Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou, 510301, China.
Microb Biotechnol. 2020 Jul;13(4):1132-1144. doi: 10.1111/1751-7915.13570. Epub 2020 Apr 4.
Pf prophages are ssDNA filamentous prophages that are prevalent among various Pseudomonas aeruginosa strains. The genomes of Pf prophages contain not only core genes encoding functions involved in phage replication, structure and assembly but also accessory genes. By studying the accessory genes in the Pf4 prophage in P. aeruginosa PAO1, we provided experimental evidence to demonstrate that PA0729 and the upstream ORF Rorf0727 near the right attachment site of Pf4 form a type II toxin/antitoxin (TA) pair. Importantly, we found that the deletion of the toxin gene PA0729 greatly increased Pf4 phage production. We thus suggest the toxin PA0729 be named PfiT for Pf4 inhibition toxin and Rorf0727 be named PfiA for PfiT antitoxin. The PfiT toxin directly binds to PfiA and functions as a corepressor of PfiA for the TA operon. The PfiAT complex exhibited autoregulation by binding to a palindrome (5'-AATTCN GTTAA-3') overlapping the -35 region of the TA operon. The deletion of pfiT disrupted TA autoregulation and activated pfiA expression. Additionally, the deletion of pfiT also activated the expression of the replication initiation factor gene PA0727. Moreover, the Pf4 phage released from the pfiT deletion mutant overcame the immunity provided by the phage repressor Pf4r. Therefore, this study reveals that the TA systems in Pf prophages can regulate phage production and phage immunity, providing new insights into the function of TAs in mobile genetic elements.
Pf 噬菌体是 ssDNA 丝状噬菌体，在各种铜绿假单胞菌菌株中普遍存在。Pf 噬菌体基因组不仅包含编码噬菌体复制、结构和组装相关功能的核心基因，还包含辅助基因。通过研究铜绿假单胞菌 PAO1 中 Pf4 噬菌体的辅助基因，我们提供了实验证据，证明 PA0729 和 Pf4 右附着位点附近的上游 ORF Rorf0727 形成了 II 型毒素/抗毒素（TA）对。重要的是，我们发现毒素基因 PA0729 的缺失大大增加了 Pf4 噬菌体的产量。因此，我们建议将毒素基因 PA0729 命名为 Pf4 抑制毒素（PfiT），将上游 ORF Rorf0727 命名为 PfiT 抗毒素（PfiA）。PfiT 毒素直接与 PfiA 结合，并作为 TA 操纵子的核心抑制剂发挥作用。PfiAT 复合物通过结合 TA 操纵子的-35 区域重叠的回文（5'-AATTCN GTTAA-3'），表现出自调控。pfiT 的缺失破坏了 TA 自调控，并激活了 pfiA 的表达。此外，pfiT 的缺失也激活了复制起始因子基因 PA0727 的表达。此外，来自 pfiT 缺失突变体的 Pf4 噬菌体克服了 Pf4r 噬菌体阻遏物提供的免疫。因此，这项研究揭示了 Pf 噬菌体中的 TA 系统可以调节噬菌体的产生和噬菌体的免疫，为移动遗传元件中 TA 的功能提供了新的见解。
