Minimal deletion of amino acids from the carboxyl terminus of the B subunit of heat-labile enterotoxin causes defects in its assembly and release from the cytoplasmic membrane of Escherichia coli.
Minimal alterations at the carboxyl terminus of the B subunit (EtxB) of heat-labile enterotoxin from Escherichia coli were found to have a marked effect on the assembly and release of this polypeptide into the periplasm. Nine mutant EtxB polypeptides were obtained by genetic manipulation of the 3'-end of the etxB gene using Bal31 nuclease digestion and codon substitution. A correlation was observed between the magnitude of the changes introduced at the carboxyl terminus and the extent to which the mutant polypeptides were defective in assembly and release. Some of the mutant B subunits, exemplified by those in which the last 2 amino acids had been deleted or in which the last 4 residues had been replaced by three different ones, were found to be only partially defective, with a proportion being associated with the periplasmic face of the cytoplasmic membrane and the remainder being exported to the periplasm. The portion associated with membranes was detected as monomers on sodium dodecyl sulfate-polyacrylamide gels, whereas the portion exported to the periplasm were detected as assembled oligomers. We conclude that the last few amino acids at the carboxyl terminus of EtxB exert a profound influence on the assembly and release of the B subunit from the cytoplasmic membrane during export in E. coli.
Sandkvist
M
M
Department of Microbiology, Michigan State University, Lansing 48909.
Hirst
T R
TR
Bagdasarian
M
M
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002872:Chromosome Deletion; D003062:Codon / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005809:Genes, Regulator; D005822:Genetic Vectors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D013728:Terminator Regions, Genetic
D001427:Bacterial Toxins; D003062:Codon; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C027538:heat-labile enterotoxin, E coli