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 |
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Authors: | Heather Macdonald and Jeff Cole |
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Institution: | (1) Department of Biochemistry, University of Birmingham, B15 2TT Birmingham, U.K. |
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Abstract: | Summary 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. |
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