The export of Escherichia coli hemolysin across the cytoplasmic and the outer membranes requires the COOH-terminal signal sequence of HlyA, the two specific translocator proteins HlyB and HlyD, and the outer membrane protein TolC. We have developed an export cloning system that is composed of two vectors: one in which the fusion of the desired gene with the 3'-end of hlyA is generated, and a second in which the sequences containing the fusion are combined with the accessory genes hlyB and hlyD, thereby reconstructing the natural organization of the hly locus. in the second vector the fusion and the accessory genes are flanked by NotI sites, allowing subcloning of the whole cluster into a variety of minitransposons to achieve the stable integration of the constructs into the chromosome of Gram-negative bacteria. Since some applications may require the production of transcriptional fusions, an alternative version of the system provides the efficient translation initiation region of T7 phage gene 10 upstream of the fusion protein coding sequence. The usefulness of the system was assessed by constructing a fusion between the gene encoding the B subunit of Shiga-like toxin lie and the 3'-end of hlyA. An attenuated Salmonella typhimurium vaccine strain harboring the resulting construct, either in multicopy or monocopy, efficiently expressed and exported the chimeric protein. We anticipate that this system will lead to a higher stability of the engineered function and permit a faithful monitoring of the export of the recombinant peptide under physiologic single-copy conditions.
