Cloning and expression of the exbB gene of Escherichia coli K-12

Abstract

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.