The multifunctional DHH-DHHA1 phosphodiesterase plays critical roles in bacterial physiological regulation and host-pathogen interactions. In Mycoplasma bovis, the DhhP-type phosphodiesterase MbovP328 exhibits both phosphodiesterase and nanoRNase activities, with the H291 residue critical for phosphodiesterase function. Transposon insertion in Mbov_0328 inactivated both activities in the mutant T9.386. Complementation restored both activities in CNT9.386, while only nanoRNase activity was recovered in CNT9.386