Absence of Thiol-Disulfide Oxidoreductase DsbA Impairs cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus

Abstract

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35°C), DsbA^- mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25°C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb₃-type cytochrome c oxidase (cbb₃-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu²⁺ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb₃-Cox production in the absence of DsbA. Indeed, addition of Cu²⁺ to the enriched medium suppresses, and conversely, omission of Cu²⁺ from the minimal medium induces, growth and cbb₃-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb₃-Cox, especially in enriched medium where bioavailable Cu²⁺ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb₃-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb₃-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb₃-Cox.

Keywords: Rhodobacter capsulatus; cbb3-type cytochrome c oxidase biogenesis; copper homeostasis; disulfide bond formation; photosynthesis; respiration.