The Paracoccus denitrificans ccmA, B and C genes: cloning and sequencing, and analysis of the potential of their products to form a haem or apo- c-type cytochrome transporter

Abstract

Two c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marker rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC (ATP-binding cassette) super-family, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 were shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)₂-CcmB-CcmC stoichiometry; it also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome c ₅₅₀ to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome c ₅₅₀-alkaline phosphatase fusion protein and apocytochrome cd ₁ were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.