title: Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus.
abstract: Staphylococcal nuclease (here termed as Nuc1) is considered an important virulence factor and a unique marker widely used in the detection of Staphylococcus aureus. A second functional thermostable nuclease (here termed as Nuc2) in S. aureus was characterized after recombinant expression in Escherichia coli. Sequence alignment and phylogenetic analysis revealed that Nuc2 was a more conserved protein in the staphylococci group compared with Nuc1. Recombinant Nuc2 showed nuclease activity in the zymogram test and was able to degrade various types of nucleic acids. The optimal reaction temperature and pH for Nuc2 were 50 °C and pH 10, respectively. The enzymatic activity of Nuc2 was stimulated in the presence of Ca(2+) (0.05 mM), Mg(2+) (0.5mM), dithiothreitol, β-mecaptoethanol, TritonX-100, Tween-20, and urea; however, activity decreased sharply when exposed to heavy metals such as Zn(2+) and Mn(2+), and in the presence of EDTA or SDS. Nuc2 showed weaker activity, lower thermostability and different sensitivity to these chemical agents compared with Nuc1, which was consistent with differences in the sequence pattern and structure predicted. Furthermore, a nuc1 and nuc2 double deletion mutant of S. aureus and respective complementation experiments suggest a major role for nuc1 in terms of thermonuclease activity in S. aureus.
authors.affiliation: MOST-USDA Joint Research Center for Food Safety & Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Hu
authors.forename: Jianghong
authors.initials: J
authors.lastname: Meng
authors.forename: Chunlei
authors.initials: C
authors.lastname: Shi
authors.forename: Kirstin
authors.initials: K
authors.lastname: Hervin
authors.forename: Pina M
authors.initials: PM
authors.lastname: Fratamico
authors.forename: Xianming
authors.initials: X
authors.lastname: Shi
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000378:metabolism; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D009696:Nucleic Acids; D011994:Recombinant Proteins; D008836:Micrococcal Nuclease