New Expression System Tightly Controlled by Zinc Availability in Lactococcus lactis

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American Society for Microbiology

RESUMO

Here we developed the new expression system PZn zitR, based on the regulatory signals (PZn promoter and zitR repressor) of the Lactococcus lactis zit operon, involved in Zn2+ high-affinity uptake and regulation. A PZn 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 PZn 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 PZn 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 PZn zitR expression system was compared to that of the well-known nisin-controlled expression (NICE) system with the same reporter genes cloned under either PZn zitR or PnisA 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 PZn 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. PZn 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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