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Biochemical Characterization of Zinc‐Responsive Transcription Factors Zur and ZntR in Klebsiella oxytoca
Author(s) -
Crawford Kyler
Publication year - 2016
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.30.1_supplement.1055.9
Subject(s) - klebsiella oxytoca , zinc , biochemistry , regulator , operon , electrophoretic mobility shift assay , biology , response regulator , bioreporter , chemistry , transcription factor , escherichia coli , reporter gene , klebsiella pneumoniae , gene , gene expression , mutant , organic chemistry
Zinc is an essential micronutrient that is sequestered by human innate immune defenses during infection. Bacterial acquisition via outer membrane transporters is necessary, but accumulation of zinc is toxic. The opportunistic human pathogen Klebsiella oxytoca is a member of the Enterobacteriaceae family that expresses the ZnuABC transporter complex to import zinc, and expresses the ZntABC transporter complex to export zinc. Though K. oxytoca infections have become an emergent problem, the regulation of this critical balance and the mechanism for zinc sensing have never been characterized. We hypothesize two transcription factors Zinc Uptake Regulator (Zur) and Znt Regulator (ZntR) are responsible for repressing import and activating export, respectively. In the current study, we applied in silico methods to identify a Zur (Zinc Uptake Regulator) and a ZntR (Znt Regulator) homologue in K. oxytoca . Both K. oxytoca Zur and ZntR were recombinantly overexpressed and purified using affinity chromatography. The target proteins aggregate in inclusion bodies found with the insoluble component of cellular fractions, so a protocol involving in vitro urea denaturation and refolding was developed. The DNA‐binding activity of the Zur and ZntR purified fractions was modeled with electrophoretic mobility shift assays (EMSA). A thorough biochemical protein characterization was conducted on Zur and ZntR, involving mass spectrometry, metal content quantification and chemical cross‐linking analyses. Additionally, we used the bacterial luxCDABE operon to create a bioluminescent reporter system that measured the expression of the znuA and zntA promoters. Overall, our results identify the presence of active Zur and ZntR regulators in K. oxytoca , and prove the metal‐dependent control of the znuA and zntA promoters. Support or Funding Information Biomedical Sciences Department ‐ Midwestern University ‐ Glendale, AZ