Isolation of the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 all components of the ubiquinol: cytochrome c2 oxidoreductase complex of Rhodopseudomonas capsulata

Abstract

The structural genes for the Rieske Fe-S protein (petA), cytochrome b (petB) and cytochrome c1 (petC) subunits of the ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodopseudomonas capsulata have been cloned by complementation, using a mutant defective in this complex. The location of these genes on the obtained plasmid, pR14A, was determined using synthetic mixed oligonucleotide probes corresponding to highly conserved amino acid sequences of these proteins from various organisms. Their correct identity was established by partial sequencing. The petA, petB and petC genes were found to lie close to each other in this order, spanning two adjacent EcoRI fragments of 2.7 X 10(3) and 1.3 X 10(3) base-pairs, respectively. An insertion-deletion mutation, covering most of petB and all of petC and an insertion mutation, located in petB were constructed in vitro and were introduced into the chromosome of an otherwise wild-type strain by gene transfer agent-mediated genetic crosses. The bc-1 mutants obtained were defective in photosynthesis but, as expected, they could grow by respiration because of a branched respiratory pathway. Therefore, in R. capsulata a functional bc1 complex is essential in vivo for photosynthesis but not for respiration. Further, in the respiratory pathway the branch point must be before the bc1 complex, most likely at the quinone pool. These mutants were also proficient in anaerobic growth in the presence of dimethylsulfoxide, indicating that a functional bc1 complex is not required for this pathway. Several other insertions and deletions, located outside of the pet gene cluster, were also constructed. The ability of these latter mutants to grow photosynthetically suggested that no other gene essential for photosynthesis is located in the proximity of the pet cluster. The plasmid pR14A was shown to complement in trans the bc-1 insertion or insertion-deletion mutants, indicating that the pet genes were expressed in R. capsulata. Cross-hybridization experiments showed that the pet cluster was quite distinct from other known genes involved in photosynthesis.