Abstract
The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds. The fhuA gene encodes the outer membrane receptor protein. Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37° C large amounts of the proFhuA protein. Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis. Despite inhibition of export proFhuA synthesis continued.
The precipitate formed was sedimented by centrifugation at 8,000xg. The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate. Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein. Binding of phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M. Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns. Addition of ferrichrome affected trypsin cleavage of both proteins in the same way. The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.
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References
Bakker EP, Randall LL (1984) The requirement for energy during export of β-lactamase in Escherichia coli is fulfilled by the total protonmotive force. EMBO J 3:895–900
Benz G, Schröder T, Kurz J, Wünsche C, Karl W, Steffens G, Pfitzner J, Schmidt D (1983) Konstitution der Desferriform der Albomycine. Angew Chem Suppl 1322–1335
Braun V, Schaller K, Wolff H (1973) A common receptor protein for phage T5 and colicin M in the outer membrane of Escherichia coli B. Biochim Biophys Acta 323:87–97
Carleman E, Garavito RM, Villiger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microscopy 126:123–143
Coulton JW, Mason P, Cameron DR, Carmels G, Jean R, Rode HV (1986) Protein fusions of β-galactosidase to the ferrichromeiron receptor of Escherichia coli K-12. J Bacteriol 165:181–192
Dierstein R, Wickner W (1985) The leader region of premaltose binding protein binds amphiphiles. Model for self-assembly in protein export. J Biol Chem 260:15919–15924
Fecker L, Braun V (1983) Cloning and expression of the fhu genes involved in iron(III)-hydroxamate uptake by Escherichia coli. J Bacteriol 156:1301–1314
Freudl R, Schwarz H, Klose M, Movva NR, Henning U (1985) The nature of information, required for export and sorting, present within the outer membrane protein OmpA of Escherichia coli K-12. EMBO J 4:3593–3598
Hoffmann H, Fischer E, Kraut H, Braun V (1986) Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli. J Bacteriol 166:144–411
Kleid DG, Yansura D, Small B, Dowbenko D, Moore DM, Grubman MJ, McKercher PD, Morgan DO, Robertson BH, Bachrach HL (1981) Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science 214: 1125–1129
Lifson ER, Lindahl L, Zengel JM (1986) Precursor for elongation factor Tu from Escherichia coli. J Bacteriol 165:474–482
Mühlpfordt H (1982) The preparation of colloidal gold particles using tannic acid as an additional reducing agent. Experimentia 38:1127–1128
Osborn MJ, Gander JE, Parisi E, Carson J (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium: isolation and characterization of cytoplasmic and outer membrane. J Biol Chem 247:3962–3972
Schilstra MJ, Skot JW, van der Meide PH, Posthma G, Cremers AFM, Bosch L (1984) Immunocytochemical localization of the elongation factor Tu in E. coli cells. FEBS Lett 165:175–179
Sung WL, Yao F-L, Zhab DM, Narang SA (1986) Short synthetic oligodeoxyribonucleotide leader sequences enhance accumulation of human proinsulin synthesized in Escherichia coli. Proc Natl Acad Sci USA 83: 561–565
Tomassen J, Leunissen J, van Damme-Jongsten M, Overduin P (1985) Failure of E. coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm. EMBO J 4:1041–1047
Uhlin BE, Nordström U (1978) A runaway-replication mutant of plasmid R1 drd-19: temperature-dependent loss of copy number control. Mol Gen Genet 165:167–179
Williams DC, van Frank RM, Muth WL, Burnett J-P (1982) Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins. Science 215:687–689
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Dedicated to Prof. G. Braunitzer on the occasion of his 60th birthday
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Hoffmann, H., Fischer, E., Schwarz, H. et al. Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation, properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane. Arch. Microbiol. 145, 334–341 (1986). https://doi.org/10.1007/BF00470867
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DOI: https://doi.org/10.1007/BF00470867