ESCHERICHIA-COLI K-12 GENES ESSENTIAL FOR THE SYNTHESIS OF C-TYPE CYTOCHROMES AND A 3RD NITRATE REDUCTASE LOCATED IN THE PERIPLASM

The 'aeg46.5' operon was originally detected as an 'anaerobically expr essed gene' located at minute 46.5 on the Escherichia coli linkage map , Subsequent results from the E. coli Genome Sequencing Project reveal ed that the 'aeg46.5' promoter was located in the centisome 49 (minute 47) region, Downstream from this promoter are 15 genes, seven of whic h are predicted to encode a periplasmic nitrate reductase and eight en code proteins homologous to proteins essential for cytochrome c assemb ly in other bacteria. All of these genes, together with the 'aeg46.5' promoter, have been subcloned on a 20 kb EcoRI fragment from Kohara ph age 19D1, Evidence is presented that, as predicted, the region include s structural genes for two c-type cytochromes of mass 16 kDa and 24 kD a, which are transcribed from the previously described 'aeg46.5' promo ter, and that the first seven genes encode a functional nitrate reduct ase, We, therefore, propose that they should be designated nap (nitrat e reductase in the periplasm) genes. Plasmids encoding the entire 20 k b region, or only the downstream eight genes, complemented five mutati ons resulting in total absence of all five known c-type cytochromes in E. coli, providing biochemical evidence that these are ccm (for cytoc hrome c maturation) genes, The ccm region was transcribed both from th e FNR-dependent, NarL- and NarP-regulated nap promoter (formerly the ' aeg46.5' promoter) and from constitutive or weakly regulated promoters apparently located within the downstream nap and ccm genes.