A RHIZOBIUM-LEGUMINOSARUM GENE REQUIRED FOR SYMBIOTIC NITROGEN-FIXATION, MELANIN SYNTHESIS AND NORMAL GROWTH ON CERTAIN GROWTH MEDIA

The gene nfrX in Azotobacter vinelandii activates transcription of other nif genes in that species. A cosmid containing cloned Rhizobium leguminosarum DNA that corrected the Nif- defect of an nfrX mutant of A. vinelandii was isolated. Following Tn5 transposon mutagenesis of the cosmid in Escherichia coli, mutant derivatives unable to correct the A. vinelandii nfrX mutants were obtained in two separate regions of DNA. In addition, mutations close to one of the nfrX regions conferred a complex phenotype when introduced into the Rhizobium genome by marker exchange. These mutants induced non-fixing nodules on peas, were slow-growing on media with succinate as C source or nitrate as N source and, when present in R. leguminosarum biovar phaseoli, they failed to make melanin, a pigment that is normally synthesized by R. l. bv. phaseoli. The mutated gene, termed melC, was fused to uidA (which encodes beta-glucuronidase); it was found that transcription of melC-uidA was enhanced in microaerobic conditions and that it was expressed at high levels in infection threads in pea nodules.