MARTELL, SAMANTHA,JANE (2022) Demonstrating the Role of Histidine as a Cytosolic Buffer of Ni(II) Ions in Escherichia coli. Masters thesis, Durham University.
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Author-imposed embargo until 11 July 2023.
Transition metals are cofactors in many essential proteins. The correct function of these proteins hinges on metalation by their cognate metal ion, regardless of its ranking in the Irving-Williams series of metal-complex affinities. In the bacterial cytosol, evidence suggests that buffer molecules compete with proteins for metal ions, which will partition according to their relative affinities for various possible binding sites. Controlling metal availability in this manner prevents mismetallation by more competitive metals. This thesis seeks to test the buffer model using a genetic approach to alter cytosolic buffer molecule levels. A key test of the hypothesis will be changes in the cellular metalation of a metal-responsive transcription factor (RcnR), as proteins responsible for regulating metal homeostasis have affinities finely poised to sense the level of buffered metal, altering gene expression to match metal supply with demand.
L-histidine (His) is a candidate buffer of Ni(II) in E. coli. His levels were altered by mutating hisG, which encodes the feedback-inhibited first enzyme in the His biosynthetic pathway. A small library of mutant strains with altered levels of cytosolic His was generated. These strains were tested for growth sensitivity to Ni(II), and changes in Ni(II) and Co(II) responsive gene regulation by the RcnR sensor. Strains that produce more His, as assessed by qualitative bioassay for His excretion, were more resistant to the effects of Ni(II) on cell growth and showed a diminished transcriptional response to Ni(II). Strains that produced less His showed increased sensitivity to Ni(II) in a growth assay. In contrast, increased cytosolic His levels did not affect the Co(II)-responsiveness of RcnR compared to the parent strain. Thus, this work shows that His is a cytosolic buffer of Ni(II) in E. coli but is not a universal metal buffer.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||12 Jul 2022 12:22|