Zhu Xifang, Li Yajiao, Lu Doukun, Zhao Gang, Liu Yankai, Wang Aiping, Guo Aizhen
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China.
The State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan 430070, China.
Int J Biol Macromol. 2025 Jul;318(Pt 4):145256. doi: 10.1016/j.ijbiomac.2025.145256. Epub 2025 Jun 16.
Mycoplasmas are host-restricted prokaryotes with nearly minimal genomes. Research on these microorganisms has been constrained by challenges in genetic manipulation and unclear gene functions. This study identified that mutation of MBOV_RS04205 (encoding MbovRecD) caused growth defects in M. bovis under cell co-culture conditions. Biochemical characterization revealed MbovRecD exhibits DNA-dependent ATPase activity, supported by molecular docking and mutagenesis of key residues (N, Q, G, G, K, S, Y, K, Q, R, and R) critical for ATP hydrolysis. Optimal enzymatic activity occurred at 40 °C, with 0.8 nM rMbovRecD and 40 μM 25- or 40-mer ssDNA. The kinetic parameters of ATPase activity in rMbovRecD were determined to be K = 8.558 μM, k = 2157.75 s, and V = 0.8631 μM·s·mg. Phylogenetically, RecD appears conserved across mycoplasmas. Transcriptomics revealed MBOV_RS04205 mutation altered 36 genes, with GO enrichment in magnesium ion binding, methyltransferase activity and phosphatase activity, while KEGG analysis linked differentially expressed genes (DEGs) to glycolysis/pyruvate metabolism. Collectively, this study identifies MbovRecD as a novel ATPase essential for M. bovis survival and elucidates its role in regulating bacterial growth through methyltransferase activity and carbohydrate metabolism. These findings provide a basis for mycoplasma prevention and control strategies and the development of new therapeutics.
支原体是宿主受限的原核生物，基因组几乎最小。对这些微生物的研究受到基因操作挑战和基因功能不明确的限制。本研究发现，MBOV_RS04205（编码MbovRecD）的突变在细胞共培养条件下导致牛支原体生长缺陷。生化特性表明，MbovRecD具有依赖DNA的ATP酶活性，分子对接和对ATP水解关键残基（N、Q、G、G、K、S、Y、K、Q、R和R）的诱变支持了这一活性。最佳酶活性发生在40°C，0.8 nM rMbovRecD和40 μM 25或40聚体单链DNA存在时。rMbovRecD中ATP酶活性的动力学参数确定为K = 8.558 μM，k = 2157.75 s，V = 0.8631 μM·s·mg。在系统发育上，RecD在支原体中似乎是保守的。转录组学显示，MBOV_RS04205突变改变了36个基因，基因本体富集于镁离子结合、甲基转移酶活性和磷酸酶活性，而京都基因与基因组百科全书分析将差异表达基因与糖酵解/丙酮酸代谢联系起来。总体而言，本研究确定MbovRecD是牛支原体生存所必需的一种新型ATP酶，并阐明了其通过甲基转移酶活性和碳水化合物代谢调节细菌生长的作用。这些发现为支原体防控策略和新疗法的开发提供了依据。
