Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins.

A simple and efficient procedure for the construction of secreted fusion proteins in Escherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) of E. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we used lacZ, encoding the cytoplasmic beta-galactosidase (beta-Gal), and phoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that all beta-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in a secA mutant strain only under SecA-deficient conditions.