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Applied and Environmental Microbiology, March 2004, p. 1600-1607, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1600-1607.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Influence of Lipoteichoic Acid D-Alanylation on Protein Secretion in Lactococcus lactis as Revealed by Random Mutagenesis

S. Nouaille,1 J. Commissaire,1 J. J. Gratadoux,1 P. Ravn,2 A. Bolotin,3 A. Gruss,1 Y. Le Loir ,1,{dagger},{ddagger} and P. Langella1,{dagger}*

Unité de Recherches Laitières et de Génétique Appliquée,1 Unité de Génétique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas cedex, France,3 Department of Lactic Acid Bacteria, Applied Molecular Biology, Biotechnological Institute, DK-2970 Hørsholm, Denmark2

Received 28 April 2003/ Accepted 5 December 2003

Lactococcus lactis, a food-grade nonpathogenic lactic acid bacterium, is a good candidate for the production of heterologous proteins of therapeutic interest. We examined host factors that affect secretion of heterologous proteins in L. lactis. Random insertional mutagenesis was performed with L. lactis strain MG1363 carrying a staphylococcal nuclease (Nuc) reporter cassette in its chromosome. This cassette encodes a fusion protein between the signal peptide of the Usp45 lactococcal protein and the mature moiety of a truncated form of Nuc (NucT). The Nuc secretion efficiency (secreted NucT versus total NucT) from this construct is low in L. lactis (~40%). Twenty mutants affected in NucT production and/or in secretion capacity were selected and identified. In these mutants, several independent insertions mapped in the dltA gene (involved in D-alanine transfer in lipoteichoic acids) and resulted in a NucT secretion defect. Characterization of the dltA mutant phenotype with respect to NucT secretion revealed that it is involved in a late secretion stage by causing mature NucT entrapment at the cell surface.

* Corresponding author. Mailing address for P. Langella: Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas cedex, France. Phone: 33 01 34 65 20 70. Fax: 33 01 34 65 20 65. E-mail: langella{at}jouy.inra.fr..

{dagger} Y.L.L. and P.L. contributed equally to this work.

{ddagger} Present address for Y. Le Loir: Laboratoire de Microbiologie, Ecole Nationale Supérieure d'Agronomie, Institut National de la Recherche Agronomique, UMR1253, 65, rue de Saint Brieuc, CS84215, 35042 Rennes cedex, France. Phone: 33 02 23 48 59 04. Fax: 33 02 23 48 59 02. E-mail: leloir{at}roazhon.inra.fr

Applied and Environmental Microbiology, March 2004, p. 1600-1607, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1600-1607.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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