Molecular cloning and expression of the spsB gene encoding an essential type I signal peptidase from Staphylococcus aureus

Abstract

The gene, spsB, encoding a type I signal peptidase has been cloned from the gram-positive eubacterium Staphylococcus aureus. The gene encodes a protein of 191 amino acid residues with a calculated molecular mass 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 the spsB gene 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 chemistry and enzymology of the type I signal peptidases

“…1A). The latter feature is shared by the signal peptidase of the cyanobacterium Phonnidium laminosum (22 kDa; Packer et al, 1995), and all known signal peptidases from Gram positive bacteria, which include enzymes from Bacillus subtilis (van Dijl et al, 1992), Bacillus amyloliquefaciens, Bacillus culdolyticus, and Bacillus lichenifonnis (for a recent compilation of sequences see Meijer et al, 1995), Staphylococcus aureus (SpsB; Cregg et al, 1996), and Mycobacterium tuberculosis (Philipp et al, 1996). All Bacillus signal peptidases and the S. uureus signal peptidase consist of 21 kDa polypeptides whereas the M. tuberculosis gene encodes a 32 kDa polypeptide.…”

“…However, it seems that the SpsA protein does not possess signal peptidase activity (see the section on the catalytic mechanism of signal peptidases). SpsA and spsB are adjacent genes on the chromosome of S. aureus (Cregg et al, 1996).…”

“…Interestingly, the SpsA protein of S. aureus, which is very similar to the Sips-like signal peptidases of Gram-positive bacteria, seems to lack the putative catalytic Ser and Lys residues (Cregg et al, 1996). The role of SpsA in the processing of secretory precursor proteins in S. aureus has not been investigated yet but it is predicted that this protein has no signal peptidase activity.…”

Evaluation of the type I signal peptidase as antibacterial target for biofilm-associated infections of Staphylococcus epidermidis

“…RT-PCR experiments showed that sip1 and sip2 belong to the same operon (data not shown), in analogy with spsA and spsB, which are also co-transcribed on the chromosome of Staphylococcus aureus (Cregg et al, 1996).…”

“…Leader peptidase (LepB) complementation assay using E. coli IT89 (Inada et al, 1989) was performed as described by Cregg et al (1996). The OD 595 was recorded at 30 min intervals for 7.5 h in 24-well plates (1 ml total volume).…”