Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus

Area of Science:

• Microbiology
• Enzymology
• Molecular Biology

Background:

• Staphylococcal nuclease (Nuc1) is a key virulence factor and marker for *Staphylococcus aureus* detection.
• A second thermostable nuclease (Nuc2) in *S. aureus* was identified and characterized.

Purpose of the Study:

• To characterize the functional and biochemical properties of the second staphylococcal nuclease (Nuc2).
• To compare Nuc2 with Nuc1 regarding sequence conservation, enzymatic activity, and stability.
• To elucidate the roles of both nucleases in *S. aureus*.

Main Methods:

• Recombinant expression of Nuc2 in *Escherichia coli*.
• Sequence alignment and phylogenetic analysis.
• Zymogram assays to confirm nuclease activity.
• Biochemical characterization of Nuc2 activity under varying conditions (temperature, pH, presence of ions and chemicals).
• Construction and analysis of *nuc1* and *nuc2* double deletion mutants in *S. aureus*.

Main Results:

• Nuc2 exhibits nuclease activity, degrading various nucleic acids with optimal activity at 50°C and pH 10.
• Nuc2 activity is modulated by specific ions and chemicals, showing different sensitivities compared to Nuc1.
• Phylogenetic analysis indicates Nuc2 is more conserved among staphylococci than Nuc1.
• A double deletion mutant confirmed Nuc1 plays a major role in *S. aureus* thermonuclease activity.

Conclusions:

• Nuc2 is a distinct functional thermostable nuclease in *S. aureus* with unique biochemical properties.
• Nuc1 is the primary contributor to thermonuclease activity in *S. aureus*.
• The characterization of Nuc2 provides insights into the enzymatic repertoire and evolution of staphylococcal nucleases.