Three new NifA-regulated genes in the Bradyrhizobium japonicum symbiotic gene region discovered by competitive DNA-RNA hybridization

The so-called symbiotic region of the Bradyrhizobium japonicum chromosome ( C. Kundig, H, Hennecke, and M. Gottfert, J. Bacteriol. 175:613-622, 1993) w as screened for the presence of genes controlled by the nitrogen fixation r egulatory protein NifA. Southern blots of restriction enzyme-digested cosmi ds that represent an ordered, overlapping library of the symbiotic region w ere competitively hybridized with in vitro-labeled RNA from anaerobically g rown wild-type cells and an excess of RNA isolated either from anaerobicall y grown nifA and rpoN mutant cells or from aerobically grown wild-type cell s. In addition to the previously characterized nif and fix gene dusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC (nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded by nrgC and nrgA exhibited amino acid s equence similarity to bacterial hydroxylases and N-acetyltransferases, resp ectively. The product of nrgB showed no significant similarity to any prote in with a database entry. Primer extension experiments and expression studi es with translational lacZ fusions revealed the presence of a functional -2 4/-12-type promoter upstream of nrgA and nrgBC and proved the NifA- and Rpo N (sigma(54))-dependent transcription of the respective genes. Null mutatio ns introduced into nrgA and nrgBC resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tes ted on soybean, cowpea, or mung bean host plants. Thus, the discovery of nr gA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.