Design of a protein-targeting system for lactic acid bacteria.
We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (approximately 95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.
Dieye
Y
Y
Unité de Recherches Laitières et Génétique Appliquée, INRA, 78352 Jouy-en-Josas Cedex, France.
Usai
S
S
Clier
F
F
Gruss
A
A
Piard
J C
JC
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D019344:Lactic Acid / Q000378:metabolism; D013294:Lactococcus lactis / Q000378:metabolism*; D008836:Micrococcal Nuclease; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D013297:Streptococcus pyogenes / Q000378:metabolism
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D021382:Protein Sorting Signals; C074002:nuc protein, staphylococcus; C014689:streptococcal M protein; C066583:usp45 protein, Lactococcus lactis; D019344:Lactic Acid; D004720:Endonucleases; D008836:Micrococcal Nuclease