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 chromosome, in the order ccmABCDEFGH, encode homologues of proteins which are essential for cytochrome c assembly in other bacteria. The ccm genes are immediately downstream from the napFDAGHBC genes encoding a periplasmic nitrate reductase. CcmH was previously shown to be essential for 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 also essential for cytochrome c assembly, and hence for cytochrome-c-dependent nitrite reduction. The ccm genes are transcribed from a ccmA promoter located within the adjacent gene, napC, which is the structural gene for a 24 kDa membrane-bound c-type cytochrome, NapC. Transcription from this ccmA promoter is induced approximately 5-fold during anaerobic 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 promoters with a TGX motif immediately upstream of the -10 sequence. Mutagenesis 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 transcription of the downstream ccmEFGH genes in the absence of transcription readthrough from the upstream napF and ccmA promoters. The results 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 CcmE, F and H are required for covalent haem attachment to cysteine-histidine motifs in cytochrome c apoproteins in the periplasm, and that CcmG is required for the reduction of cysteine residues on apocytochromes c in preparation for haem ligation.
Tanapongpipat
S
S
School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK.
Reid
E
E
Cole
J A
JA
Crooke
H
H
D000693:Anaerobiosis; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics; D003574:Cytochrome c Group / Q000235:genetics*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005798:Genes, Bacterial; D009573:Nitrites / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D003574:Cytochrome c Group; D017931:DNA Primers; D009573:Nitrites; D011994:Recombinant Proteins; D001616:beta-Galactosidase