Title

Genetic Manipulation of a Cyanobacterium for Enhanced Toxic Heavy Metal Sequestration

Date of Award

1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Sabine Heinhorst

Advisor Department

Chemistry and Biochemistry

Abstract

A study was undertaken to genetically manipulate a cyanobacterium to generate a recombinant organism capable of increased metal sequestration. The work represents one of the first reports of expression of a non-photosynthetic eukaryotic gene in cyanobacteria. A cloned copy of the Saccharomyces cerevisiae copper metallothionein gene CUPI, responsible for enhanced metal sequestration in yeast, was employed in a vector construct developed for a naturally competent, unicellular cyanobacterium. For utilization in PCR amplification, forward and reverse primers containing restriction endonuclease sequences were designed to allow directional cloning into the cyanobacterial shuttle vector pTrcIK. The recombinant plasmid was incorporated via homologous recombination into the genome of R2-PIM8 $smtA\sp-,$ a modified strain of Synechococcus sp. PCC 7942 developed with a genomic platform to direct this integration and mutant for expression of the endogenous metallothionein. The cyanobacterial vector construct carrying the cloned CUPI gene, pMcK2, was shown to be capable of directing in vitro inducible protein expression as determined by studies utilizing $\sp{35}$S-cysteine labeling with an E. coli extract. Stable integration of the construct into the cyanobacterial genome via recombination was verified by Southern blot hybridization and confirmed by DNA sequence analysis. Northern blot analysis indicated constant high titers of the mRNA transcript three hours following induction with IPTG. Recombinant yeast metallothionein protein expression was demonstrated directly by in vivo $\sp{35}$S-cysteine labeling and, indirectly, by in vitro $\rm\sp{109}Cd\sp{2+}$-labeling studies. Evidence for increased copper tolerance and sequestration was not obtained.