Evidence for a ninth gene, ptlI in the locus encoding the pertussis toxin secretion system of Bordetella pertussis and formation of a PtlI-PtlF complex

The pertussis toxin secretion system of Bordetella pertussi initially was thought to comprise eight proteins, Pt1A-Pt1H. We have investigated the existence of another protein, PtlI, encoded by a putative gene located between ptlD and ptlE. A B. pertussis strain expressing a ptlI::phoA translational fusion possessed alkaline phosphatase activity, suggesting that ptlI encodes a protein. In B. pertussis, a protein with an apparent molecular weight of similar to 5,200 (similar to that predicted by the ptlI sequence) was immunoreactive with an antibody raised to a PtlI-maltose-binding protein fusion protein, PtlE expression in a mutant sustaining an in-frame deletion in ptlI indicated that ptlE starts further downstream than initially predicted. Pt1F, not detected in the ptlI deletion mutant, was restored partially by expressing ptlI in trans, A 36-kDa species, consistent with a Pt1I-Pt1F complex, was immunoreactive with antibodies to Pt1I and Pt1F in nonreduced cell extracts of a Bordetella bronchiseptica strain which overexpresses the Pt1 proteins. Upon dithiothreitol treatment, the 36-kDa species was diminished greatly or undetectable. In B. pertussis, Pt1I and Pt1F co-precipitated with antibody to Pt1F. These findings demonstrate the existence of Pt1I and a Pt1I-Pt1F complex, providing the first description of an interaction between Pt1 proteins.