FIG 6.

VciB is an inner membrane protein with topology consistent with predictions. (A) The V5 epitope-tagged vciB plasmid construct and point mutant derivatives were assessed for the ability to complement the colony formation defect of the Feo⁺ VciB⁻ strain (EPV153) in a colony size assay. All constructs had the C41S mutation, in addition to the mutations indicated. (B) Subcellular fractionation of bacterial cells using ultracentrifugation. Total lysate (total), cytoplasmic (cyto), inner membrane (inner), and outer membrane (outer) fractions are marked. (Top) Western blot using anti-V5 antibody. (Bottom) Coomassie blue-stained SDS-PAGE gel. (C) Representative Western blots of residues probed using SCAM. Lanes: –, negative control (no treatment); +, positive control (MalPEG treated); N, samples pretreated with NEM; M, samples pretreated with MTSES. (D) Supported topology model with V5 epitope tag (blue), Cys mutants (orange circles), and point mutations introduced in conserved residues (green squares). Transmembrane regions were predicted using TMHMM (39). The topology map was generated using the TeXtopo package in the LaTeX program (55).