Rhizobium purine auxotrophs, perturbed in nodulation, have multiple changes in protein synthesis

We investigated the infectivity, growth ability and the overall biochemical status of Rhizobium purine auxotrophs to determine why these strains show varied nodulation phenotypes on their host plants. Strains ANU2861 (purM::T n5) and ANU2866 (purY::Tn5), both derivatives of ANU280, are unable to nodu late siratro plants. In contrast, strain L1 (purF::Tn5ssgusA40), a derivati ve of ANU843, is able to induce fully-developed nodules and normal levels o f nodulation on several clover hosts. To our knowledge, strain L1 is the on ly genetically-defined pur(-) mutant capable of inducing fully-developed no dules on a legume host. None of the pur(-) mutants are able to grow at norm al rates in vitro even in the presence of purine pathway supplements includ ing AICA-riboside. Further, the nodulation ability of the strain ANU280 pur (-) mutants is unable to be restored by the addition of AICA-riboside. Full growth and nodulation ability can only be restored to the pur(-) mutants b y genetic complementation. We confirmed that supplementation of pur interme diates failed to fully restore wild-type growth by using proteome analysis to examine the overall biochemical status of the mutants. Proteome analysis demonstrated that the purine mutants possess multiple changes in the prote in species present. In addition, strain ANU2866 but not strains ANU2861 or L1 could not tolerate the effects of nutrient step-up or step-down transiti ons, and failed to produce any colonies on laboratory media. Collectively, these data show that purine auxotrophs suffer pleiotropic effects at the le vel of protein synthesis and their overall metabolism is compromised. There fore, care should be exercised in concluding that purine intermediates or b y-products of this pathway are required per se for nodule development.