Changes in the Rhizobium meliloti Genome and the Ability to Detect Supercoiled Plasmids During Bacteroid Development. Roger Wheatcroft. Plant Research Centre, Agriculture Canada, Ottawa, Ontario K1A OC6, Canada. D. G. McRae, and R. W. Miller. Plant Research Centre, Agriculture Canada, Ottawa, Ontario K1A OC6, Canada.. MPMI 3:9-17. Accepted 15 August 1989. Copyright 1990 The American Phytopathological Society.

Medicago sativa (alfalfa) roots were infected with Rhizobium meliloti strain Balsac. Nodule extracts were fractionated isopycnically on Percoll density gradients into nodule bacteria, transforming bacteria, and mature bacteroids. The average DNA content of mature bacteroids was found to be double that of free-living cells grown in liquid culture to stationary phase. This corresponded to the ratio of discrete nucleoid structures observed in stained cells of the two types. Probing blots of restricted total cellular DNA with plasmid-specific and other DNA probes, including nod, nif, exo, dct, and other fix gene regions, gave no indication of structural rearrangement or unequal amplification of the genome during bacteroid development. Supercoiled plasmids of Balsac were detected in Eckhardt gels and purified on ethidium bromide-CsCl density gradients from free-living cells but not from mature bacteroids. Transforming bacteria were intermediate in that plasmids other than megaplasmids were detected in gels. We conclude that plasmid supercoiling is not maintained in nitrogen-fixing bacteroids in the nodule or is readily lost upon their extraction, or both. We suggest that modification of the DNA superhelical structure may be important in bacteroid development and for the expression of symbiotic genes in R. meliloti.