Characterization of a functional insertion sequence ISSau2 from Staphylococcus aureus.

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• ISSau2, a member of the IS150 f subgroup in the IS3 family, encodes a fusion transposase OrfAB.
• OrfAB is produced via programmed -1 translational frameshifting of two overlapping reading frames, orfA and orfB.

Purpose of the Study:

• To characterize the binding and cleaving activities of the ISSau2 transposase.
• To determine the transposition frequency of ISSau2 in vivo.

Main Methods:

• Purification of ISSau2 transposase OrfAB.
• In vitro binding and cleavage assays using terminal inverted repeat sequences (IRs).
• In vivo GFP hop-on assay to measure transposition frequency.

Main Results:

• Purified OrfAB specifically bound and cleaved ISSau2 IRs in vitro.
• The in vivo transposition frequency of ISSau2 was approximately 1.76 × 10^-3.
• OrfB exhibited catalytic activity similar to OrfAB, while OrfA did not; both OrfA and OrfB inhibited OrfAB-induced transposition.

Conclusions:

• ISSau2 is a functional member of the IS150/IS3 family.
• ISSau2 transposase OrfAB binds, cleaves IRs, and induces transposition.
• OrfA and OrfB inhibit ISSau2 transposition, contributing to understanding its biological roles in *S. aureus*.