Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710;
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom.
Proc Natl Acad Sci U S A. 2021 Jul 27;118(30). doi: 10.1073/pnas.2105447118.
Filamentous actinobacteria of the genus have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The RsiG-(c-di-GMP)-WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)S(X)R(X)Q(X)D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a (RsiG)-(c-di-GMP)-WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium , the (RsiG)-(c-di-GMP)-WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.
具有复杂生命周期的丝状放线菌属，其生殖气生菌丝分化为孢子的过程涉及到这一过程。我们最近的研究表明，c-di-GMP 通过武装一种独特的反σ因子 RsiG 来控制这一转变，使其与专性孢子形成σ因子 WhiG 结合。RsiG-(c-di-GMP)-WhiG 结构揭示了单体 RsiG 通过两个 E(X)S(X)R(X)Q(X)D 重复基序结合 c-di-GMP，每个螺旋的一个基序都位于一个反平行的螺旋卷曲结构中。在这里，我们发现 RsiG 同源物广泛存在于放线菌中。引人注目的是，来自放线菌最基部分支的单细胞细菌的 RsiG 是小蛋白，仅包含一个 c-di-GMP 结合基序，但仍能与它们的 WhiG 伴侣结合。我们的结构显示，一个（RsiG）-（c-di-GMP）-WhiG 复合物通过同源二聚化形成反平行螺旋卷曲，从而使它们能够结合 c-di-GMP，类似于单体双基序 RsiG。进一步的数据表明，在单细胞放线菌中，（RsiG）-（c-di-GMP）-WhiG 调控开关控制着 IV 型菌毛的表达。系统发育分析表明，单基序 RsiG 可能代表原始状态，而内部基因复制事件产生了在放线菌其他地方遗传的双基序 RsiG。因此，这些研究表明，反σ因子 RsiG 是如何通过基因内重复事件从携带一个 c-di-GMP 结合基序的小蛋白进化而来的，该蛋白作为同源二聚体发挥作用，到携带两个 c-di-GMP 结合基序的较大蛋白，作为单体发挥作用。与这一观点一致的是，我们的结构揭示了单体双基序反σ因子的潜在选择性优势。
