ABSTRACT
A total of 96 independent Tn5 insertions within a 39-kilobase-pair (kbp) segment of chromosomal DNA containing the three structural genes for nitrogenase (nifH, nifD, and nifK) from Bradyhizobium japonicum I110 were obtained in Escherichia coli and transferred to the wild-type strain by marker exchange. Individual transconjugants containing a Tn5 insertion were inoculated onto Glycine max cv. Wilkin (soybeans) and analyzed for their effect on symbiotic nitrogen fixation. In addition to the three structural genes, genes essential for nitrogen fixation (fix genes) were located in three separate regions: 9 kbp upstream of the nifDK operon; 1.5 kbp downstream of the nifDK operon; 4.5 kbp upstream of nifH. All of the fix::Tn5 insertion strains formed nodules which contained low or undetectable levels of nitrogenase activity. Bacteroids isolated from these nodules had approximately the same levels of the nifDK and nifH transcripts as those detectable from nodules formed by the wild-type strain. Western blot analysis of bacteroid proteins from nodules formed by the fix::Tn5 mutants or the wild-type strain showed the presence of similar levels of the nitrogenase protein subunits. The region upstream of nifH was characterized further by DNA sequence analysis and was shown to contain the nifB gene. The coding sequence of the nifB gene consisted of 1,494 nucleotides and was preceded by putative promoter (5' GTGG-10 base pairs [bp] TTGCA 3') and upstream activator (5' TGT-4 bp-T-5 bp-ACA 3') sequences.
