Isolation of fla-lacZ fusions in Escherichia coli K-12: most fusions result in soluble beta-galactosidase.

Abstract

A series of fusions of flagellar genes to the lacZ gene was generated by insertion of Mu dII301 (Apr lac) bacteriophage into the genome of Escherichia coli. The beta-galactosidase activity in each resulting mutant was measured, and the location of the activity in the membrane, periplasmic, or cytoplasmic fraction of the cell was determined. There were three classes of mutants: those which had beta-galactosidase activity mainly in the membrane fraction, those which had it distributed in the soluble and membrane fractions, and those which had it in the cytoplasmic fraction only. The last, soluble-fraction-only, class was predominant in fla-lac gene fusions. In particular, the following mutants were shown to have beta-galactosidase activity in the membrane fractions: on the inner membrane, mutants with flaB fusions, and on the inner and outer membranes, mutants with flaA4850, flaM, and flaU4849 fusions. These results suggest that fla-lacZ gene fusions produce proteins which are able to detect the signals of the leader sequence and the membrane-anchoring region of the flagellar system.