Previous Article | Next Article ![]()
Journal of Bacteriology, July 2001, p. 4157-4166, Vol. 183, No. 14
Unité de Recherches Laitières et
Génétique Appliquée, INRA, 78352 Jouy-en-Josas Cedex,
France
Received 5 December 2000/Accepted 16 April 2001
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 (~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.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.14.4157-4166.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Design of a Protein-Targeting System for Lactic
Acid Bacteria
*
Corresponding author. Mailing address: Unité de
Recherches Laitières et Génétique Appliquée,
INRA, 78352 Jouy-en-Josas Cedex, France. Phone: 33-1-34 65 20 67. Fax:
33-1-34 65 20 65. E-mail: piard{at}jouy.inra.fr.
This article has been cited by other articles:
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |