Disruption of the Membrane Nuclease Gene (MBOVPG45_0215) of Mycoplasma bovis Greatly Reduces Cellular Nuclease Activity

Abstract

ABSTRACTAlthough the complete genome sequences of three strains ofMycoplasma bovisare available, few studies have examined gene function in this important pathogen. Mycoplasmas lack the biosynthetic machinery for thede novosynthesis of nucleic acid precursors, so nucleases are likely to be essential for them to acquire nucleotide precursors. Three putative membrane nucleases have been annotated in the genome ofM. bovisstrain PG45, MBOVPG45_0089 and MBOVPG45_0310, both of which have the thermonuclease (TNASE_3) functional domain, and MBOVPG45_0215 (mnuA), which has an exonuclease/endonuclease/phosphatase domain. While previous studies have demonstrated the function of TNASE_3 domain nucleases in several mycoplasmas, quantitative comparisons of the contributions of different nucleases to cellular nuclease activity have been lacking. Mapping of a library of 319 transposon mutants ofM. bovisPG45 by direct genome sequencing identified mutants with insertions in MBOVPG45_0310 (the Δ0310 mutant) and MBOVPG45_0215 (the Δ0215 mutant). In this study, the detection of the product of MBOVPG45_0215 in the Triton X-114 fraction ofM. boviscell lysates, its cell surface exposure, and its predicted signal peptide suggested that it is a surface-exposed lipoprotein nuclease. Comparison of a ΔmnuAmutant with wild-typeM. bovison native and denatured DNA gels and in digestion assays using double-stranded phage λ DNA and closed circular plasmid DNA demonstrated that inactivation of this gene abolishes most of the cellular exonuclease and endonuclease activity ofM. bovis. This activity could be fully restored by complementation with the wild-typemnuAgene, demonstrating that MnuA is the major cellular nuclease ofM. bovis.IMPORTANCENucleases are thought to be important contributors to virulence and crucial for the maintenance of a nutritional supply of nucleotides in mycoplasmas that are pathogenic in animals. This study demonstrates for the first time that of the three annotated cell surface nuclease genes in an important pathogenic mycoplasma, the homologue of the thermostable nuclease identified in Gram-positive bacteria is responsible for the majority of the nuclease activity detectablein vitro.