PMH2, A SMALL PLASMID BEARING THE NIF GENE-CLUSTER OF ENTEROBACTER-AGGLOMERANS-333 AS AN EXCISABLE CASSETTE

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E . agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nif A,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carr ies the complete nif gene cluster as a nif cassette on a single XbaI f ragment. The nif construct pMH2 in Escherichia coli strains has signif icant nitrogenase activity compared to wild-type E. agglomerans 333. T he nif gene cluster construct was found to be very stable.