Premium
Biochemical Analysis of the Zinc Uptake Regulator (Zur) from Klebsiella oxytoca
Author(s) -
Khacheryan Levon,
Xie Yujie,
Gallas Genna,
Hernandez Jose
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.756.1
Subject(s) - klebsiella oxytoca , zinc , mutant , biochemistry , biology , transcription (linguistics) , intracellular , regulator , chemistry , transcriptional regulation , gene , transcription factor , escherichia coli , enterobacteriaceae , linguistics , philosophy , organic chemistry
Zinc metabolism is critical to maintain bacterial pathogenesis in their respective hosts. Research has shown that altering the zinc homeostasis of bacteria via mutation of genes critical for regulation, export or uptake functions, such as Zur, ZntR, ZntA or ZnuABC, induces metal toxicity (excessive increase in a metal concentration) or insufficient intracellular zinc concentration. Prolonged disruption of intracellular zinc ion concentration homeostasis leads to attenuated bacteria and ultimately, cell death. The Zinc uptake regulator (Zur) protein has been shown to act as a master regulator of gene transcription of proteins associated with zinc uptake transporters such as ZnuABC. A particular intracellular zinc concentration is necessary in bacteria for proper structure and function of essential biological enzymes. When zinc levels within the cell are sufficiently high, Zn 2+ binds with Zur inducing a conformational change, which allows the Zn 2+ ‐Zur complex to bind to the promoter region of its target DNA. This promoter region is upstream of the gene for the uptake transporter (ZnuABC); thus, causing repression of transcription of genes necessary for zinc acquisition. When zinc levels drop, the Zn 2+ is released from Zur; thus reducing Zur's affinity for the DNA and normal transcription activity of ZnuABC ensues. In our research, we aimed to identify conserved amino acids that are essential for Zur functioning in Klebsiella oxytoca . We performed site‐directed mutagenesis and used an in silico analysis to identify a target mutation in the protein, which resulted in the H159A mutant. The effects of the mutation were studied in vivo with β‐galactosidase assays, which showed no effect of H159A mutation on the Zur protein function. Metal and oxidative stress viability effects were tested on a strain overproducing the Zur protein. Zur overexpression resulted in increased sensitivity to oxidative stress with H 2 O 2 and Zn 2+ compared to wild type due to increased cell inhibition with increasing concentrations of each condition. On the other hand, there was no increase in sensitivity with Zur overexpression for varying concentrations of Fe 2+ , Mn 2+ , and methyl viologen exposure. Finally, the effects of zinc availability on Zur and Superoxide Dismutase expression were also evaluated. Three isoforms of superoxide dismutase (MnSOD, Cu/ZnSOD and FeSOD) were expressed in K. oxytoca , which were grown in rich‐medium. In‐gel activity assays indicate cells overexpressing Zur lower Cu/ZnSOD and increase MnSOD activity compared to wild type. These results suggest Zur plays a crucial role in altering SOD activity in various isoforms to ultimately, inhibit oxidative stress and prolong cell survival. Overall, our results identify the presence of active Zur and three isoforms of SOD in K. oxytoca , and prove the relevance of Zur in the sensitivity to oxidative stress. Support or Funding Information ORSP Summer fellowship. Arizona College of Osteopathic Medicine. Midwestern University.