From intracellular bacteria to differentiated bacteroids: transcriptome and metabolome analysis in Aeschynomene nodules using the Bradyrhizobium sp. ORS285 bclA mutant

During the legume-rhizobium symbiosis, free-living soil bacteria known as rhizobia trigger the formation of root nodules. The rhizobia infect these organs and adopt an intracellular lifestyle within the symbiotic nodule cells where they become nitrogen-fixing bacteroids. Several legume lineages enforce their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this differentiation process in Bradyrhizobium sp. ORS285, a symbiont of Aeschynomene spp.. In the absence of BclA, Bradyrhizobium sp. ORS285 proceeds until the intracellular infection of nodule cells but the bacteria cannot differentiate into enlarged polyploid bacteroids and fix nitrogen. The nodule bacteria of the bclA mutant constitute thus an intermediate stage between the free-living soil bacteria and the intracellular nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the ORS285 wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules discriminated nodule-induced genes that are specific to differentiated and nitrogen-fixing bacteroids and others that are activated in the host microenvironment irrespective of bacterial differentiation and nitrogen fixation. These analyses demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied with a first transcriptome switch involving several hundreds of upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving less genes but that are expressed to extremely elevated levels. The transcriptomes further highlighted the dynamics of oxygen and redox regulation of gene expression during nodule formation and we discovered that bclA represses the expression of non-ribosomal peptide synthetase gene clusters suggesting a non-symbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids. 3 conditions with 3 replicates each for each condition are analyzed. Two nodule bacteria conditions are considered : one from A. indica nodules and from A. afraspera nodules, as well as a culture reference in rich medium

在豆科植物-根瘤菌（rhizobia）共生过程中，被称为根瘤菌的自由生活土壤细菌会诱导根瘤形成。根瘤菌会侵染这些器官，并在共生根瘤细胞内采取胞内生活方式，进而转变为具有固氮能力的类菌体（bacteroids）。多个豆科植物谱系会促使其共生菌发生极端细胞分化，包括细胞体积增大和基因组内复制。抗菌肽转运蛋白BclA是慢生根瘤菌属（Bradyrhizobium sp.）ORS285菌株——一种田菁属（Aeschynomene spp.）共生菌——的该分化过程的主要决定因子。当缺失BclA时，慢生根瘤菌ORS285仍能完成对根瘤细胞的胞内侵染，但无法分化为体积增大的多倍体类菌体并固氮。因此，bclA突变体的根瘤细菌处于自由生活土壤细菌与胞内固氮类菌体之间的中间阶段。对感染ORS285野生型或bclA突变体的非洲田菁（Aeschynomene afraspera）和印度田菁（Aeschynomene indica）的完整根瘤进行代谢组学分析，共鉴定出47种伴随类菌体分化差异积累的代谢物。对这些根瘤的细菌进行转录组分析，可区分出两类根瘤诱导基因：一类仅在分化完成且具备固氮能力的类菌体中特异性表达，另一类则在宿主微环境中被激活，与细菌分化和固氮状态无关。这些分析表明，根瘤菌在共生根瘤细胞内的定殖伴随两次转录组转变：第一次转变涉及数百个上调与下调基因，第二次转变伴随类菌体分化，涉及的基因数量更少，但表达水平会大幅升高。转录组数据还揭示了根瘤形成过程中氧气与氧化还原对基因表达的调控动态，我们还发现BclA会抑制非核糖体肽合成酶基因簇的表达，这提示BclA存在非共生功能。综上，我们的研究揭示了从胞内细菌到分化完全的固氮类菌体转变过程中伴随的代谢与基因表达变化。本研究共分析3种实验条件，每种条件设置3次生物学重复；所涉及的根瘤细菌条件包括两类：分别取自印度田菁和非洲田菁的根瘤细菌，以及富培养基中的培养对照。