Two thermostable nucleases coexisted in Staphylococcus aureus: evidence from mutagenesis and in vitro expression

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same genetic origin. However, two ORFs in S. aureus genomes were predicted to encode nucleases. One encoded an unnamed nuclease A (SNase) (termed nuc1), and the other encoded a thermonuclease (TNase) named nuc (termed nuc2). In order to verify whether the two thermostable nuclease proteins are coexpressed in S. aureus, the nuc1 and nuc2 genes were cloned and expressed in Escherichia coli, and both of the recombinant proteins showed thermostable nuclease activity in a toluidine blue-DNA assay. Furthermore, a nuc1-deleted mutant of S. aureus strain RN4220 (termed RNDeltanuc1) was successfully constructed by homologous recombination. Selection and characterization of this mutant strain revealed that it still exhibited thermostable nuclease activity, but at a relative lower level than that of the parent strain. The nucleases secreted by the parent strain and nuc1-deleted strain still showed functional activity after 30 min at 121 degrees C. The findings indicated that two types of thermostable nucleases, encoded by two different genes, coexisted in S. aureus.