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

Autor: Deshmukh, Meenal, May, Matthew, Zhang, Yan, Gabbert, Karen K., Karberg, Katherine A., Kranz, Robert G., Daldal, Fevzi
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Zdroj: Molecular Microbiology; Nov2002, Vol. 46 Issue 4, p1069, 12p
Abstrakt: 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[sub 1] and c[sub 2] or c[sub y]. However, Ps[sup +] 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[sup +] growth by a genomic library constructed using DNA from a Ps[sup +] 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 coliσ[sup 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[sup +] 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[sup +] phenotype on a CycH-null mutant. These findings therefore demonstrate that, during R. capsulatus growth on minimal medium, the requirement for... [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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