Molecular cloning, expression, and characterization of a Ca2+-dependent, membrane-associated nuclease of Mycoplasma genitalium.
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 Mn2+. 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.
Li
Linbo
L
Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
Krishnan
Manickam
M
Baseman
Joel B
JB
Kannan
T R
TR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D045704:Mycoplasma genitalium / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D004720:Endonucleases