An Investigation into Mechanisms of Metal Tolerance and Accumulation by Seaweed at the Phycospheric and Genomic Levels

O'Neill, David (2012) An Investigation into Mechanisms of Metal Tolerance and Accumulation by Seaweed at the Phycospheric and Genomic Levels. PhD thesis, Waterford Institute of Technology.

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Abstract

This thesis outlines the characterization of a novel metallothionein (MT)-like gene-encoded protein from the red seaweed Polysiphonia lanosa. The bioinformatic analysis of a cDNA library identified a 90 bp putative open reading frame (G3AORF1) encoding a 29 amino acid protein exhibiting several characteristic features of MT proteins, including 9 cysteine residues, no aromatic residues and a 60% amino acid sequence identity to two MTs. Relative Quantification by real-time PCR confirmed its increase in expression in response to Copper (Cu) exposure. Genomic DNA (gDNA) sequence data obtained by a T/A-based cloning protocol of PCR-amplified gDNA fragments proved this coding region to be a novel DNA sequence uninterrupted by introns. Southern blot analysis demonstrated the target sequence to be located on an EcoRI restriction fragment of approximately 3.5 kb in length, suggesting either (i) a single copy of this sequence, (ii) multiple copies of the target clustered on a single restriction fragment, or (iii) multiple copies of the target located on fragments of equal size. Inductive coupled plasma (ICP) optical emission spectroscopy determined Escherichia coli cells expressing His•tagged and untagged forms of the recombinant protein to remove a greater amount of Cu from solution than host and non-recombinant control cultures. A qualitative indirect Enzyme Linked Immunosorbent Assay indicated stabilization and protection against proteolysis of the recombinant protein by Cu-binding. Recombinant bacterial clones acquired an enhanced tolerance to ≤ 20 mg.L-1 of Cu, Nickel (Ni) and Lead (Pb) when cultured on solid media. The role of seaweed surface-bound bacteria and their contribution to metal accumulation by the algal host was also investigated in this study. The growth and accumulation capability of cultured bacterial isolates was seen to reduce upon increases from 2.0 to 3.0 mg.L-1Cu and Ni in the growth medium. Maximum removal of Cu was recorded at 22-38% and 6-12% for Ni. The growth of two isolates was not affected by ≤ 10.0 mg.L-1 Ni possibly indicating the presence of Ni resistance genes or a Ni efflux system within these strains. The construction of 16S rRNA gene libraries from DNA isolated from the surfaces of Ulva sp. and P. lanosa harvested from a polluted site indicated host-specific bacterial communities but with common -Proteobacteria group dominance. Bacterial community members identified were related to species known to possess a range of significant traits that would be of clear benefit to the defense and physiological state of the algal hosts in polluted marine environments. The production of Mn oxides by these surface-bound bacteria was also highlighted as a potential mechanism of heavy metal accumulation.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Metal tolerance,
Departments or Groups: *NONE OF THESE*
Divisions: School of Science > Department of Chemical and Life Sciences
Depositing User: Derek Langford
Date Deposited: 20 Dec 2013 10:28
Last Modified: 22 Sep 2018 08:27
URI: http://repository.wit.ie/id/eprint/2755

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