Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan, USA.
J Bacteriol. 2018 Nov 26;200(24). doi: 10.1128/JB.00300-18. Print 2018 Dec 15.
Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in as the 3'-to-5' exoribonuclease oligoribonuclease (Orn). Mutants of accumulated pGpG, which inhibited the linearization of c-di-GMP. This product inhibition led to elevated c-di-GMP levels, resulting in increased aggregate and biofilm formation. Thus, the hydrolysis of pGpG is crucial to the maintenance of c-di-GMP homeostasis. How species that utilize c-di-GMP signaling but lack an ortholog hydrolyze pGpG remains unknown. Because Orn is an exoribonuclease, we asked whether pGpG hydrolysis can be carried out by genes that encode protein domains found in exoribonucleases. From a screen of these genes from and , we found that only enzymes known to cleave oligoribonucleotides ( and ) rescued the Δ mutant phenotypes to the wild type. Thus, we tested additional RNases with demonstrated activity against short oligoribonucleotides. These experiments show that only exoribonucleases previously reported to degrade short RNAs (, , , and ) can also hydrolyze pGpG. A mutant had elevated c-di-GMP, suggesting that these two genes serve as the primary enzymes to degrade pGpG. These results indicate that the requirement for pGpG hydrolysis to complete c-di-GMP signaling is conserved across species. The final steps of RNA turnover and c-di-GMP turnover appear to converge at a subset of RNases specific for short oligoribonucleotides. The bacterial bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling molecule regulates complex processes, such as biofilm formation. c-di-GMP is degraded in two-steps, linearization into pGpG and subsequent cleavage to two GMPs. The 3'-to-5' exonuclease oligoribonuclease (Orn) serves as the enzyme that degrades pGpG in Many phyla contain species that utilize c-di-GMP signaling but lack an Orn homolog, and the protein that functions to degrade pGpG remains uncharacterized. Here, systematic screening of genes encoding proteins containing domains found in exoribonucleases revealed a subset of genes encoded within the genomes of and that degrade pGpG to GMP and are functionally analogous to Orn. Feedback inhibition by pGpG is a conserved process, as strains lacking these genes accumulate c-di-GMP.
双(3'-5')-环二鸟苷酸(c-di-GMP)是一种细菌第二信使，调节生物膜形成和毒力等过程。在降解过程中，c-di-GMP 首先线性化为 5'-磷酸鸟苷酰-(3',5')-鸟苷(pGpG)，随后被一种先前未知的酶水解为两个 GMP，这种酶最近在[1]中被鉴定为 3'-5'外切核糖核酸酶寡核糖核酸酶(Orn)。[1]的突变体积累了 pGpG，抑制了 c-di-GMP 的线性化。这种产物抑制导致 c-di-GMP 水平升高，导致聚集物和生物膜形成增加。因此，pGpG 的水解对于 c-di-GMP 动态平衡的维持至关重要。那些利用 c-di-GMP 信号但缺乏 Orn 同源物的物种如何水解 pGpG 仍然未知。由于 Orn 是一种外切核糖核酸酶，我们询问是否可以通过编码外切核糖核酸酶中发现的蛋白结构域的基因来进行 pGpG 水解。从[1]和[2]的这些基因筛选中，我们发现只有已知能切割寡核苷酸的酶（和）能使[1]的Δ突变体表型恢复到野生型。因此，我们测试了其他具有针对短寡核苷酸的证明活性的 RNases。这些实验表明，只有先前报道能降解短 RNA 的外切核糖核酸酶（、、、和）也能水解 pGpG。[1]的突变体 c-di-GMP 水平升高，表明这两个基因是降解 pGpG 的主要酶。这些结果表明，完成 c-di-GMP 信号所需的 pGpG 水解在物种间是保守的。RNA 周转和 c-di-GMP 周转的最后步骤似乎集中在一组特定于短寡核苷酸的 RNases 上。细菌双-(3'-5')-环二鸟苷酸(c-di-GMP)信号分子调节复杂过程，如生物膜形成。c-di-GMP 分两步降解，线性化为 pGpG，然后进一步切割为两个 GMP。3'-5'外切核糖核酸酶寡核糖核酸酶(Orn)是降解 pGpG 的酶[1]。许多门包含利用 c-di-GMP 信号但缺乏 Orn 同源物的物种，而负责降解 pGpG 的蛋白质仍未被描述。在这里，系统筛选编码含有外切核糖核酸酶中发现的蛋白结构域的基因，揭示了[1]和[2]基因组中编码一组基因的子集，这些基因能将 pGpG 降解为 GMP，并且与 Orn 功能上类似。pGpG 的反馈抑制是一个保守的过程，因为缺乏这些基因的菌株会积累 c-di-GMP。
