Studying Zinc Homeostasis in Escherichia Coli Using Carbonic Anhydrase-based Ratiometric Sensors.

[摘要] Zinc is an essential element for numerous cellular processes.In this work, we developed a series of highly selective, sensitive and quantitative zinc sensors based on carbonic anhydrase (CA).An aryl sulfonamide ligand of CA exhibits a large fluorescence signal coupled to zinc binding, forming a Förster resonance energy transfer (FRET) pair with a red fluorescent protein fused to CA.These sensors respond to alterations in physiological free zinc concentrations, and are applied to Escherichia coli to quantify the readily exchangeable zinc concentration.In minimal media, E. coli BL21(DE3) cells expressing the CA sensors exhibit a median intracellular readily exchangeable zinc concentration of 20 pM.To understand the functions of ZitB and ZntA, important transporters in zinc detoxification in E. coli, we applied the sensor H94N CA_TagRFP to monitor the intracellular free zinc changes in wild type, ∆zitB and ∆zntA strains upon sudden exposure to toxic levels of zinc (;;zinc shock”). The intracellular free zinc increasestransiently from picomolar to nanomolar levels.The corresponding zinc-dependent changes in the zntA mRNA level indicate that ZntR-mediated transcription of zntA exhibits an apparent K1/2 for zinc activation in the nanomolar range in vivo.In the ∆zitB strain the free zinc concentration rises more rapidly after zinc shock compared to wild-type cells, while a prolonged accumulation of free zinc is observed in the ∆zntA strain. Based on these results, we propose that ZitB functions as a constitutive, first-line defense against toxic zinc influx, while ZntA is up-regulated to efficiently lower the free zinc concentration.The zinc detoxification functions of three transporters and a periplasmic protein regulated by the BaeS/R two-component system were explored.The effects of single gene knockouts in the BaeS/R regulon on changes in cell growth rate, free zinc, total zinc and total copper after zinc shock were investigated to evaluate their functions.Two exporters, MdtABC and MdtD, and the periplasmic protein, Spy, are involved in zinc detoxification based on the growth defects and increases in free and total zinc/copper that were observed in the single knockout strains.These proteins potentially complement the ATP-driven zinc export mediated by ZntA in E. coli.

[发布机构] University of Michigan

[效力级别] E. Coli

[关键词] Intracellular Free Zinc;E. Coli;Carbonic Anhydrase;ZitB;ZntA and ZntR;BaeS/R;Biological Chemistry;Science;Chemical Biology