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Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12. Identification of the pit+ gene product and physical mapping of the pit-gor region of the chromosome
Authors:Christopher M Elvin  Nicholas E Dixon and Harry Rosenberg
Institution:(1) Department of Biochemistry, John Curtin School of Medical Research, Australian National University, P.O. Box 334, 2601 Canberra City, A.C.T., Australia;(2) Present address: Research School of Chemistry, Australian National University, P.O. Box 4, 2601 Canberra City, A.C.T., Australia
Abstract:Summary The pit + gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E. coli genes inserted in the cosmid vector pHC79. A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase. Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region. Subcloning and deletion analysis indicated that the entire pit + gene was located within a 2.2-kb Sal1-Ava1 fragment. The pit + gene product was identified by SDS-polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: (i) 35S-methionine-labelling of minicells carrying pit + plasmids or plasmids from which all or part of the pit + gene was deleted. (ii) Overproduction of the Pit protein using a thermoinducible ldquorunawayrdquo replication plasmid.Complementation of the pit-1 mutant allele using a unit-copy-number pit + plasmid indicated that the pit-1 mutation is recessive.Strains carrying a multicopy pit + plasmid show a 10-fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of 32Pi accumulation.Abbreviations kb kilobase-pairs - kdal kilodalton - Pi inorganic phosphate - G3P sn-glycerol-3-phosphate - LB Luria broth - Tc tetracycline - Cm chloramphenicol - Ap ampicillin - UV ultraviolet light - TE 10 mM Tris.HCl, pH 8.0, 1 mM EDTA - HEPES N-2-hydroxyethylpiperazine-Nprime-2-ethanesulphonic acid
Keywords:Escherichia coli  Phosphate transport  Membrane protein  Gene cloning  Glutathione oxidoreductase
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