Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12.
Gene lamB encodes an outer membrane protein involved in maltose and maltodextrin transport as well as phage adsorption. The active form is a trimer. We characterized 11 mutations in lamB, obtained after random insertion of a BamH1 linker and screening for stable immunodetectable mutant proteins. Six mutations resulted in the loss of the distal part of the LamB protein either by deletion (five cases) or frameshift (one case). The six corresponding proteins had all lost the ability to confer phage sensitivity and the capacity to grow on dextrins, and to yield immunodetectable oligomers. Induction of a high level of the four longest of these proteins was toxic to the cell. Five other mutations were due to in-frame insertions. In four cases, the corresponding proteins still had the ability to yield immunodetectable oligomers, to confer phage sensitivity and the capacity to grow on dextrins and were not toxic on induction. In one case (AJC73), the mutant protein had lost the first three properties and was toxic on induction. Deletions and duplications between some of the inserts were also constructed and studied. To account for our results we present a hypothetical scheme in which trimerization would not only be needed for phage sensitivity and growth on dextrins but also for proper insertion into the outer membrane. The C-terminus of the protein, as well as other regions such as the site of mutation AJC73, would be required for the formation of stable trimers. We tentatively interpret toxicity as due to improper insertion into the outer membrane. Our results also show that it is possible to insert several amino acids (up to 11 in one case) at a number of positions in LamB without appreciably affecting its export and activities.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids; D018272:Porins; D011991:Receptors, Virus / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D004262:DNA Restriction Enzymes