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

Abstract

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::Tn5) and ANU2866 (purY::Tn5), both derivatives of ANU280, are unable to nodulate siratro plants. In contrast, strain L1 (purF::Tn5ssgusA40), a derivative of ANU843, is able to induce fully-developed nodules and normal levels of nodulation on several clover hosts. To our knowledge, strain L1 is the only genetically-defined pur^– mutant capable of inducing fully-developed nodules on a legume host. None of the pur^– mutants are able to grow at normal rates in vitro even in the presence of purine pathway supplements including 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 by genetic complementation. We confirmed that supplementation of pur intermediates 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 protein species present. In addition, strain ANU2866 but not strains ANU2861 or L1 could not tolerate the effects of nutrient step-up or step-down transitions, and failed to produce any colonies on laboratory media. Collectively, these data show that purine auxotrophs suffer pleiotropic effects at the level of protein synthesis and their overall metabolism is compromised. Therefore, care should be exercised in concluding that purine intermediates or by-products of this pathway are required per se for nodule development.