Structural prediction and mutational analysis of the Gifsy-I Xis protein

The Gifsy-I phage integrates into the Salmonella Typhimurium chromosome via an integrase mediated, site-specific recombination mechanism. Excision of the Gifsy-I phage requires three proteins, the Gifsy-I integrase (Int), the Gifsy-I excisionase (Xis) protein, and host encoded Integration Host Factor (IHF). The Gifsy-I xis gene encodes the 94-residue Gifsy-I excisionase protein that has a molecular weight of 11.2 kDa and a pI of 10.2. Electrophoretic Mobility Shift Assays (EMSA) suggested at least one region of the protein is responsible for protein-DNA interactions with a tripartite DNA binding site composed of three direct imperfect repeats.

Here we have undertaken experiments to dissect and model the structural motifs of Gifsy-I Xis necessary for its observed DNA binding activity. Diethyl sulfate mutagenesis (DES) and mutagenic PCR techniques were used to generate Gifsy-I xis mutants. Mutant Xis proteins that lacked activity in vivo were purified and tested by EMSA for binding to the Gifsy-I Xis attP attachment site. Results from mutagenesis experiments and EMSA were compared to results of structural predictions and sequence analyses.

Sequence comparisons revealed evidence for three distinct structural motifs in the Gifsy-I Xis protein. Multiple sequence alignments revealed unexpected homologies between the Gifsy-I Xis protein and two distinct subsets of polynucleotide binding proteins. Our data may suggest a role for the Gifsy-I Xis in the regulation of the Gifsy-I phage excision beyond that of DNA binding and possible interactions with the Gifsy-I Int protein.