Symbiotic and Nutritional Phenotypes of Rhizobium Etli PurH Mutants

The main goal of this project was to determine if the rhizobial production of 5-aminoimidazole-4-carboxamide ribotide (AI CAR) is required for the infection and nodulation of bean plants by rhizobia bacteria. One possibility suggested by the symbiotic deficiency of previously isolated purine auxotrophs is that perhaps AI CAR or a derivative is an infection factor. As the first step in an approach to test the hypothesis that AI CAR is an infection factor, mutants that produce AI CAR but cannot use it for purine synthesis were isolated. Such mutants were obtained by site-directed mutagenesis of the Rhizobium etli purH gene. The mutants that resulted were the starting material for this project. 0 The first objectives were to verify that these mutants had the expected genotype and phenotype. The genotypes of the purR mutants were confirmed by Southern hybridization using apurH-specific probe. As expected, the mutants accumulated AICAr (AICA riboside), and, surprisingly, did so even under nonstarvation conditions. However, the purH mutants had an unexpected nutritional phenotype. They did not grow on adenine or hypoxanthine as sole purine sources except when histidine was also added. This phenotype was designated PurH· Hhr+ (purH histidine r equirement). Secondary mutants were isolated that grew on purines without histidine, but were still purine auxotrophs and could not use AICAr as a purine source. The PurH· Hhr· double mutants were used in nodulation tests, certain results of which would have ruled out the hypothesis that AI CAR is an infection factor. These muf.a nts were symbiotically defective without supplementation and with only AICAr supplementation. However, the mutants elicited enhanced nodule development when a purine source was provided. These results showed that the plant does not supply enough purine to allow infection. On the main question, the results neither supported nor ruled out that AICAR is a specifically required factor for infection. A more definitive test will require a PurE· PurH· Hhr- His· mutant