Summary
The exbB locus of Escherichia coli is involved in the uptake of certain iron(III) siderophore compounds, of vitamin B12 and of certain colicins. Outer membrane receptor proteins are essential constituents of the corresponding uptake systems. The DNA carrying the exbB locus was cloned into pACYC184 and subcloned into pUC18. With the use of insertion mutagenesis employing transposon Tn1000 and by deletion analysis, the exbB locus was confined to a 1.9 kb DNA fragment. An in vitro transcription/translation system and minicells programmed by exbB + plasmids expressed a protein with an apparent molecular weight of 26,000. One plasmid, designated pKE7, expressed this protein to an extent that it became a prominent band in the membrane fraction of transformants. In contrast, chromosomally encoded ExbB protein could not be detected. The plasmid-encoded ExbB protein was mainly localized in the cytoplasmic membrane. Ferrichrome transport in exbB mutants was restored by exbB + plasmids. Moderate overexpression of ExbB resulted in an enhanced ferrichrome transport, strong overexpression reduced the transport rate compared to a wild-type strain. The ExbB function shares some properties with the TonB function.
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References
Birnboim HC (1983) A rapid method for the isolation of plasmid-DNA. Methods Enzymol 100:243–255
Dougan G, Kehoe M (1984) The minicell system as a method for studying expression from plasmid DNA. Methods Enzymol 17:233–257
Gutermann S, Dann L (1973) Excretion of enterochelin by exbA and exbB mutants of Escherichia coli. J Bacteriol 114:1225–1230
Guyer M (1983) Uses of transposon γδ in the analysis of cloned genes. Methods Enzymol 101:362–369
Hancock REW, Hantke K, Braun V (1976) Iron transport in Escherichia coli K12: Involvement of the colicin B receptor and of a citrate-inducible protein. J Bacteriol 127:1270–1375
Hantke K, Zimmermann L (1981) The importance of the exbB gene for vitamin B12 and ferric iron transport. FEMS Microbiol Lett 12:31–35
Hoffmann H, Fischer E, Schwarz H, Braun V (1986) 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
Lugtenberg B, Meijers J, Peters R, van der Hoek P, van Alphen L (1975) Electrophoretic resolution of the “major outer membrane protein” of Escherichia coli K12 into four bands. FEBS Lett 58:254–258
Maniatis T, Fritsch EF, Sambrook H (1982) Molecular cloning. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York
Mann BJ, Holroyd CD, Bradbeer C, Kadner RJ (1986) Reduced activity of TonB-dependent functions in strains of Escherichia coli. FEMS Microbiol Lett 33:255–260
O'Brien JG, Cox GB, Gibson F (1970) Biological active compounds containing 2,3 dihydroxybenzoic acid and serine formed by Escherichia coli. Biochim Biophys Acta 201:453–460
Plastow GS, Holland JB (1979) Identification of the Escherichia coli inner membrane peptide specified by a lambda tonB transducing bacteriphage. Biochem Biophys Res Commun 90:1007–1014
Pugsley AP, Reeves P (1976a) Characterization of group B colicinresistant mutants of Escherichia coli K12: colicin resistance and the role of enterochelin. J Bacteriol 127:218–228
Pugsley AP, Reeves P (1976b) Iron uptake in colicin B-resistant mutants of Escherichia coli K12. J Bacteriol 126:1052–1062
Schnaitman C (1971) Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol 108:545–552
Silhavy TJ, Bermann ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York
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Communicated by J. Lengeler
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Eick-Helmerich, K., Hantke, K. & Braun, V. Cloning and expression of the exbB gene of Escherichia coli K-12. Mol Gen Genet 206, 246–251 (1987). https://doi.org/10.1007/BF00333580
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DOI: https://doi.org/10.1007/BF00333580