RHIZOBIUM-MELILOTI MUTANTS DEFECTIVE IN SYMBIOTIC NITROGEN-FIXATION AFFECT THE OXYGEN GRADIENT IN ALFALFA (MEDICAGO-SATIVA) ROOT-NODULES

MASEPOHL B, WITTY JF, RIEDEL KU, KLIPP W, Pühler A. RHIZOBIUM-MELILOTI MUTANTS DEFECTIVE IN SYMBIOTIC NITROGEN-FIXATION AFFECT THE OXYGEN GRADIENT IN ALFALFA (MEDICAGO-SATIVA) ROOT-NODULES. JOURNAL OF EXPERIMENTAL BOTANY. 1993;44(2):419-426.Rhizobium meliloti bacteroids carrying mutations in either fdxN or fixX isolated from alfalfa root nodules were shown to contain the nitrogenase proteins NifH, NifD and NifK. In contrast to an in vitro system or N2-fixation based on R. meliloti wild-type bacteroids, nitrogenase activity could not be restored in crude extracts of these mutant bacteroids by the addition of an artificial electron donor, indicating that the nitrogenase proteins were present but not functional. ESR-studies revealed that both mutants lacked the FeMo-cofactor of nitrogenase. To analyse the role of free O2 on the damage of the nitrogenase components and the FeMo-cofactor in these mutant bacteroids, microelectrode studies of O2 concentrations and gradients within alfalfa root nodules were carried out. R. meliloti mutants defective in other genes necessary for symbiotic N2-fixation were also included in this study. Four distinct types of O2 gradients were defined by the apparent presence or absence of an O2 diffusion barrier and by the minimum internal O2 concentration. These data clearly demonstrated the influence of the microsymbiont on the O2 gradients within the nodules. Nodules induced by Rm0540, an R. meliloti mutant with altered exopolysaccharide production, which is not able to infect plant cells, did not contain an O2 diffusion barrier. In contrast, nodules containing a mutant defective in dicarboxylate transport (dctA-), produced an O2 gradient similar to the wild-type. Microelectrode measurements revealed H-2 concentrations in alfalfa wild-type nodules comparable to soyabean. whereas no hydrogen could be detected in nodules harbouring the dctA mutant or any other mutant strain