Export defect adjacent to the processing site of staphylococcal nuclease is suppressed by a prlA mutation

Abstract

Plasmids have been constructed in which the Escherichia coli alkaline phosphatase promoter and signal sequence have been fused to the staphylococcal nuclease gene to promote the high-level expression and secretion of this gene product in E. coli. We determined that the first amino acid residue after the signal sequence can determine whether this protein was processed and exported to the periplasmic space. Fractionation and protease accessibility studies were used to show that the export-defective, nuclease precursor is internal to the cytoplasmic membrane barrier of the cell. Furthernore, this export defect was suppressed in a strain containing a prUi mutation. These findings are novel in that this region of the polypeptide chain has been implicated in processing but not export and that priA mutations have not been previously known to suppress such defects.While engineering the high-level expression and secretion of the nuclease from Staphylococcus aureus Foggi in Escherichia coli, we noticed that the first amino acid after the signal sequence can determine whether staphylococcal nuclease was processed and exported to the periplasmic space. Furthermore, thiS export defect was suppressed in a strain containing a prlA mutation (4). These findings are novel since mutations adjacent to the processing site generally affect signal peptide cleavage but not export (8,16), and prlA mutations have been shown to suppress defects in the hydrophobic core region of the signal sequence (3, 4) but not defects adjacent to the processing site. Therefore, we characterized this phenomenon in greater detail, and the results are described below.To promote the high-level expression and secretion of staphylococcal nuclease in E. coli, plasmids have been constructed in which this gene lacking its own signal sequence (18) was fused to the E. coli alkaline phosphatase promoter and signal sequence (7). pFOG403 is a pBR322 derivative plasmid containing a precise fusion of these elements (Fig. 1). To maintain an amino acid sequence adjacent to the processing site similar to that found in the alkaline phosphatase precursor, oligonucleotide-directed mutagenesis was performed on pFOG403 to insert the triplet CGG encoding an arginine residue between the alkaline phosphatase signal sequence and the staphylococcal nuclease gene (Fig. 1). This procedure resulted in the creation of pFOG406. The details of these plasmid constructions will be described elsewhere (D. Shortle, manuscript in preparation).Strain MC4100 (F-AlacUJ69 araD136 relA rpsL thi) (10) or its isogenic derivative SE6004 containing the prlA4 mutation t3) was used as a host for pFOG403, pFOG406, or pBR322. Overnight cultures grown in L broth were diluted 50-fold into MOPS (morpholinopropanesulfonic acid) medium (14) containing 1.0% glucose, 0.15% vitamin-free casein hydrolysate, 0.0001% thiamine, and 0.1 mM KH2PO4 and grown at 37°C with shaking for 7 h to allow induction and accumulation of the plasmid-encoded staphylococcal nuclease. For radiolabeling protein, strains were grown for 4 ...

The complete general secretory pathway in gram-negative bacteria.

“…The fact that prl mutations in secE or secY facilitate the export of proteins with debilitated signal peptides (149,330,445,474,524) or export initiation domains (330,445) has been interpreted to indicate that weakened signals can be recognized by receptors rendered less stringent by the prl mutation (244). Genetic evidence suggests that SecE is required before SecY (37) and hence is more likely than SecY to be the signal peptide receptor.…”