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) superfamily, 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),-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 css0 to much lower levels than other c-type cytochrome deficient mutants of P. denifrificans but expression and translocation of an apocytochrome c5%-alkaline phosphatase fusion protein and apocytochrome cd, were unaffected in HN53. The results suggest that the substrate for the putative CcmABCtransporter is probably neither haem nor c-type apocytochromes.

Apocytochrome ~5 5 0 was detected in the periplasm of a new mutant of Paracoccus denitrificans, HN48, that is pleiotropically lacking c-type cytochromes, produces reduced levels of siderophores and carries a T n 5 insertion in the ccmF gene for which sequence data, along with that for the contiguous ccmH, are reported. A counterpart to the ccmF gene was found in an archaebacterium but could not be located in the yeast genome, whereas mitochondria1 haem lyases in the latter were not present in an archaeobacterial or in eubacterial genomes. A topological analysis for CcmF is presented which indicates a t least eleven transmembrane helices, suggesting a role as a transporter; evidence against the substrate being haem is presented but sequence similarity with Escherichia coli y-aminobutyric acid transporter was identified. Analysis by pulse-chase methodology has shown that, in this and another cytochrome-c-def icient mutant, the apo form of P. denitrificans cytochrome css0 is much less stable than the holo form, directly demonstrating the presence of a periplasmic degradation system in P. denitrificans that removes non-functional proteins. A variety of phenotypes are observed for P. denitrificans mutated in different ccm genes, thus indicating that the stability of the ccm gene products does not require assembly of a complex of all the Ccm proteins.