Bradyrhizobium ORS285 forms a nitrogen-fixating symbiosis with both Nod factor (NF)-dependent and NF-independent Aeschynomene spp. The Bradyrhizobium ORS285 ribBA gene encodes for a putative bifunctional enzyme with 3,4-dihydroxybutanone phosphate (3,4-DHBP) synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps in the riboflavin biosynthesis pathway. In this study, we show that inactivating the ribBA gene does not cause riboflavin auxotrophy under free-living conditions and that, as shown for RibBAs from other bacteria, the GTP cyclohydrolase II domain has no enzymatic activity. For this reason, we have renamed the annotated ribBA as ribBX. Because we were unable to identify other ribBA or ribA and ribB homologs in the genome of Bradyrhizobium ORS285, we hypothesize that the ORS285 strain can use unconventional enzymes or an alternative pathway for the initial steps of riboflavin biosynthesis. Inactivating ribBX has a drastic impact on the interaction of Bradyrhizobium ORS285 with many of the tested Aeschynomene spp. In these Aeschynomene spp., the ORS285 ribBX mutant is able to infect the plant host cells but the intracellular infection is not maintained and the nodules senesce early. This phenotype can be complemented by reintroduction of the 3,4-DHBP synthase domain alone. Our results indicate that, in Bradyrhizobium ORS285, the RibBX protein is not essential for riboflavin biosynthesis under free-living conditions and we hypothesize that its activity is needed to sustain riboflavin biosynthesis under certain symbiotic conditions.

Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

缓生根瘤菌ORS285与依赖Nod因子（NF）和不依赖NF的Aeschynomene spp形成固氮共生。在慢生ORS285 RIBBA基因编码推定的双功能酶与3,4- dihydroxybutanone磷酸盐（3,4- DHBP）合酶和鸟苷三磷酸（GTP）环化II活性，催化在核黄素生物合成途径的初始步骤。在这项研究中，我们显示灭活ribBA基因在自由生活条件下不会引起核黄素营养缺陷，并且，如来自其他细菌的RibBAs所示，GTP环水解酶II结构域没有酶活性。因此，我们将带注释的ribBA重命名为ribBX。由于我们无法鉴定缓生根瘤菌ORS285基因组中的其他ribBA或ribA和ribB同源物，我们假设ORS285菌株可以使用非常规酶或核黄素生物合成初始步骤的替代途径。沉静ribBX对的相互作用产生极大的影响根瘤菌与许多测试的ORS285合萌属。在这些Aeschynomene物种中，ORS285 ribBX该突变体能够感染植物宿主细胞，但不能维持细胞内感染并且结节较早衰老。该表型可以通过单独引入3，4-DHBP合酶结构域来补充。我们的结果表明，在缓生根瘤菌ORS285中，RibBX蛋白对于自由生活条件下的核黄素生物合成不是必需的，我们假设在某些共生条件下需要其活性来维持核黄素的生物合成。

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