Fig 2.

Analysis of GerH_B topology. (A) Model of GerH_B with 11 putative transmembrane domains as determined by topology prediction. To test these putative TMs, C-terminal GFP fusions were made to full-length GerH_B (N364) and nine GerH_B proteins truncated at the indicated locations. TM8 was excluded because of its low predictive value. (B) Expression of GerH_B fusion proteins in B. anthracis. Whole-cell lysates from bacteria expressing a vector control (lane C), GFP, or GerH_B fusion proteins were analyzed by immunoblotting with anti-GFP antibodies to assess protein expression and stability. The predicted sizes of GFP and full-length GerH_B-364::GFP are indicated. (C) Analysis of GerH_B fusion proteins by flow cytometry and protease sensitivity assays. Flow cytometry was used to measure the fluorescence intensities of bacteria expressing control proteins (GFP or FpuA::GFP) or the GerH_B fusion proteins. The percentage of GFP⁺ cells in each sample was determined as described for Fig. 1B (gray bars). Protease sensitivity assays were performed with proteinase K. The percentage of each fusion protein that was protease sensitive was determined as described for Fig. 1C (black bars). Each bar represents the mean ± standard error of the mean. ND, not determined.