Sloboda Seada, Ge Xinwei, Jiang Daqing, Su Lin, Wiens Gregory D, Beveridge Carly A, Duchaud Eric, McBride Mark J, Rochat Tatiana, Zhu Yongtao
Department of Biological Sciences, Minnesota State University Mankato, Mankato, Minnesota, USA.
Department of Biosciences and Bioinformatics, School of Science, Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu, China.
Appl Environ Microbiol. 2025 Feb 19;91(2):e0144824. doi: 10.1128/aem.01448-24. Epub 2025 Jan 10.
causes bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanisms uses to cause disease are poorly understood. Despite considerable effort, most strains of have resisted attempts at genetic manipulation. restriction-modification (R-M) systems may contribute to this resistance. Restriction endonucleases (REases) rapidly degrade nonself DNA if it is not properly methylated by their cognate DNA methyltransferases (MTases). We used comparative genomics to show that R-M systems are abundant in and that strain-specific variations partially align with phylogeny. We identified two critical type II R-M systems, HpaII-like (FpsJI) and ScrFI-like (FpsJVI), that are conserved in most of the sequenced strains. Protection of foreign DNA against HpaII and ScrFI was achieved by expression of the MTases M.FpsJI and M.FpsJVI in . Furthermore, deleting the two REase genes from resulted in efficient conjugative DNA transfer from into the otherwise genetically intractable strain CSF259-93. This allowed us to construct a CSF259-93 mutant lacking , a core component of the type IX protein secretion system. The pre-methylation system developed in this study functions in all tested strains harboring HpaII-like and ScrFI-like REases. These newly developed genetic tools may allow the identification of key virulence factors and facilitate the development of live attenuated vaccines or other measures to control BCWD.
Bacterial cold-water disease (BCWD) caused by is a problem for salmonid aquaculture worldwide, and current control measures are inadequate. An obstacle in understanding and controlling BCWD is that most strains resist DNA transfer, thus limiting genetic studies of their virulence mechanisms. restriction enzymes that destroy foreign DNA were suspected to contribute to this problem. Here, we used DNA methyltransferases to modify and protect foreign DNA from digestion. This allowed efficient conjugative DNA transfer into nine diverse strains that had previously resisted DNA transfer. Using this approach, we constructed a gene deletion mutant that failed to cause disease in rainbow trout. Further genetic studies could help determine the molecular factors involved in pathogenesis and may aid development of innovative BCWD control strategies.
在鲑科鱼类和其他鱼类中引发细菌性冷水病（BCWD），给全球水产养殖业造成重大经济损失。其致病机制尚不清楚。尽管付出了巨大努力，但大多数菌株仍抗拒基因操作。限制修饰（R-M）系统可能导致了这种抗性。如果非自身DNA未被其同源DNA甲基转移酶（MTases）正确甲基化，限制性内切酶（REases）会迅速降解它。我们通过比较基因组学表明，R-M系统在该菌中很丰富，且菌株特异性变异部分与系统发育一致。我们鉴定出两个关键的II型R-M系统，HpaII样（FpsJI）和ScrFI样（FpsJVI），它们在大多数已测序菌株中保守。通过在该菌中表达MTases M.FpsJI和M.FpsJVI，实现了对外源DNA的HpaII和ScrFI保护。此外，从该菌中删除两个REase基因，使得DNA能从该菌高效接合转移到原本遗传上难以操作的菌株CSF259-93中。这使我们能够构建一个缺失该菌的基因的CSF259-93突变体，该菌是IX型蛋白分泌系统的核心成分。本研究中开发的预甲基化系统在所有携带HpaII样和ScrFI样REases的受试菌株中均起作用。这些新开发的遗传工具可能有助于鉴定关键毒力因子，并促进减毒活疫苗或其他控制BCWD措施的开发。
由该菌引起的细菌性冷水病（BCWD）是全球鲑科鱼类养殖面临的一个问题，目前的控制措施并不充分。理解和控制BCWD的一个障碍是大多数该菌菌株抗拒DNA转移，从而限制了对其毒力机制的遗传学研究。怀疑破坏外源DNA的限制性酶导致了这个问题。在这里，我们使用该菌的DNA甲基转移酶来修饰和保护外源DNA不被消化。这使得DNA能高效接合转移到九个先前抗拒DNA转移的不同该菌菌株中。利用这种方法，我们构建了一个在虹鳟中不致病的基因缺失突变体。进一步的遗传学研究有助于确定参与发病机制的分子因素，并可能有助于开发创新的BCWD控制策略。
