Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Mol Plant Microbe Interact. 2010 Dec;23(12):1643-54. doi: 10.1094/MPMI-07-10-0151.
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 Sinorhizobium meliloti requires cobalamin to establish a symbiotic relationship with its plant host, Medicago sativa (alfalfa). Here, the specific requirement for cobalamin in the S. meliloti-alfalfa symbiosis was investigated. Of the three known cobalamin-dependent enzymes in S. meliloti, 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 coli cobalamin-independent (class Ia) 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 bacteroids 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 Ia RNR by reactive oxygen species, inadequate oxygen availability in the nodule, or both. These results 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.
维生素 B(12)(钴胺素)是动物和原生生物的关键辅助因子,但它的生物合成仅限于原核生物。我们之前曾表明,共生固氮α变形菌苜蓿中华根瘤菌 (Sinorhizobium meliloti) 需要钴胺素来与其植物宿主紫花苜蓿(苜蓿)建立共生关系。在这里,研究了钴胺素在 S. meliloti-苜蓿共生中的具体需求。在 S. meliloti 中,三种已知的钴胺素依赖性酶中,甲基丙二酰辅酶 A 变位酶 (BhbA) 不会影响共生,而编码钴胺素依赖性蛋氨酸合酶的 metH 基因的破坏会导致共生严重缺陷。钴胺素非依赖性蛋氨酸合酶 MetE 的表达缓解了这种共生缺陷,表明对蛋氨酸合成的需求并不反映对钴胺素依赖性酶的需求。为了研究由 nrdJ 编码的钴胺素依赖性核糖核苷酸还原酶 (RNR) 的功能,工程改造 S. meliloti 以表达大肠杆菌的钴胺素非依赖性(Ia 类)RNR 代替 nrdJ。该菌株在共生中严重缺陷。电子显微镜照片显示,这些细胞可以穿透苜蓿根瘤,但无法分化成固氮类菌体,而是在植物细胞质中溶解。流式细胞术分析表明,这些细菌基本上无法进行内复制。这些表型可能是由于 Ia 类 RNR 被活性氧失活、根瘤中氧气供应不足或两者兼而有之。这些结果表明,钴胺素依赖性 RNR 对 S. meliloti 在其植物宿主中的存活的关键作用可以解释 S. meliloti 为钴胺素生物合成所投入的大量资源。