Rhizobium sp. NGR234 is a broad-host range strain. The rpoN gene of this organism encodes a sigma factor which is a primary co-regulator of endosymbiosis. We characterized the locus upstream of rpoN, and identified a contiguous open reading frame, here termed ORF1. DNA sequence analysis of this ORF showed that it encoded a polypeptide highly conserved with a corresponding ORF of Rhizobium meliloti. The gene product contained two ATP/GTP binding pockets. Codon usage in the ORF and the nitrogenase operon nifKDH of NGR234 was similar. Although we used a non-transposable cassette flanked by appropriate sized DNA fragments, we were unable to isolate site-directed mutants in the ORF, whose ATP/GTP binding protein product is thus probably of essential biological function. ORF1 and rpoN exhibited conserved linkage among diverse rhizobia, and in Azotobacter vinelandii. Intragenomic and interspecific homology studies confirmed directly that ORF1 (NGR234) belonged to a large family of ATP-binding protein genes.