Staphylococcal nuclease (here termed as Nucl) 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 coll. Sequence alignment and phylogenetic analysis revealed that Nuc2 was a more conserved protein in the staphylococci group compared with Nucl. 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 Ca2+ (0.05 mM), Mg2+ (0.5 mM), dithiothreitol, beta-mecaptoethanol, TritonX-100, Tween-20, and urea; however, activity decreased sharply when exposed to heavy metals such as Zn2+ and Mn2+, and in the presence of EDTA or SDS. Nuc2 showed weaker activity, lower thermostability and different sensitivity to these chemical agents compared with Nucl, 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. (C) 2012 Elsevier GmbH. All rights reserved.