The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.

In this paper, we examine the importance of glutathione in symbiosis, using a glutathione biosynthetic gshB mutant derived from Rhizobium tropici CIAT899, a common bean (Phaseolus vulgaris) endosymbiont. Plants infected with the mutant strain presented a delayed nodulation phenotype and a reduction in the dry weight of aerial part of plants, suggesting diminished nitrogen-fixation activity. In addition, bacterial gshB expression was assayed in wild-type infected nodules, during the different steps of nodulation, and found to increase in mature and early senescent nodules. Conspicuously, nodules induced by gshB mutant bacteria presented an early senescent pattern, which was associated with increased levels of superoxide accumulation. These results provide a direct evidence of the role of bacterial glutathione in protecting nodules from reactive oxygen species, which may determine nodule senescence.