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  • Title: Overexpression of ccl1-2 can bypass the need for the putative apocytochrome chaperone CycH during the biogenesis of c-type cytochromes.
    Author: Deshmukh M, May M, Zhang Y, Gabbert KK, Karberg KA, Kranz RG, Daldal F.
    Journal: Mol Microbiol; 2002 Nov; 46(4):1069-80. PubMed ID: 12421312.
    Abstract:
    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 c1 and c2 or cy. 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 TTGGCC to TTGACC, improving its similarity to the consensus sequence of Escherichia colisigma 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 beta-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.
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