USE OF THE BLUE HALO ASSAY IN THE IDENTIFICATION OF GENES ENCODING EXPORTED PROTEINS WITH CLEAVABLE SIGNAL PEPTIDES - CLONING OF A BORRELIA-BURGDORFERI PLASMID GENE WITH A SIGNAL PEPTIDE

We have recently reported a phoA expression vector, termed pMG, which, like TnphoA, is useful in identifying genes encoding membrane-spannin g sequences or signal peptides. This cloning system has been modified to facilitate the distinction of outer membrane and periplasmic alkali ne phosphatase (AP) fusion proteins from inner membrane AP fusion prot eins by transforming pMG recombinants into Escherichia coli KS330, the strain utilized in the ''blue halo'' assay first described by Strauch and Beckwith (Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988). The lip oprotein mutation lpp-5508 of KS330 results in an outer membrane that is leaky to macromolecules, and its degP4 mutation greatly reduces per iplasmic proteolytic degradation of AP fusion proteins. pMG AP fusions containing cleavable signal peptides, including the E. coli periplasm ic protein beta-lactamase, the E. coli and Chlamydia trachomatis outer membrane proteins OmpA and MOMP, respectively, and Tp 9, a Treponema pallidum AP recombinant, diffused through the leaky outer membrane of KS330 and resulted in blue colonies with blue halos. In contrast, inne r membrane AP fusions derived from E. coli proteins, including leader peptidase, SecY, and the tetracycline resistance gene product, as well as Tp 70, a T. pallidum AP recombinant which does not contain a signa l peptide, resulted in blue colonies without blue halos. Lipoprotein-A P fusions, including the Borrelia burgdorferi OspA and T. pallidum Tp 75 and TmpA showed halo formation, although there was significantly le ss halo formation than that produced by either periplasmic or outer me mbrane AP fusions. In addition, we applied this approach to screen rec ombinants constructed from a 9.0-kb plasmid isolated from the B31 viru lent strain of B. burgdorferi. One of the blue halo colonies identifie d produced an AP fusion protein which contained a signal peptide with a leader peptidase I cleavage recognition site. The pMG/KS330r- clonin g and screening approach can identify genes encoding proteins with cle avable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors.