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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

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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 “runaway” 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.

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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-N′-2-ethanesulphonic acid

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Author notes

  1. Christopher M. Elvin & Nicholas E. Dixon

    Present address: Research School of Chemistry, Australian National University, P.O. Box 4, 2601, Canberra City, A.C.T., Australia

Authors and Affiliations

  1. Department of Biochemistry, John Curtin School of Medical Research, Australian National University, P.O. Box 334, 2601, Canberra City, A.C.T., Australia

    Christopher M. Elvin, Nicholas E. Dixon & Harry Rosenberg

Authors
  1. Christopher M. Elvin
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  2. Nicholas E. Dixon
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  3. Harry Rosenberg
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Communicated by J.W. Lengeler

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Elvin, C.M., Dixon, N.E. & Rosenberg, H. Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12. Molec Gen Genet 204, 477–484 (1986). https://doi.org/10.1007/BF00331028

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  • DOI: https://doi.org/10.1007/BF00331028

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