Identification of two classes of Rhizobium phaseoli genes required for melanin synthesis, one of which is required for nitrogen fixation and activates the transcription of the other

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Abstract
The symbiotic plasmid pRP2JI of Rhizobium phaseoli strain 8002 was shown to contain two separate regions of DNA which are required and sufficient for the synthesis of the pigment melanin. One of these regions containing the class II mel gene(s) was located to other genes involved in nodulation and in nitrogen fixation. Mutations in this region abolished both the ability to synthesize melanin and to fix nitrogen in Phaseolus bean root nodules. Mutations in the other, unlinked region, containing class I mel gene(s), also abolished melanin synthesis but did not affect symbiotic nitrogen fixation. Transcriptional fusions between the class I mel gene and the Escherichia coli lacZ gene were constructed and it was demonstrated that the class II mel gene(s) activated their transcription in free-living culture. Further, strains containing the cloned regulatory class II gene(s) synthesized melanin when growing in minimal medium, in contrast to wild-type strains which became pigmented only in complete medium containing yeast extract and tryptone. It was shown by hybridization experiments that the regulatory mel gene was closely linked to or may correspond to the regulatory nifA gene; a fragment of R. phaseoli DNA which included the class II gene(s) of R. phaseoli hybridized to a previously identified nifA-like gene of R. leguminosarum, the species that nodulates peas.