Site-specific mutagenesis in Enterobacter agglomerans: construction of nif B mutants and analysis of the gene's structure and function.

Abstract

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured 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, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB- mutants with the kanamycin cassette inserted in either orientation showed a nif- phenotype. Further, we determined the nucleotide sequence of nifB. A typical sigma 54-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.