Date of Award

Spring 5-13-2017

Degree Type

Dissertation

Degree Name

PhD Molecular Bioscience

Department

Biology

Advisor

Tinchun Chu, Ph.D

Committee Member

Caroyln Bentivegna, Ph.D

Committee Member

Daniel B. Nichols, Ph.D

Committee Member

Angela Klaus, Ph.D

Committee Member

Lee H. Lee, Ph.D

Keywords

Stress Response, Metal Toxicity, Cyanobacteria physiology

Abstract

The way that cyanobacteria process and interact with heavy metals is an important part of their physiology. Metals like zinc play a role in many essential cell processes; while metals such as cadmium appear to be universally toxic. Little is known about how these metals play a role in the harmful overgrowth of cyanobacteria called blooms. While individual mechanisms have been elucidated, a complete response for metals in cyanobacteria has not yet been proposed. To establish a proposed mechanism of response in cyanobacteria, work was undertaken studying the response to zinc and cadmium in Synechococcus sp. IU 625 (S. IU 625). We investigated the effect that 0, 10, 25 and 50 mg/L ZnCl2 had on the growth of S. IU 625 to mimic zinc stressed conditions. From these concentrations, it was observed that 10 mg/L ZnCl2 had no detectable differences in growth rate; 25 mg/L ZnCl2 resulted in a cell number reduction of ~50%, but not a reduction in optical density. The 50 mg/L ZnCl2 was completely lethal. Following growth monitoring, imaging analysis including viability staining and scanning electron microscopy, was performed. Metal allocation analysis was performed by using inductively coupled plasma – mass spectrometry to determine zinc concentration intra and extracellularly. Bioinformatics performed on known metal genes in other species of prokaryotes resulted in several potential candidates for response. Quantitative real-time polymerase chain reaction results indicate of the 3 candidate genes for zinc response, metallothionein smtA was expressed early and transcript levels remained high. The hyper active efflux pump pacS, and the cation transport anti-porter catT were not significantly expressed until after 21 days of metal exposure in the 10 and 25 mg/L ZnCl2 culture. Following this analysis, a more thorough study of the transcriptome was conducted for control, 10 and 25 mg/L ZnCl2 cultures on days 4 and 7. This analysis revealed that zinc exposure causes differential expression in 10% of the genome. The novel use of flow xi cytometry to track pigment and size changes in the cell populations in the control, 10, 25, and 50 mg/L ZnCl2 cultures was employed to characterize these changes. These findings allows the proposal of a mechanism for response for zinc stress response. S. IU 625 is much more cadmium sensitive, with the range of survival only up to 20 mg/L CdCl2. Like observed with the ZnCl2 culture, 10 mg/L CdCl2 exposed cells do not exhibit a difference when compared to the control. 30 mg/L CdCl2 proved completely lethal following 4 days of exposure. Unlike observed in the ZnCl2 cultured cells, morphological differences were not observed in the CdCl2 cell populations. Pigment fluorescent changes were detected in these cells, like the ZnCl2 cultured cells, indicating that both metals adversely affect photosynthesis in S. IU 625. These results give a comprehensive overview of the effects of zinc and cadmium early in the cell cycle to death phase. The results indicate that S. IU 625 may be of interest for future bioremediation strategies or for providing markers for metal stress studies in other organisms.

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