Abstract
Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a beta-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.
Highlights
The recently revealed Omp85/TpsB superfamily of outer membrane proteins is dedicated to protein transport in most major kingdoms of life, no members have been identified in Archaebacteria yet [8, 9]
TpsB transporters are found in two-partner secretion systems, developed by Gram-negative bacteria for the secretion of large “TpsA” proteins destined to the cell surface or the milieu and serving mostly as virulence factors [10]
All the mutant genes were coexpressed with the fha44 gene in E. coli UT5600 to determine the secretion activities of the corresponding FhaC proteins, as described previously [24]
Summary
Plasmid Construction—pFJD118 was constructed as follows. pFcc3 [24] was linearized by restriction with BamHI, which cleaves at a unique site positioned after the third codon into the sequence of mature FhaC. The orientation of that linker was verified by sequencing, and the resulting plasmid was called pFcc3-His6 The latter plasmid was restricted with XbaI and SacI, and the fhaC-containing DNA fragment was inserted into the same sites of pET24d (Novagen), resulting in pFJD118. To construct pFJD150, the plasmid encoding the C-terminal truncate FhaC-⌬3–206, pFcc206 was restricted with BamHI and HindIII, and the resulting fragment coding for the last 349 residues of FhaC was inserted into a pFJD118 derivative called pFJD138 in replacement of its own BamHI-HindIII fragment. The pellets were resuspended in 20 mM Hepes (pH 7), and membrane aliquots from each strain were analyzed by SDS-PAGE and immunoblotting using anti-FhaC antibodies [24].
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