New Expression System Tightly Controlled by Zinc Availability in Lactococcus lactis

Abstract

ABSTRACT 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 β-galactosidase. Nuclease and β-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.