Molecular cloning, expression, and characterization of a Ca 2+-dependent, membrane-associated nuclease of Mycoplasma genitalium

Linbo Li, Manickam Krishnan, Joel B. Baseman, T. R. Kannan

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

In this study, we identified and characterized the enzymatic properties of MG-186, a calcium-dependent Mycoplasma genitalium nuclease. MG-186 displays the hallmarks of nucleases, as indicated by its amino acid sequence similarity to other nucleases. We cloned, UGA corrected, expressed, purified, and demonstrated that recombinant MG-186 (rMG-186) exhibits nuclease activity similar to that of typical sugar-nonspecific endonucleases and exonucleases. Biochemical characterization indicated that Ca2+ alone enhances its activity, which was inhibited by divalent cations, such as Zn2+ and Mn 2+. Chelating agents EGTA and EDTA also inhibited nuclease activity. Mycoplasma membrane fractionation and Triton X-114 phase separation showed that MG-186 was a membrane-associated lipoprotein, and electron microscopy revealed its surface membrane location. Incubation of purified human endometrial cell nuclei with rMG-186 resulted in DNA degradation and morphological changes typical of apoptosis. Further, immunofluorescence analysis of rMG-186-treated nuclei indicated that morphological changes were linked to the disintegration of lamin and the internalization of rMG-186. Since M. genitalium has the capacity to invade eukaryotic cells and localize to the perinuclear and nuclear region of parasitized target cells, MG-186 has the potential to provide M. genitalium, which possesses the smallest genome of any self-replicating cell, with the ability to degrade host nucleic acids both as a source of nucleotide precursors for growth and for pathogenic purposes.

Original languageEnglish (US)
Pages (from-to)4876-4884
Number of pages9
JournalJournal of bacteriology
Volume192
Issue number19
DOIs
StatePublished - Oct 2010

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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