Graduate School of Science and Engineering, Kagoshima University.
Microbes Environ. 2023;38(1). doi: 10.1264/jsme2.ME22093.
Frankia spp. are multicellular actinobacteria that fix atmospheric dinitrogen (N) not only in the free-living state, but also in root-nodule symbioses with more than 200 plant species, called actinorhizal plants. To identify novel Frankia genes involved in N fixation, we previously isolated mutants of Frankia casuarinae that cannot fix N. One of these genes, mutant N3H4, did not induce nodulation when inoculated into the host plant Casuarina glauca. Cell lineages that regained the ability to fix N as free-living cells were isolated from the mutant cell population. These restored strains also regained the ability to stimulate nodulation. A comparative ana-lysis of the genomes of mutant N3H4 and restored strains revealed that the mutant carried a mutation (Thr584Ile) in the glutamine-dependent NAD synthetase gene (Francci3_3146), while restored strains carried an additional suppressor mutation (Asp478Asn) in the same gene. Under nitrogen-depleted conditions, the concentration of NAD(H) was markedly lower in the mutant strain than in the wild type, whereas it was higher in restored strains. These results indicate that glutamine-dependent NAD synthetase plays critical roles in both free-living and symbiotic N fixation in Frankia.
弗兰克氏菌属是多细胞放线菌，不仅能在自由生活状态下固定大气中的二氮（N），还能与 200 多种被称为根瘤菌的植物形成根瘤共生关系。为了鉴定参与固氮的新弗兰克氏菌基因，我们之前分离出了不能固定 N 的 Casuarina 根瘤菌突变体。其中一个基因，突变体 N3H4，在接种到宿主植物木麻黄时不会诱导结瘤。从突变体细胞群体中分离出了重新获得自由生活细胞固氮能力的细胞谱系。这些恢复的菌株也恢复了刺激结瘤的能力。对突变体 N3H4 和恢复菌株的基因组进行比较分析表明，突变体携带了一个突变（Thr584Ile）在谷氨酰胺依赖的 NAD 合成酶基因（Francci3_3146）中，而恢复的菌株在同一基因中携带了一个额外的抑制突变（Asp478Asn）。在氮饥饿条件下，突变株中 NAD(H)的浓度明显低于野生型，而在恢复株中则更高。这些结果表明，谷氨酰胺依赖的 NAD 合成酶在弗兰克氏菌的自由生活和共生固氮中都起着关键作用。
