Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, a-4 (RpoN NtrA GlnF). In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoNj and rpoN2) were identified and sequenced. The two predicted B. japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to NGR234 (81,88), Rhodobacter capsulatus (1,44,47), and Thiobacillus ferrooxidans (7). The DNA sequences of the rpoN genes of several species have been determined, and the deduced protein sequences were clearly homologous (see Fig. 4). Interestingly, no substantial homology to other known bacterial u factors was observed. Consistent with the finding that, within a given species, -24/-12 promoters were associated with functionally unrelated genes, many rpoN mutants showed a pleiotropic phenotype. For example, an R. meliloti rpoN mutant was not only unable to fix nitrogen symbiotically but also was affected in nitrate assimilation, transport of C4-dicarboxylates, and nodulation efficiency (22,73).In the soybean root nodule endosymbiont Bradyrhizobium japonicum, numerous genes involved in nitrogen fixation (nif and fix genes) have been identified in at least two chromosomal gene clusters (35). Most of them were shown by sequence analysis and transcript mapping to be preceded by -24/-12 promoters (36). In addition, -24/-12 promoters were identified upstream of the B. japonicum glnII and glnB
A special sequence motif in the Bradyrhizobium japonicum NifA protein, consisting of two functionally essential cysteines separated by four other amino acids (Cys-aa4-Cys), has been proposed to be part of a potential metal-binding site ) Nucleic Acids Res. 16, 2207-2224. Using the techniques of oligonucleotide-directed mutagenesis, we report here that several of the four intervening amino acids can be replaced by others without loss of NifA function. The deletion of one amino acid to give a Cys-aa3-Cys motif renders the protein inactive whereas the creation of a Cys-aa 5-Cys motif (one amino acid inserted) still leads to a partially active NifA protein.
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