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

Abstract

The pertussis toxin secretion system of Bordetella pertussis initially was thought to comprise eight proteins, PtlA-PtlH. 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 ~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. PtlF, not detected in the ptlI deletion mutant, was restored partially by expressing ptlI in trans. A 36-kDa species, consistent with a PtlI-PtlF complex, was immunoreactive with antibodies to PtlI and PtlF in nonreduced cell extracts of a Bordetella bronchiseptica strain which overexpresses the Ptl proteins. Upon dithiothreitol treatment, the 36-kDa species was diminished greatly or undetectable. In B. pertussis, PtlI and PtlF co-precipitated with antibody to PtlF. These findings demonstrate the existence of PtlI and a PtlI-PtlF complex, providing the first description of an interaction between Ptl proteins.