Dissection of the Bradyrhizobium japonicum NifA+sigma54 regulon, and identification of a ferredoxin gene (fdxN) for symbiotic nitrogen fixation.

Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+sigma(54) regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN (1/2) mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and sigma(54)-binding sites plus a simple operon definition. One of the newly identified NifA+sigma(54)-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional sigma(54 )promoter with the dinucleotide GA instead of the consensus GC motif at position -12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+sigma(54) targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+sigma(54 )regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processes.