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. 2013 Nov 25;8(11):e81074. doi: 10.1371/journal.pone.0081074

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© 2013 Zhang et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Hcp release assay. Hcp_FLAG release was assessed by separating whole cells (C) and culture supernatant (SN) fractions from 2×10⁹ ΔtssB1 cells producing TssB1 (tssB⁺) or TssB1 bearing double or single substitutions within the hydrophobic motif (tssB^VI, V106W-I110W; tssB^IL, I110W-L117W; tssB^VL, V106W-L117W; tssB^V, V106W; tssB^I, I110W; tssB^L, L117W). Proteins were separated by 12.5%-acrylamide SDS-PAGE and Hcp and TolB were immunodetected using anti-FLAG monoclonal (lower panel) and anti-TolB polyclonal (upper panel) antibodies. (B) Bacterial two-hybrid assay. BTH101 reporter cells producing the T25 domain of the Bordetella adenylate cyclase fused to TssC1 (T25-C) and the T18 domain fused to TssB1 (T18-B) or TssB1 variants bearing substitutions within the hydrophobic motif (T18-B^VI, V106W-I110W; T18-B^IL, I110W-L117W; T18-B^VL, V106W-L117W; T18-B^V, V106W; T18-B^I, I110W; T18-B^L, L117W) were spotted on X-Gal indicator LB agar plates.