Molecular Cloning and Expression of thespsBGene Encoding an Essential Type I Signal Peptidase fromStaphylococcus aureus

The gene, spsB, encoding a type I signal peptidase has been cloned from the gram-positive eubacterium Staphylococcusaureus.Thegeneencodesaproteinof191aminoacidresidueswithacalculatedmolecularmass of 21,692 Da. Comparison of the protein sequence with those of known type I signal peptidases indicates conservation of amino acid residues known to be important or essential for catalytic activity. The enzyme has been expressed to high levels in Escherichia coli and has been demonstrated to possess enzymatic activity against E. coli preproteins in vivo. Experiments whereby the spsB gene was transferred to a plasmid that is temperature sensitive for replication indicate that spsB is an essential gene. We identified an open reading frame immediately upstream of thespsBgene which encodes a type I signal peptidase homolog of 174 amino acid residues with a calculated molecular mass of 20,146 Da that is predicted to be devoid of catalytic activity. The majority of proteins that are translocated across one or more membranes from the site of synthesis are initially syn- thesized with an N-terminal extension known as a signal, or leader, peptide (42). Proteolytic cleavage of the signal se- quence to yield the mature protein occurs during or shortly after the translocation event and is catalyzed in both pro- karyotesandeukaryotesbyenzymesknownassignal,orleader, peptidases (SPases). The bacterial SPases are membrane pro- teins consisting of a single polypeptide anchored to the mem-