Overexpression of ccl1 −2 can bypass the need for the putative apocytochrome chaperone CycH during the biogenesis of c ‐type cytochromes

Summary In Gram‐negative bacteria, including Rhodobacter capsulatus , the membrane protein CycH acts as a putative apocytochrome chaperone during the biogenesis of c ‐type cytochromes. CycH‐null mutants are unable to produce various c ‐type cytochromes and sustain photosynthetic (Ps) growth that requires the cytochromes c 1 and c 2 or c y . However, Ps + revertants are readily obtained only on minimal, but not on enriched, medium. To obtain further information about the biogenesis of c ‐type cytochromes, these suppressor mutants were studied. Complementation of a CycH‐null mutant for Ps + growth by a genomic library constructed using DNA from a Ps + suppressor yielded a plasmid carrying the ccl1–2 operon, the products of which, Ccl1 and Ccl2, are also involved in the biogenesis of c ‐type cytochromes. DNA sequence analysis revealed that the complementing activity resulted from a single point mutation, G488A, located upstream of the coding region of ccl1–2 . This mutation changed the −35 region of the ccl1–2 promoter from TTG G CC to TTG A CC, improving its similarity to the consensus sequence of Escherichia coli σ 70 ‐dependent promoters. That the G488A mutation indeed enhanced transcription of ccl1–2 was demonstrated by the use of reporter gene fusions. An appropriate ccl1‐2::lacZ transcriptional–translational fusion carrying the G488A mutation produced in R. capsulatus over 30‐fold higher β‐galactosidase activity than a wild‐type construct. Immunoblot analyses confirmed that Ccl1 and Ccl2 were overproduced in the Ps + suppressors. Deletion of either ccl1 or ccl2 , from the ccl1–2 cluster carrying the G488A mutation abolished the complementing ability, indicating that overexpression of both ccl1 and ccl2 was required to confer the Ps + phenotype on a CycH‐null mutant. These findings therefore demonstrate that, during R. capsulatus growth on minimal medium, the requirement for CycH in c ‐type cytochrome biogenesis could be bypassed by overexpressing the ccl1–2 operon.