Division of Microbiology, Department of Pre-Clinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Front Cell Infect Microbiol. 2018 Jan 18;7:533. doi: 10.3389/fcimb.2017.00533. eCollection 2017.
Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2'--methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2'--methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both and also when expressed in . Deletion of the gene in or substitution with alanine of K, D, or K in the catalytic center of the enzyme cause complete loss of 2'--methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2'--methylation function of Cj0588/TlyA from any other putative roles that the protein might play. strains expressing catalytically inactive versions of Cj0588 have the same phenotype as -null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in is a consequence of the enzyme's ability to methylate its rRNA.
许多细菌病原体表达一种与环肽抗生素（如卷曲霉素）耐药相关的酶 TlyA 的同源物，该酶是 rRNA 2'--甲基转移酶。TlyA 还与其他几种毒力特性相关联，而这些特性似乎与其甲基转移酶活性无关。细菌病原体 拥有 TlyA 同源物 Cj0588，已证明该同源物有助于毒力。在这里，我们研究了 Cj0588 的作用机制，并证明它是 TlyA 的一种 I 型同源物，可使 23S rRNA 核苷酸 C1920 2'--甲基化。Cj0588 保留了相同的特定功能 ，并且当在 中表达时也是如此。在 或用丙氨酸取代酶催化中心的 K、D 或 K 时，缺失 基因会导致完全丧失 2'--甲基化活性。辅因子相互作用保持不变，与核糖体底物的结合亲和力仅略有降低，表明失活蛋白正确折叠。取代突变因此将 Cj0588/TlyA 的 2'--甲基化功能与其可能发挥的任何其他假定作用分离。表达 Cj0588 催化失活版本的 菌株与 -null 突变体具有相同的表型，并且由于核糖体亚基结合受到干扰、运动能力降低以及形成生物膜的能力受损，对卷曲霉素的耐受性降低。当恢复甲基转移酶活性时，这些功能得以重建，我们得出结论，Cj0588 对 的毒力的贡献是由于该酶能够甲基化其 rRNA。
