TRANSCRIPTIONAL CONTROL AND ESSENTIAL ROLES OF THE ESCHERICHIA-COLI CCM GENE-PRODUCTS IN FORMATE-DEPENDENT NITRITE REDUCTION AND CYTOCHROME-C SYNTHESIS

The eight ccm genes located at minute 47 on the Escherichia coli chrom osome, in the order ccmABCDEFGH, encode homologues of proteins which a re essential for cytochrome c assembly in other bacteria. The ccm gene s are immediately downstream from the napFDAGHBC genes encoding a peri plasmic nitrate reductase. CcmH was previously shown to be essential f or cytochrome c assembly. Deletion analysis and a two-plasmid strategy have now been used to demonstrate that CcmA, B, D, E, F and G are als o essential for cytochrome c assembly, and hence for cytochrome-c-depe ndent nitrite reduction. The ccm genes are transcribed from a ccmA pro moter located within the adjacent gene, napC, which is the structural gene for a 24 kDa membrane-bound c-type cytochrome, NapC. Transcriptio n from this ccmA promoter is induced approximately 5-fold during anaer obic growth, independently of a functional Fnr protein: it is also not regulated by the ArcB-ArcA two-component regulatory system. The ccmA promoter is an example of the 'extended -10 sequence' group of promote rs with a TGX motif immediately upstream of the -10 sequence. Mutagene sis of the TG motif to TC, CT or CC resulted in loss of about 50 % of the promoter activity. A weak second promoter is suggested to permit t ranscription of the downstream ccmEFGH genes in the absence of transcr iption readthrough from the upstream napF and ccmA promoters. The resu lts are consistent with, but do not prove, the current view that CcmA, B, C and D are part of an essential haem transport mechanism, that Cc mE, F and H are required for covalent haem attachment to cysteine-hist idine motifs in cytochrome c apoproteins in the periplasm, and that Cc mG is required for the reduction of cysteine residues on apocytochrome s c in preparation for haem ligation.