Mycoplasma hyopneumoniae mhp379 Is a Ca ²⁺ -Dependent, Sugar-Nonspecific Exonuclease Exposed on the Cell Surface

Abstract

Mycoplasma hyopneumoniae mhp379 is a putative lipoprotein that shares significant amino acid sequence similarity with a family of bacterial thermostable nucleases. To examine the nuclease activity of mhp379, the gene was cloned and expressed in Escherichia coli following the deletion of the amino-terminal signal sequence and prokaryotic lipoprotein cleavage site and mutagenesis of the mycoplasma TGA tryptophan codons to TGG. The recombinant fusion protein yielded a 33-kDa thrombin cleavage product, corresponding in size to the mature mhp379 protein. Exonuclease activity was indicated by agarose gel electrophoresis analysis of the reaction products that were released when different nucleic acid substrates were used. Endonuclease activity was also indicated by the digestion of closed circular plasmid DNA. The recombinant mhp379 fusion protein completely digested single-stranded DNA, double-stranded DNA (dsDNA), and RNA. The optimal reaction conditions were determined with a novel nuclease assay based on the enhancement of fluorescence of SYBR green I bound to dsDNA. Optimal activity was observed in the presence of calcium ions at a concentration of 15 mM and a pH of 9.5. No nuclease activity was observed in the absence of calcium ions. Mycoplasmas do not have the ability to synthesize nucleic acid precursors, and thus, nucleases are likely to be important in the acquisition of precursors for the synthesis of nucleic acids. Homologs of an ATP-binding cassette (ABC) transport system were identified immediately downstream of the gene encoding mhp379, and two homologs of M. pneumoniae lipoprotein multigene family 2 were also identified immediately upstream. Homologs of mhp379 were identified in the sequenced genomes of a number of mycoplasma species, and in most cases the homologous ABC transport system was identified immediately downstream of the homologous gene; in several cases a homolog of M. pneumoniae lipoprotein multigene family 2 was also identified immediately upstream. These observations suggest that mhp379 comprises part of a conserved ABC transport operon in mycoplasmas and that the exonuclease activity of mhp379 may be associated with the conserved function of the ABC transport system in the import of nucleic acid precursors. This is the first study to identify the gene and characterize the activity of a mycoplasma exonuclease.Neither the genes nor the enzymes for the biosynthesis of nucleic acid precursors in mycoplasmas have been identified (5,21,24,34,42,46,47,51,57,59). However, genomic sequence analyses of a number of mycoplasma species have identified novel enzymes associated with salvage pathways for the biosynthesis of nucleic acids from nucleotides (5,21,24,34,42,44,51,(57)(58)(59). These salvage pathways have been functionally characterized in Mycoplasma mycoides subspecies mycoides (35-37, 58). Mycoplasma nucleases are thought to play a metabolic role in the production of nucleotide substrates from host or microbial nucleic acids released through natural and induced cell death. These nucleotide...

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

“…The nuclease activity of wild-type M. bovis and the putative nuclease mutants was analyzed by 1% agarose gel electrophoresis as described previously (12). The exonuclease and endonuclease activities of the cells were determined by incubating proteins from cells from late-log-phase cultures suspended in 50 l of nuclease reaction buffer (25 mM Tris-HCl, pH 8.8, 10 mM CaCl 2 , 10 mM MgCl 2 ) at 37°C with 500 ng of double-stranded phage DNA (New England BioLabs) or 2.0 g of closed circular plasmid DNA (plasmid pRecAGKIRRPG2, which was generated to disrupt a specific gene target by homologous recombination in M. bovis) as the substrates.…”

“…Intact M. bovis cells were treated with trypsin to partially digest cell surface proteins, as described previously (12,21). M. bovis cells were cultured, and the cell pellet was washed in 50 mM Tris, 0.145 M NaCl, pH 7.4 (Tris-salt [TS] buffer).…”

“…Orthologues of the three annotated putative membrane nucleases in the genome of M. bovis strain PG45, MBOVPG45_0089, MBOVPG45_0215, and MBOVPG45_0310, were identified in a number of phylogenetically distant mycoplasmas belonging to the M. hominis and M. pneumoniae clusters and are listed in Table 1 Genomic location and features of membrane nucleases in M. bovis. MBOVPG45_0310 is part of a 7.6-kbp ABC transporter operon that is predicted to be involved in nucleotide transport (12,13,16,29,30). However, MBOVPG45_0089 and MBOVPG45_0215 do not appear to lie within operons.…”

“…The predicted molecular mass of the product of MBOVPG45_0215 is 44.13 kDa, that of the product of MBOVPG45_0310 is 41.56 kDa, and that of the product of MBOVPG45_0089 is 20.51 kDa. Mapping of a library of 319 transposon mutants of the M. bovis PG45 genome identified 1 mutant (the ⌬0310 mutant) with a disruption in MBOVPG45_0310, which codes for the nuclease of a putative nucleotide transporter operon (12,13,29,30). Another mutant (the ⌬0215 mutant) had a disruption in MBOVPG45_0215, which encodes a membrane nuclease, MnuA.…”

“…Nuclease activity has also been demonstrated in vitro following cloning, expression, and purification of predicted membrane nucleases from M. hyopneumoniae (12), M. gallisepticum (13), M. genitalium (14), M. pneumoniae (15), and M. agalactiae (16). However, most studies on membrane nucleases of mycoplasmas have been based on an examination of in vitro profiles of activity following protein purification (12)(13)(14)(15)(16). Genome sequencing has identified genes encoding many additional putative nucleases in mycoplasmas that are predicted to be located either on the cell surface or within the cytoplasm, but these studies cannot assess the relative significance of a particular nuclease in the cell.…”