Effects of inserting eight amino acid residues into the major lipoprotein on its assembly in the outer membrane of Escherichia coli.
A DNA sequence consisting of 24 base pairs was inserted into the structural gene (lpp) coding for the major lipoprotein of the Escherichia coli outer membrane which was carried on a high-copy-number plasmid in which expression was regulated through a lac promoter-operator region. This modification resulted in the insertion of eight amino acid residues, Glu-Glu-Phe-Leu-Glu-Glu-Phe-Leu, between the glutamine residue at position 9 and the leucine residue at position 10 of the wild-type lipoprotein sequence. When production of the mutant lipoprotein was induced by a lac inducer, the cells became swollen, showed unusual morphology, and eventually lysed. When the membrane fraction was analyzed after the induction, the mutant lipoprotein was found to have been normally secreted across the cytoplasmic membrane and assembled in the outer membrane. This lipoprotein was modified with glycerol and palmitic acid and even formed the bound form, which was linked covalently to peptidoglycan. The major difference between the membrane-associated mutant lipoprotein and the wild-type lipoprotein was that the mutant lipoprotein became sensitive to trypsin treatment. These results indicate that the substantial alteration in mutant lipoprotein structure near the amino-terminal end does not interfere with modification of the amino-terminal cysteine residue or cleavage of the signal peptide by the prolipoprotein-specific signal peptidase. However, this mutant lipoprotein assembled in the outer membrane appears to have deleterious effects with respect to envelope structure and cellular morphology and viability.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D007700:Kinetics; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids
D000596:Amino Acids; D008074:Lipoproteins