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 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, P-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 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.