Photosynthetic Bradyrhizobium strains possess the unusual ability to form nitrogen-fixing nodules on a specific group of legumes in the absence of Nod factors. To obtain insight into the bacterial genes involved in this Nod-independent symbiosis, we screened 15,648 Tn5 mutants of Bradyrhizobium sp. strain ORS278 for clones affected in root symbiosis with Aeschynomene indica. From the 268 isolated mutants, 120 mutants were altered in nodule development (Ndv-) and 148 mutants were found to be deficient in nitrogen fixation (Fix-). More than 50% of the Ndv- mutants were found to be altered in purine biosynthesis, strengthening the previous hypothesis of a symbiotic role of a bacterial purine derivative during the Nod-independent symbiosis. The other Ndv- mutants were auxotrophic for pyrimidines and amino acids (leucine, glutamate, and lysine) or impaired in genes encoding proteins of unknown function. The Fix- mutants were found to be affected in a wide variety of cellular processes, including both novel (n = 56) and previously identified (n = 31) genes important in symbiosis. Among the novel genes identified, several were involved in the Calvin cycle, suggesting that CO2 fixation could play an important role during this symbiosis.