Diversity and Evolution of Hydrogenase Systems in Rhizobia

Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) strains and homogeneous intraspecific patterns within R. tropici and A. caulinodans strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of hupS and hupL clustered R. leguminosarum and R. tropici hup sequences together with those from B. japonicum and Bradyrhizobium sp. (Lupinus) strains, suggesting a common origin. In contrast, Bradyrhizobium sp. (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.

摄取型氢化酶可使根瘤菌回收豆科根瘤内固氮过程中产生的氢气。大豆慢生根瘤菌（Bradyrhizobium japonicum）与豌豆根瘤菌豌豆生物型（Rhizobium leguminosarum bv. viciae）的氢化酶摄取系统（hydrogenase uptake system，hup系统）展现出高度保守的序列与基因组织结构，但二者在调控机制及特定基因携带情况上存在显著差异。本研究对羽扇豆慢生根瘤菌（待定种，Bradyrhizobium sp. (Lupinus)）、豇豆慢生根瘤菌（待定种，Bradyrhizobium sp. (Vigna)）、热带根瘤菌（Rhizobium tropici）及茎瘤固氮根瘤菌（Azorhizobium caulinodans）的菌株的hup基因簇进行功能表征，旨在明确内共生细菌中hup基因组成与调控机制的多样性范围。采用hupS、hupE、hupUV、hypB及hoxA探针进行的基因组DNA杂交实验显示，羽扇豆慢生根瘤菌（待定种）与豇豆慢生根瘤菌（待定种）菌株的种内hup图谱存在多样性，而热带根瘤菌与茎瘤固氮根瘤菌菌株的种内模式则较为均一。该分析还揭示了氢化酶调控基因携带情况的差异。基于hupS与hupL部分序列的系统发育分析显示，豌豆根瘤菌、热带根瘤菌的hup序列与大豆慢生根瘤菌及羽扇豆慢生根瘤菌（待定种）菌株的hup序列聚为一支，表明二者具有共同的起源。与之相反，豇豆慢生根瘤菌（待定种）的hup序列与其他根瘤菌序列存在显著分化，这表明该类群可能独立演化，并可能通过水平基因转移从未知来源获得了该序列。