Sinorhizobium meliloti Requires a Cobalamin-Dependent Ribonucleotide Reductase for Symbiosis With Its Plant Host

Vitamin B-12 (cobalamin) is a critical cofactor for animals and protists, yet its biosynthesis is limited to prokaryotes We previously showed that the symbiotic nitrogen-fixing alphaproteobacterium Sinorhtzobium melilott requires cobalamin to establish a symbiotic relationship with its plant host, Medicago saliva (alfalfa) Here, the specific requirement for cobalamin in the S melilott alfalfa symbiosis was investigated Of the three known cobalamin-dependent enzymes in S melilott, the methylmalonyl CoA mutase (BhbA) does not affect symbiosis, whereas disruption of the metH gene encoding the cobalamin-dependent methionine synthase causes a significant defect in symbiosis Expression of the cobalamin-Independent methionine synthase MetE alleviates this symbiotic defect, indicating that the requirement for methionine synthesis does not reflect a need for the cobalamin-dependent enzyme To investigate the function of the cobalamin-dependent ribonucleotide reductase (RNR) encoded by nrdJ, S meliloti was engineered to express an Escherichia colt cobalamin-independent (class la) RNR instead of nrdJ This strain is severely defective in symbiosis Electron micrographs show that these cells can penetrate alfalfa nodules but are unable to differentiate Into nitrogen-fixing bacterolds and, Instead, are lysed in the plant cytoplasm Flow cytometry analysis indicates that these bacteria are largely unable to undergo endoreduplication These phenotypes may be due either to the inactivation of the class la RNR by reactive oxygen species, inadequate oxygen availability in the nodule, or both These re sults show that the critical role of the cobalamin-dependent RNR for survival of S meliloti in its plant host can account for the considerable resources that S meliloti dedicates to cobalamin biosynthesis