Roles for the Rhodobacter sphaeroides CcmA and CcmG proteins

Abstract

Rhodobacter sphaeroides cells containing an in-frame deletion within ccmA lack detectable soluble and membrane-bound c-type cytochromes and are unable to grow under conditions where these proteins are required. Only strains merodiploid for ccmABCDG were found after attempting to generate cells containing either a ccmG null mutation or a ccmA allele that should be polar on to expression of ccmBCDG, suggesting that CcmG has another important role in R. sphaeroides.

FIG. 1

The R. sphaeroides ccmABCDG locus. Shown is the genetic organization of this locus (not drawn to scale; see below), the predicted direction of transcription (arrows), and the function of presumed gene products (bottom). Shaded boxes indicate where the predicted termination codon of one gene (ccmA, ccmC, and ccmD) overlaps the presumed initiation codon of the next gene (ccmB, ccmD, and ccmG, respectively).

FIG. 2

Heme peroxidase staining of soluble and membrane samples from aerobically grown cells. For each sample, ∼400 ug of protein was heated at 70°C in the presence of 5% 2-mercaptoethanol and separated by sodium dodecyl sulfate–15% polyacrylamide gel electrophoresis. Lanes 1 and 3 contain soluble and membrane samples from wild-type cells (2.4.1), respectively; lanes 2 and 4 contain soluble and membrane samples from cells lacking CcmA (CCMA4), respectively. Western blot analysis with antiserum against individual c-type cytochromes (date not shown) indicates that the soluble heme-staining protein in wild-type cells is mostly a combination of cytochrome c₂ and cytochrome c₅₅₄. Numbers at left are molecular weight markers (in thousands).