PIERGENTILI, CECILIA (2016) Exploring the determinants of metal sensing in Salmonella typhimurium using FrmR, a non-metal sensing RcnR/CsoR family member. Doctoral thesis, Durham University.
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Salmonella FrmR, a member of the RcnR/CsoR family of metalloregulators, has been characterised during the course of this work and was coincidentally confirmed to bind specifically to the frmRA operon, which encodes a putative Zn(II)-requiring class III alcohol dehydrogenase. FrmR shares a high degree of similarity with Ni(II)/Co(II)-sensing RcnR, in particular conserving two residues of a so-called WXYZ motif required to detect metals. Metal-binding properties of FrmR were therefore extensively investigated in vitro and its ability, or otherwise, to respond to metals explored in vivo.
FrmR binds Zn(II), Cu(I), Co(II) and Ni(II), adopting different geometries, and always involving a mercapto group from the only cysteine residue (Cys35). Moreover, KZn(II)FrmR is only slightly below the range of affinity found for other zinc sensors.
Since FrmR fails to sense metals in cells, where only formaldehyde is detected, questions about which parameters are required in metal regulation in Salmonella and, in general, in bacteria were investigated. A single-point mutation (Glu64 His) allows FrmR to sense cellular zinc and cobalt. FrmR and E64HFrmR have been consequently used as a case of study to test hypotheses about the mechanisms determining which metals are detected by a given sensor in cells.
In addition, the ability of FrmR to detect cellular formaldehyde has been investigated, and a reaction mechanism tested by site-directed mutagenesis in vitro. Salmonella Ni(II)/Co(II)-sensing RcnR has been characterised, and employed to test the specificity of formaldehyde responsiveness of FrmR. By a single point-mutation (Ser2 Pro), the Ni(II)/Co(II)-sensing RcnR has been successfully switched to a formaldehyde sensor in vitro, further endorsing the proposed mechanism.
Investigation of FrmR structure has been pursued by producing apo- and Zn(II)-bounded FrmR and E64HFrmR crystals, which were then analysed at the Diamond Light Source. The best dataset has been processed to obtain a 3D-structure.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||FrmR; RcnR-CsoR; Salmonella enterica;formaldehyde detoxification; formaldehyde; cobalt; copper; metal; metal homeostasis; metal-sensor; metalloprotein; zinc|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||25 Apr 2016 10:37|