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

Abstract

Although the complete genome sequences of three strains of Mycoplasma bovis are available, few studies have examined gene function in this important pathogen. Mycoplasmas lack the biosynthetic machinery for the de novo synthesis 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 of M. bovis strain 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 of M. bovis PG45 by direct genome sequencing identified mutants with insertions in MBOVPG45_0310 (the ⌬0310 mutant) and MBOVPG45_0215 (the ⌬0215 mutant). In this study, the detection of the product of MBOVPG45_0215 in the Triton X-114 fraction of M. bovis cell lysates, its cell surface exposure, and its predicted signal peptide suggested that it is a surface-exposed lipoprotein nuclease. Comparison of a ⌬mnuA mutant with wild-type M. bovis on native and denatured DNA gels and in digestion assays using double-stranded phage DNA and closed circular plasmid DNA demonstrated that inactivation of this gene abolishes most of the cellular exonuclease and endonuclease activity of M. bovis. This activity could be fully restored by complementation with the wild-type mnuA gene, demonstrating that MnuA is the major cellular nuclease of M. 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 detectable in vitro.A lthough the complete genomes of many Mycoplasma species are now available, the scarcity of tools for exploring their molecular biology has resulted in an understanding of the function of their genes more rudimentary than that which is available for many other prokaryotes. The genomes of three strains of the important bovine pathogen Mycoplasma bovis, the type strain PG45, the Hubei-1 strain, and the HB0801 strain, have been sequenced and published recently (1-3), but apart from several reports on phase switching in M. bovis (4-7), few studies have examined the molecular mechanisms underlying their pathogenicity and virulence or the metabolic functions of the products of their genes.Mycoplasmas evolved from Clostridium-like bacteria by a process of reductive evolution and are believed to have retained only the regulatory and ...

Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity

“…Zymogram analysis involves inhibition of nuclease activity by sodium dodecylsulfate (SDS) and renaturation following SDS removal by diffusion [ 10 ]. Briefly, total cellular proteins of M. bovis HB0801, rMbovNase, or rMbovNase Δ181–342 protein were mixed with SDS loading buffer, heated to 100 °C for 10 min, and loaded onto 12% SDS-PAGE gels saturated in advance with herring sperm DNA (160 μg·mL −1 ; Sigma–Aldrich, St. Louis, MO, USA).…”

“…Moreover, several nucleases have been implicated in mycoplasma-mediated host pathogenicity and cytotoxicity [ 7 , 8 , 9 ]. Based on the sequence alignment, there is a highly conserved region named TNASE_3 belonging to micrococcal nuclease (thermonuclease) COG1525 [ 10 ]. The conserved region has several catalytic sites involved in the nuclease activity and binding of calcium ions [ 11 ].…”

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…Multiple predicted nuclease genes are present in the M. bovis genome, including three in the PG45 type strain. In vitro studies using transposon mutants disclosed MBOVPG45_0215 as exhibiting the majority of nuclease activity for the DNA substrates tested (Sharma, Tivendale, Markham, & Browning, ). A recombinant form of a second nuclease MBOV_RS02825 (equivalent to MBOVPG45_0310) was shown to degrade the DNA component of NETs and to induce apoptosis of macrophages (Zhang et al., ).…”

Mpn491, a secreted nuclease ofMycoplasma pneumoniae, plays a critical role in evading killing by neutrophil extracellular traps

“…The results suggested that hydrolytic activity or the expression level of Mpn133 may be apparently lower than that of Mpn491, and it might be possible that Mpn133 is less important for the resistance of M. pneumoniae to NETs‐mediated killing than Mpn491. Meanwhile, another study reported that Mpn491 possesses glutamic acid‐lysine‐serine region‐like amino‐acid sequence between residues 175 and 218 (Sharma, Tivendale, Markham, & Browning, ), raising a possibility that the nuclease might also exert a cytotoxic activity toward mammalian cells. Further research is required to fully elucidate the additional function of the nuclease.…”