Molecular cloning and functional analysis of the cysG and nirB genes of Escherichia coli K12, two closely-linked genes required for NADH-dependent nitrite reductase activity

Abstract

We have cloned two genes, nirB+ and cysG+ which are required for NADH-dependent nitrite reductase to be active, from the 74 min region of the Escherichia coli chromosome. Restriction mapping and complementation analysis establish the gene order crp-nirB-cysG-aroB. Both genes are trans-dominant in merodiploids and, under some conditions, can be expressed independently. The cysG+ gene can be expressed from both high and low copy number plasmids carrying a 3.6 kb PstI-EcoRI restriction fragment. Attempts to sub-clone the nirB+ gene into pBR322 on a 14.5 kb EcoRI fragment were unsuccessful, but this fragment was readily sub-cloned into and expressed from the low copy number plasmid pLG338 (Stoker et al. 1982). Overproduction of the 88 kDa nitrite reductase apoprotein by strains carrying a functional nirB+ gene suggests that nirB is the structural gene for this enzyme.