FIG 5 .
Rickettsia typhi expresses two structurally divergent VirB8-like proteins. (A) Structural model for R. typhi RvhB8-1 (RT0280; YP_067242). (Left) RvhB8-I monomer in ribbon representation with nine residues involved in the dimerization interface shown in stick representation. For clarity, residues colored red are not shown in the dimer. (Center) RvhB8-I dimer in ribbon representation with seven residues involved in the dimerization interface shown in stick representation. Green indicates a minor dimerization site involving residues within the loop between α-helices α1 and α2. Red indicates a major dimerization site involving residues of α-helix α1 and the NPXG motif. (Right) Higher magnification of the RvhB8-I subunit interface. (B) Ribbon representation for the monomer and dimer of the crystal structure (PDB ID 4O3V) of R. typhi RvhB8-II (RT0278; YP_067240). Depiction of dimerization scheme follows the layout shown for RvhB8-I in panel A, except that the minor dimerization site (green) involved residues between β-strands β2 and β3. The brown star denotes the break in the structure. (C) Sequence alignment of RvhB8-I and RvhB8-II proteins and secondary structure assignment. Sequences are those of the globular domains depicted in panels A and B. For each protein, residues involved in the dimerization interface are within black (or red) boxes. Invariant residues are highlighted yellow. Magenta cylinders and gray arrows depict the α-helices and β-strands, respectively, for structures shown in panels A and B. For RvhB8-II, the brown star denotes the break in the structure, with missing residues colored white. (D) rvhB8-I and rvhB8-II are arrayed in tandem operons within the R. typhi genome. The schema shows nucleotide coordinates 351539 to 360917 from the R. typhi strain Wilmington genome (NC_006142) (46). Nine genes are encoded within three predicted operons: 1, rvhB9-I and rvhB8-II (red); 2, rvhB7, rvhB8-I (blue), rvhB9-II, rvhB10, and rvhB11; 3, rvhD4 and gppA (guanosine-5′-triphosphate,3′-diphosphate pyrophosphatase). The direction of transcription for each operon is shown with green arrows. Operons were predicted with fgenesb (90). Other rvh genes are encoded in separate clusters within the R. typhi genome (41). (E) Expression of the R. typhi rvhB8-I and rvhB8-II genes during early host cell infection. RNA was extracted from HeLa cells infected with R. typhi, and gene expression of RT0280 (encoding RvhB8-I) and RT0278 (encoding RvhB8-II) was measured by reverse transcription-quantitative PCR (RT-qPCR). Gene expression was normalized to R. typhi reference genes adr1 and sca5 (2ΔCT). Infections were repeated in triplicate with technical duplicate readings for RT-qPCR. Values are means and standard errors of the means.