title: ZntR-mediated transcription of zntA responds to nanomolar intracellular free zinc.
abstract: In E. coli, ZitB and ZntA are important metal exporters that enhance cell viability under high environmental zinc. To understand their functions in maintaining zinc homeostasis, we applied a novel genetically-encoded fluorescent zinc sensor to monitor the intracellular free zinc changes in wild type, ∆zitB and ∆zntA E. coli cells upon sudden exposure to toxic levels of zinc ("zinc shock"). The intracellular readily exchangeable zinc concentration (or "free" zinc) increases transiently from picomolar to nanomolar levels, accelerating zinc-activated gene transcription. After zinc shock, the zitB mRNA level is constant while the zntA mRNA increases substantially in a zinc-dependent manner. In the ∆zitB E. coli 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. Furthermore, the ZntR-mediated transcription of zntA exhibits an apparent K(1/2) for zinc activation in the nanomolar range in vivo, significantly higher than the femtomolar affinity for zinc binding and transcription activation previously measured in vitro. A kinetically-controlled transcription model is sufficient to explain the observed regulation of intracellular free zinc concentration by ZntR and ZntA after zinc shock.
authors.affiliation: Program in Chemical Biology & Department of Chemistry, The University of Michigan, 930 N University Ave, Ann Arbor, MI 48109, USA.
authors.forename: Da
authors.initials: D
authors.lastname: Wang
authors.forename: Olijahwon
authors.initials: O
authors.lastname: Hosteen
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C492246:ZntR protein, E coli; C017800:zinc-binding protein; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase; D015032:Zinc