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Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane‐spanning sequences using a novel alkaline phosphatase expression vector

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence‐encoding region. Library construction with Sau3A‐ digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum‐AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non‐cleavable hydrophobic membrane‐spanning sequences. Triton X‐114 detergent phase partitioning of individual T. pallidum‐AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]‐palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N‐terminal signal sequence containing a consensus