Annotating the CIA7 Gene: Comparing Lead Bioaccumulation, Cell Growth and Morphology in Two Strains of Chlamydomonas reinhardtii

Abstract

As industrialization increases to meet the demands of 21st century life, incidences of heavy metal contamination increases as well. Non‐essential heavy metals serve no biological function and can have devastating effects across all spectra of life. Lead (Pb) is of significant research importance due to its toxicity at both acute and chronic levels of exposure. Industrial sources of lead contamination include production of crude oils, and paints. Bioavailable Pb contamination can damage key metabolic processes in eukaryotic and prokaryotic organisms. Plants and algal cells are particularly affected by Pb, as unchecked exposure can cause metabolic dysfunction and eventually damage the chloroplast's photosystems. Alga and plants employ an array of physical and chemical defenses to maintain heavy metal homeostasis. Among these are the production of non‐specific heavy metal binding proteins known as phytochelatins (PC) and metallothioneins (MT). Both protein families share similar amino acid motifs, particularly moieties of cysteine, whose sulfhydryl (‐SH) active site has a high affinity for heavy metal ions. MT and PC aid in the chelation and eventual removal of heavy metal ions. Investigation of heavy metal tolerance genes in green algae is of importance due to their position as primary producers in most aquatic ecosystems. Chlamydomonas reinhardtii, a unicellular green alga, has been a model organism for the study of photosynthesis, bioremediation, and heavy metal toxicity. Though its genome has been sequenced, several genes and their protein products remain unannotated. One such gene, Cia7, is hypothesized to play a role in maintaining C. reinhardtii's heavy metal homeostasis. Preliminary evidence shows that CIA7 contains a highly conserved motif similar to other metal sequestering proteins. To aid in confirming CIA7's ability to sequester heavy metals, this study presents the results of a bioaccumulation experiment involving a wild type (CC4425) and a Cia7 mutant (CC5013). CC4425 and the CC5013 were grown simultaneously in separate treatment flasks containing either 0uM/150 uM PbCl2 in replete TAP media for 4 days under constant lighting. Pb ion bioaccumulation was measured utilizing an Induced Coupled Plasma Optical Emission Spectrophotometer (ICP‐OES) following a microwave acid digestion step. A 2×2 factorial analysis of variance was conducted to determine the significance of the data obtained. Results of bioaccumulation experiments indicated that there is a significant difference in Pb bioaccumulation between the mutant cia7 and wild‐type at 150 μM Pb treatment. The mutant cia7 bioaccumulated higher concentration of Pb compared to wild‐type. This difference is significant at 95% confidence interval. Flow cytometry and fluorescent microscopy experiments were conducted to further explore the differences in morphology and cell count between strains across both treatments.Support or Funding InformationNorman Hackerman Advanced Research Program