title: Overexpression of ccl1-2 can bypass the need for the putative apocytochrome chaperone CycH during the biogenesis of c-type cytochromes.
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.
authors.affiliation: Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Meenal
authors.initials: M
authors.lastname: Deshmukh
authors.forename: Matthew
authors.initials: M
authors.lastname: May
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Karen K
authors.initials: KK
authors.lastname: Gabbert
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Karberg
authors.forename: Robert G
authors.initials: RG
authors.lastname: Kranz
authors.forename: Fevzi
authors.initials: F
authors.lastname: Daldal
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009876:Operon; D010940:Plant Proteins; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics; D013489:Suppression, Genetic
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D008565:Membrane Proteins; D018832:Molecular Chaperones; D010940:Plant Proteins; D011506:Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases