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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 approximately 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.
Highlights
Introduction of the ptlIphoA Fusion into B. pertussis—First, pSZH4, a plasmid containing the entire ptx-ptl region from B. pertussis [18] was digested with NotI and HindIII, resulting in removal of nucleotides 7977–13025 of the ptl region
The B. pertussis strain expressing the ptlI::phoA fusion [BP536(pKMF8)], but not a control strain which lacked the ptlI::phoA fusion [BP536(pUFR047)], possessed alkaline phosphatase activity, as deduced by the development of a dark blue color when cells were incubated in the presence of 5-bromo-4-chloro-3-indolyl phosphate
These results suggest that ptlI encodes a protein
Summary
31643–31649, 1996 Printed in U.S.A. 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*. A 36-kDa species, consistent with a PtlIPtlF complex, was immunoreactive with antibodies to PtlI and PtlF in nonreduced cell extracts of a Bordetella bronchiseptica strain which overexpresses the Ptl proteins. 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. We present evidence for the existence of an additional ptl gene, ptlI (nucleotides 8192– 8377), situated immediately downstream of ptlD, and for the existence of a PtlI-PtlF complex, providing the first description of a structural interaction between Ptl proteins
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