2ND SITE MUTATIONS SPECIFICALLY SUPPRESS THE FIX(-) PHENOTYPE OF RHIZOBIUM-MELILOTI NDVF MUTATIONS ON ALFALFA - IDENTIFICATION OF A CONDITIONAL NDVF-DEPENDENT MUCOID COLONY PHENOTYPE

Rhizobium meliloti mutants carrying ndvF insertion or deletion mutatio ns induce nodules on alfalfa which contain very few infected cells and fail to fix N-2 (Fix(-)). We have characterized five independent seco nd site mutations (designated sfx) which completely suppress the Fix(- ) phenotype of ndvF mutants on Medicago sativa but not on another R. m eliloti host Melilotus alba. Genetic mapping and phenotypic analysis r evealed that the suppressor mutations sfx-1, sfx-4 and sfx-4 mapped to a single locus which was distinct from another locus defined by the s fx-2 and sfx-3 mutations. Tn5-mob-mediated conjugal mapping experiment s showed that the sfx-1 locus was located clockwise from trp-33 on the R. meliloti chromosome and a detailed cotransduction map of this regi on was generated. To clone the sfx-1 locus, we prepared a cosmid libra ry from total DNA obtained from an sfx-1, ndvF deletion strain. From t his library, a cosmid pTH56, which converted Fix(-) ndvF mutants to Fi x(+), was isolated. Southern blot analysis provided direct physical ev idence that the insert DNA in plasmid pTH56 was contiguous with the sf x-1 region. On low osmolarity glutamate-yeast extract-mannitol-salts m edium (GYM) agar medium, ndvF insertion and deletion mutants were foun d to have a mucoid colony phenotype, as opposed to the dry colony phen otype of the wild-type strain. This phenotype was shown to be dependen t on the exoB and expE genes required for synthesis of exopolysacchari de II in R. meliloti but not to be dependent on genes required exclusi vely for the synthesis of the succinoglycan or exopolysaccharide I. Tr ansduction of either sfx-1 or sfx-2 or transfer of the cosmid pTH56 in to the ndvF mutants restored them to a wild-type dry colony phenotype. The mucoid phenotype is not responsible for the Fix phenotype of ndvF mutants as the Fix(-), ndvF exp double mutants can be complemented to Fix(+) by introducing plasmids which carry only the wild-type ndvF ge nes.