ proteins. J Bacteriol 164:578–584
Geerse RH, Ruig CR, Schnitema ARJ, Postma PR (1986) Relationship between pseudo-HPr and the PEP: fructose phosphotransferase system in Salmonella typhimurium and Escherichia coli. Mol Gen Genet 203:435–444
Gharbi S, Belaich A, Murgier M, Lazdunski A (1985) Multiple controls exerted on in vivo expression of the pepN gene in Escherichia coli. J Bacteriol 163:1191–1195
Gibson MM, Ellis EM, Graeme-Cook KA, Higgins CF (1987) OmpR and EnvZ are pleiotropic regulatory proteins: positive regulation of the tripeptide permease (tppB) of Salmonella typhimurium. Mol Gen Genet 207:120–129
Goldie AH, Sanwal BD (1980a) Genetic and physiological characterization of mutants deficient in phosphoenolpyruvate carboxykinase activity. J Bacteriol 141:1115–1121
Goldie AH, Sanwal BD (1980b) Allosteric control by calcium and mechanism of desensitization of phosphoenolpyruvate carboxykinase of Escherichia coli. J Biol Chem 255:1399–1405
Goldie H (1984) Regulation of transcription of the Escherichia coli phosphoenolpyruvate carboxykinase locus: studies with pck-lacZ operon fusions. J Bacteriol 159:832–838
Gottesman S (1984) Bacterial regulation: global regulatory mechanisms. Annu Rev Genet 18:415–441
Hernandez-Chico C, San Millan JL, Kolter R, Moreno F (1986) Growth phase and ompR regulation of transcription of microcin B17 genes. J Bacteriol 167:1058–1065
Kohara Y, Akiyama K, Isono K (1987) The physical map of the whole E. coli chromosome: Application of a strategy for rapid analysis and sorting of a large genomic library. Cell 50:495–508
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 165:265–275
Mizuno T, Wurtzel ET, Inouye M (1982) Cloning of the regulatory genes (ompR and envZ) for the matrix proteins of the Escherichia coli outer membrane. J Bacteriol 150:1462–1466
Sancar A, Hack A, Rupp WD (1979) Simple method for identifying plasmid-coded proteins. J Bacteriol 137:692–693
Stueber D, Bujard H (1982) Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J 1:1399–1404
Sutcliffe JG (1978) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90
Yamada M, Feucht BU, Saier MH Jr (1987) Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli. J Bacteriol 169:5416–5422
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Goldie, H., Medina, V. Physical and genetic analysis of the phosphoenolpyruvate carboxykinase (pckA) locus from Escherichia coli K12. Molec. Gen. Genet. 220, 191–196 (1990). https://doi.org/10.1007/BF00260481
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DOI: https://doi.org/10.1007/BF00260481