Transcriptomic profiles reveal DNA-dependent ATPase MbovRecD modulates Mycoplasma bovis survival through...

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 (N³⁵⁰, Q³⁵⁵, G³⁷⁵, G³⁷⁷, K³⁷⁸, S³⁷⁹, Y³⁸⁰, K⁴¹⁶, Q⁴⁸⁶, R⁵¹⁵, and R⁷¹⁶) 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_m = 8.558 μM, k_cat = 2157.75 s^-1, and V_max = 0.8631 μM·s^-1·mg^-1. 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.