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Applied and Environmental Microbiology, September 2004, p. 5398-5406, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5398-5406.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Expression System Tightly Controlled by Zinc Availability in Lactococcus lactis

D. Llull and I. Poquet^*

Unité de Recherches Laitières et Génétique Appliquée, INRA, Jouy-en-Josas, France

Received 21 January 2004/ Accepted 10 May 2004

Here we developed the new expression system P_Zn zitR, based on the regulatory signals (P_Zn promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn²⁺ high-affinity uptake and regulation. A P_Zn zitR-controlled expression vector was constructed, and expression regulation was studied with two reporter genes, uspnuc and lacLM; these genes encode, respectively, a protein derived from Staphylococcus aureus secreted nuclease and Leuconostoc mesenteroides cytoplasmic ß-galactosidase. Nuclease and ß-galactosidase activities of L. lactis MG1363 cells expressing either uspnuc or lacLM under the control of P_Zn zitR were evaluated on plates and quantified from liquid cultures as a function of divalent metal ion, particularly Zn²⁺, availability in the environment. Our results demonstrate that P_Zn zitR is highly inducible upon divalent cation starvation, obtained either through EDTA addition or during growth in chemically defined medium, and is strongly repressed in the presence of excess Zn²⁺. The efficiency of the P_Zn zitR expression system was compared to that of the well-known nisin-controlled expression (NICE) system with the same reporter genes cloned under either P_Zn zitR or P_nisA nisRK control. lacLM induction levels reached with both systems were on the same order of magnitude, even though the NICE system is fivefold more efficient than the P_Zn zitR system. An even smaller difference or no difference was observed after 3 h of induction when nuclease was used as a reporter for Western blotting detection. P_Zn zitR proved to be a powerful expression system for L. lactis, as it is tightly controlled by the zinc concentration in the medium.

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* Corresponding author. Mailing address: Unité de Recherches Laitières et Génétique Appliquée, INRA, 78352 Jouy-en-Josas Cedex, France. Phone: 33-1-34 65 20 74. Fax: 33-1-34 65 20 65. E-mail: poquet{at}jouy.inra.fr.

Present address: Laboratorio de Genética Bacteriana, Centro de Investigaciones Biológicas, Madrid, Spain.

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Applied and Environmental Microbiology, September 2004, p. 5398-5406, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5398-5406.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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