In vivo selection and characterization of internal deletions in the lamB::lacZ gene fusion

Strain Pop3299 contains the lamB:: lacZ42-12 gene fusion that encodes a hybrid protein that is efficiently exported to the cellular envelope of Escherichia coli. As a result of this efficient export, this strain is killed by the inducer maltose and unable to grow on minimal media supplemented with lactose. In an attempt to isolate mutants in which export of the hybrid protein is altered, we selected Lac + mutants of strain Pop3299 on lactose tetrazolium media. Unlike mutants previously isolated on lactose minimal media, all the mutants we obtained carried large deletions within the lamB:: lacZ gene fusion. Thus, it appears that the type of selection employed affects the type of mutations obtained. We have analyzed the nucleotide sequences of representative mutants, and demonstrate a correlation between the deletion size and the export-related maltose and lactose phenotypes. In addition, we demonstrate that the deletions do not appear to arise from regions of micro-homology.