Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12.
The gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO2H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005804:Genes, Lethal; D005838:Genotype; D009154:Mutation; D010957:Plasmids
D001425:Bacterial Outer Membrane Proteins; D004262:DNA Restriction Enzymes