Date of Award
Campus Access Dissertation
Chemistry and Biochemistry
F Wayne Outten
Metals are important for maintaining proper cellular functions. Cells evolved metal
homeostasis systems to control metal acquisition and utilization in response to environmental changes in order to fulfill the cellular metal demand under different situations. Extensive studies have been carried out to elucidate metal homeostasis systems for individual metals. However, the cross-regulation between different metals is not well-understood. In our study, we focus on the cross-regulation of iron and nickel in bacteria by studying the yqjH-yqjI gene pair in Escherichia coli (E. coli).
In E. coli, the yqjH gene encodes a putative ferric siderophore reductase that is also part of the Fur regulon. The yqjI gene, divergently transcribed from yqjH, encodes a novel member of the winged-helix family of transcriptional regulators and also contains an N-terminal extension similar to the Ni2+-binding C-terminal tail of SlyD. We found that YqjH has ferric reductase activity and is required for iron homeostasis in E. coli. In addition, elevated nickel stress levels disrupt iron homeostasis in E. coli and deletion of yqjH increases nickel toxicity. YqjI represses the expression of yqjH and its own gene, and nickel is able to reverse this inhibition effect. Purified YqjI has at least two oligomeric forms in solution (monomer, and most probably hexamer), and shows multiple oligomeric forms in the protein gel (monomer, dimer, trimer, tetramer, etc.). The interchange of the oligomeric states is regulated by oxygen but not by nickel. Only
“hexameric” apo-YqjI has DNA affinity and binding to nickel or iron reduces YqjI DNA-binding activity in vitro. The above results suggest that the YqjI protein controls expression of yqjH to help maintain iron homeostasis under conditions (such as elevated cellular nickel levels) that disrupt iron metabolism.
By optimizing the protocol for fluorescence-labeled DNase I footprinting, we improved the data quality and reproducibility, which allows it to be used for the quantification of protein-DNA binding affinity. We then used this technique to characterize the mechanism of YqjI regulation in the yqjH-yqjI intergenic region, including definitive mapping of the YqjI binding sites at the yqjH and yqjI promoters. DNase I footprinting revealed that the YqjI binding sites at the yqjH and yqjI promoters are not equivalent. YqjI binding results in an extended footprint at the yqjI promoter compared to the yqjH promoter. A series of point mutations, insertions, or deletions in the yqjH-yqjI intergenic region were generated and analyzed through a combination of in vitro DNase I footprinting and in vivo gene reporter constructs in order to characterize YqjI-dependent transcriptional regulation at each promoter in the yqjH-yqjI intergenic region. The results of these studies indicate that the two YqjI binding sites, while separated by nearly 200 bp, appear to communicate in order to provide full YqjI-dependent regulation. YqjI binding at both sites is required for full repression of either promoter suggesting a model where YqjI induces or stabilizes a bent conformation in the DNA in order to create a “repressor knot” in the yqjH-yqjI intergenic region. These studies provide a complex picture of novel YqjI transcriptional regulation within the yqjH-yqjI intergenic region.
Wang, S.(2012). Genetic and Biochemical Studies of YqjH and YqjI: Cross-talk between Iron Homeostasis and Nickel Homeostasis in Escherichia Coli. (Doctoral dissertation). Retrieved from http://scholarcommons.sc.edu/etd/741