Abstract
The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.
Highlights
The filamentous hemagglutinin (FHA) of the whooping cough agent Bordetella pertussis requires the outer membrane protein FhaC for secretion
One aliquot was directly precipitated with trichloroacetic acid and prepared for SDSPAGE, and the other one was first digested with 500 g/ml proteinase K (PK) to remove all soluble proteins
The FhaB*-secreting spheroplasts were incubated with proteoliposomes that had been reconstituted from outer membrane proteins of an FhaC-expressing E. coli strain, a significant fraction of FhaB* became PK-resistant (Fig. 1A, lanes 5 and 6)
Summary
The filamentous hemagglutinin (FHA) of the whooping cough agent Bordetella pertussis requires the outer membrane protein FhaC for secretion. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of Gram-negative bacteria. TpsB proteins are members of the superfamily of Omp protein transporters [8, 9] Some of these transporters, including TpsB of Gram-negative bacteria and Toc75-III of the chloroplast outer envelope, mediate the translocation of their protein substrates across a membrane [10]. Our studies clearly indicate that FhaC is the sole translocator of the passenger protein FhaB
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