Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease

Abstract

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon , heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca 2؉ /Mg 2؉

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

“…This is supported by the identification of one or more nucleases in most mycoplasma species studied so far (33) and the concomitant identification of multiple genes encoding putative nucleases in genomic sequence analyses (5,21,24,34,42,51,57,59). However, many of these nucleases do not appear to be conserved between mycoplasma species, based on differences in size and reaction conditions (2,25,33,45). In particular, these nucleases differ in the requirement for specific divalent cations, which is an important criterion in their classification (6).…”

“…One or more transport mechanisms for the import of intact exogenous nucleotides have been identified in M. mycoides subspecies mycoides (38,39,60). Membrane-associated nuclease activity has been identified in all mycoplasma species studied so far (33), and a comparatively large number of mycoplasmas have additionally been shown to produce extracellular nuclease activity (33).The identification of intracellular, extracellular, and membrane-associated nuclease activities in a number of mycoplasma species suggests the involvement of nucleases in a variety of cellular processes (2,25,33,45). This is supported by the identification of one or more nucleases in most mycoplasma species studied so far (33) and the concomitant identification of multiple genes encoding putative nucleases in genomic sequence analyses (5,21,24,34,42,51,57,59).…”

“…For Mycoplasma pulmonis, the biochemical properties of membrane-associated nucleases have been partially characterized (25,43) and the gene encoding the nuclease that is reportedly associated with the majority of this membrane-associated activity, designated mnuA, has been cloned and expressed (25). Only the major membrane-associated nuclease of Mycoplasma penetrans has been purified to homogeneity and the biochemical properties completely characterized (2). The nucleases of M. penetrans and M. pulmonis displayed endonuclease activity that was strictly dependent on the presence of calcium and/or magnesium ions (2,43).…”

“…Only the major membrane-associated nuclease of Mycoplasma penetrans has been purified to homogeneity and the biochemical properties completely characterized (2). The nucleases of M. penetrans and M. pulmonis displayed endonuclease activity that was strictly dependent on the presence of calcium and/or magnesium ions (2,43). Calcium and magnesium ion-dependent endonuclease activity has also been found in a number of other mycoplasma species (43).…”

Degradation of Nuclease-Stabilized RNA Oligonucleotides in Mycoplasma-Contaminated Cell Culture Media

“…Some mycoplasma species, notably Mycoplasma pneumoniae, are human pathogens (Cunha, 2010). Various mycoplasma species are known to produce ribonucleases and deoxyribonucleases (e.g., Minion and Goguen, 1986;Marcus and Yoshida, 1990;Minion et al, 1993;el-Farrash et al, 1994;Shang et al, 1995;Bendjennat et al, 1997); however, the ability of these nucleases to degrade chemically modified RNA has not been explored.…”