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J Bacteriol, April 1998, p. 1904-1912, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

Isabelle Poquet,1,* S. Dusko Ehrlich,2 and Alexandra Gruss1

Laboratoire de Génétique Appliquée-URLGA,1 and Laboratoire de Génétique Microbienne,2 Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France

Received 8 October 1997/Accepted 16 January 1998

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called Delta SPNuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of Delta SPNuc to report protein export. The shuttle vector pFUN was designed to construct Delta SPNuc translational fusions whose expression signals are provided by inserted DNA. The capacity of Delta SPNuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All Delta SPNuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that Delta SPNuc is well suited to report both protein export and membrane protein topology.

* Corresponding author. Mailing address: Laboratoire de Génétique Appliquée-URLGA, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France. Phone: 33 01 34 65 20 74. Fax: 33 01 34 65 20 65. E-mail: poquet{at}biotec.jouy.inra.fr.




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