Lindsay, William Pirie (1992) Expression of recombinant metal-binding proteins in E. Coli and in Synechococcus PCC7942: examination of metal binding in vivo and vitro. Doctoral thesis, Durham University.
Metallothioneins (MTs), cysteine-rich proteins and polypeptides, are proposed to detoxify excess intracellular metal ions via sequestration. Three genes, each encoding a protein related to this group of molecules, were expressed in Escherichia coli and in Synechococcus PCC7942 (variant PIM8) in order to examine the metal binding properties of their products. Phenotypic alterations, in terms of metal-tolerance and - accumulation, were assessed in cells expressing these genes. The genes which were expressed were: (1) smtA from Synechococcus PCC7942, which is designated to be the first isolated prokaryotic MT gene; (2) PsMT(_A), a gene from pea (Pisum sativum L) which encodes a protein with similarity to class I MT; and (3) a synthetic gene encoding(Glu-Cys)(_3)Gly, an analogue of the phytochelatin (PC; class III MT) molecule (γGlu-Cys)(_3)Gly. The protein encoded by smtA was shown to have high affinity for metal ions (Hg, Cd, Cu, Zn), supporting the designation of smtA as a prokaryotic MT gene. Comparison with mammalian MT revealed that the affinity of the product of smtA for Zn was higher than that of the mammalian protein, suggesting a role for this protein in Zn homoeostasis and/or detoxification in Synechococcus sp. E. coli cells expressing smtA exhibited increased accumulation of Zn and Cd (3-fold and 1.4-fold respectively relative to control cells), but no increase in tolerance toward Zn, Cd or Cu. Comparison of the metal-affinity of the product of PsMT(_A) with that of mammalian MT revealed that this protein also has high affinities for Cu, Cd and Zn. These data support the hypothesis that PsMTp^ is a higher plant MT gene. Affinity of the product of this gene for Cu was higher than that of mammalian MT, suggesting a role for this protein in Cu homoeostasis and/or detoxification. Expression of PsMT(_A) in E. coli resulted in increased accumulation of Cu, Cd and Zn. Cu accumulation was increased more substantially than either Zn or Cd accumulation in cells expressing PsMT(_A). No increase in tolerance toward any of these metals was observed in E. coli expressing this gene. There is evidence that PCs are involved in Cd detoxification in higher plants. Genes encoding enzymes involved in the synthesis of these molecules have not been isolated, precluding gene transfer experiments for investigation of their function. Expression of a gene encoding (Glu-Cys) gGly in E. coli resulted in increased tolerance toward Cd, but not Cu or Zn. Thus, a predicted function of a secondary metabolite (PC) was observed when a gene product based on the structure of this molecule was expressed in a heterologous system. No significant increase in accumulation of Cd, Cu or Zn was detected in cells expressing this gene.smtA transcripts were shown to increase in abundance in response to elevated concentrations of Cd in Synechococcus PCC7942 (variant PIM8). Sequences derived from the smt locus were fused to a synthetic gene encoding (Glu-Cys)(_3)Gly, and introduced into Synechococcus PCC7942(variant PIM8). Transcripts encoding (Glu-Cys)gGly increased in response to exposure of these cells to Cd. Cells containing this construct exhibited increased tolerance toward Cd. Data concerning expression of(Glu-Cys)(_3)Gly in E. coli and Synechococcal cells support the hypothesis that PCs may have a role in detoxification of excess intracellular Cd. Comparison of the data obtained in these studies has been used to assess the factors affecting metal-accumulation and -tolerance as a result of expression of heterologous metal-ligands in microbial cells.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||16 Nov 2012 10:56|