New expression system tightly controlled by zinc availability in Lactococcus lactis.
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 Zn(2+) 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 beta-galactosidase. Nuclease and beta-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 Zn(2+), 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 Zn(2+). 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.
Llull
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Unité de Recherches Laitières et Génétique Appliquée, INRA, 78352 Jouy-en-Josas Cedex, France.
Poquet
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D002413:Cations, Divalent / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D007700:Kinetics; D013294:Lactococcus lactis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D015183:Restriction Mapping; D015032:Zinc / Q000378:metabolism*
D002413:Cations, Divalent; D004492:Edetic Acid; D015032:Zinc