Summary
The lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants. The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions. Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB + allele was dominant over the lkyB207 mutant allele. Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB +), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the lkyB gene product was a membrane protein of molecular weight 42,000.
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References
Bachmann BJ (1983) Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev 47:180–230
Bayer ME (1968) Areas of adhesion between wall and membrane of Escherichia coli. J Gen Microbiol 53:395–404
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Blobel G, Dobberstein B (1975a) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67:835–851
Blobel G, Dobberstein B (1975b) Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol 67:852–862
Clarke L, Carbon J (1976) A colony bank containing synthetic ColE1 plasmids representative of the entire Escherichia coli genome. Cell 9:91–99
Costerton JW, Ingram JM, Cheng KJ (1974) Structure and function of the cell envelope of gram negative bacteria. Bacteriol Rev 38:87–110
Fognini-Lefebvre N, Portalier R (1984) Isolation and preliminary characterization of beta-lactamase excretory mutants of Escherichia coli K-12. FEMS Microbiol Lett 21:323–328
Hall MN, Silhavy TJ (1981) Genetic analysis of the ompB locus in Escherichia coli K-12. J Mol Biol 151:1–15
Heppel LA (1971) The concept of periplasmic enzymes. In: Rothfield LI (ed) Structure and function of biological membranes. Academic Press, New York, pp 223–247
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685
Lazzaroni JC, Portalier RC (1979) Isolation and preliminary characterization of periplasmic-leaky mutants of Escherichia coli K-12. FEMS Microbiol Lett 5:411–416
Lazzaroni JC, Portalier RC (1981) Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12. J Bacteriol 145:1351–1358
Lazzaroni JC, Portalier RC (1985) Regulation of the lkyB gene expression in Escherichia coli K-12 strains carrying an lkyB-lacZ gene fusion. Mol Gen Genet 201:323–328
Lazzaroni JC, Fognini-Lefebvre N, Portalier R (1986) Effects of lkyB mutations on the expression of ompF, ompC and lamB porin structural genes in Escherichia coli K-12. FEMS Microbiol Lett 33:235–239
Lugtenberg B, Van Alphen L (1983) Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram negative bacteria. Biochim Biophys Acta 737:51–115
Malamy MH, Horecker B (1961) The localization of alkaline phosphatase in Escherichia coli K-12. Biochim Biophys Res Commun 5:104–108
Miller JH (1972) In: Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Maniatis T, Fritsch EF, Sambrook J (1982) In: Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Randall LL, Hardy SJ (1984) Export of proteins in bacteria. Microbiol Rev 48:290–298
Rothstein SJ, Jorgensen RA, Postle K, Reznikoff WS (1980) The inverted repeats of Tn5 are functionally different. Cell 19:795–805
Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Silhavy TJ, Bassford PJ Jr, Beckwith JR (1979) A genetic approach to the study of protein localization in Escherichia coli. In: Inouye M (ed) Bacterial outer membrane, biogenesis and function. Wiley and Sons, New York, pp 203–254
Tommassen J, De Geus P, Lugtenberg B, Hacket J, Reeves P (1982) Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products. J Mol Biol 157:265–274
torriani AM (1967) Alkaline phosphatase from Escherichia coli. In: Cantoni GL, Davies DL (eds), Procedures in nucleic acid research, Harper and Row Publishers, Inc, New York, pp 224–235
Wickner W (1979) Assembly of proteins into biological membranes: membrane trigger hypothesis. Annu Rev Biochem 48:23–45
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Communicated by J. Lengeler
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Lazzaroni, J.C., Fognini-Lefebvre, N. & Portalier, R.C. Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product. Molec. Gen. Genet. 204, 285–288 (1986). https://doi.org/10.1007/BF00425511
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DOI: https://doi.org/10.1007/BF00425511