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Journal of Bacteriology, July 2001, p. 4157-4166, Vol. 183, No. 14
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

Y. Dieye, S. Usai, F. Clier, A. Gruss, and J.-C. Piard*

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.

* 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.

Journal of Bacteriology, July 2001, p. 4157-4166, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4157-4166.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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