SITE-SPECIFIC MUTAGENESIS IN ENTEROBACTER-AGGLOMERANS - CONSTRUCTION OF NIFB MUTANTS AND ANALYSIS OF THE GENES STRUCTURE AND FUNCTION

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomeran s. The method is based on the observation that E. agglomerans can be c ured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique , we inserted a kanamycin cassette into the cloned nifB gene, transfer red it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transforma nts on citrate medium with kanamycin, The nifB(-) mutants with the kan amycin cassette inserted in either orientation showed a nif(-) phenoty pe. Further, we determined the nucleotide sequence of nifB. A typical sigma(54)-dependent promoter and a consensus NifA binding site were fo und upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amin o acid sequence of the NifB protein showed strong similarity to the Ni fB sequences of other diazotrophic bacteria, The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.