Date of Award

Spring 4-29-2020

Degree Type

Dissertation

Degree Name

PhD Molecular Bioscience

Department

Biology

Advisor

Tinchun Chu, Ph.D.

Committee Member

Jane L. Ko, Ph.D.

Committee Member

Daniel B. Nichols, Ph.D.

Committee Member

Angela V. Klaus, Ph.D.

Committee Member

Lee H. Lee, Ph.D.

Keywords

cyanobacteria, cyanotoxins, microcystin, metallothionein, zinc chloride, Microcystis

Abstract

Cyanobacteria harmful algal blooms (CHABs) are globally increasing biomasses of detrimental cyanobacteria due to anthropogenic water phosphorous and nitrogen loading and climate change. CHABs often produce secondary metabolites, cyanotoxins, that cause harmful effects to organisms, most water systems, and socioeconomic infrastructures. Additionally, the presence of heavy metal pollutant runoff in CHAB affected environments may result in CHAB population changes – aggravating toxigenicity. Zinc metal resistance and stress response were studied in microcystin (MC) cyanotoxin-producing Microcystis aeruginosa UTEX LB 2385 (M. aeruginosa UTEX LB 2385) and non-MC producing Microcystis aeruginosa UTEX LB 2386 (M. aeruginosa UTEX LB 2386) cyanobacteria. Molecular analysis of zinc treated M. aeruginosa UTEX LB 2385 was performed for mcy genes and intracellular metallothionein (mt) gene. An ELISA assay was used to examine MC concentrations in zinc treated M. aeruginosa UTEX LB 2385 cultures.
Zinc metal response within M. aeruginosa strains was evaluated through 15 days via growth monitoring studies, by treating cultures with ZnCl2 (0, 0.1, 0.25, and 0.5 mg/L) concentrations. M. aeruginosa UTEX LB 2385 and M. aeruginosa UTEX LB 2386 survived 0.25 mg/L ZnCl2 treatment, with increasing biomass through 15 days. Phase contrast microscopy showed morphological responses in mostly inhibited 0.5 ZnCl2 mg/L treated M. aeruginosa strains. The 0.25 mg/L ZnCl2 cultures presented larger FSC flow cytometry profiles in toxigenic M. aeruginosa UTEX LB 2385 vs. M. aeruginosa UTEX LB 2386 through 8 days. Greater chlorophyll a concentration sensitivity was shown in ZnCl2 treated non-toxigenic vs. toxigenic cultures.
Molecular stress response studies in ZnCl2 treated M. aeruginosa UTEX LB 2385 were initiated by designing quantitative PCR (qPCR) oligonucleotides via phylogenetic analysis of MC synthetase and MT amino acid sequences. qPCR of specific cDNA samples showed significant upregulation of Mmt throughout all time points and significant upregulation of mcyC at a later time point (day 12). This data showed ZnCl2 treated M. aeruginosa UTEX LB 2385 cultures possessed several zinc metal response mechanisms.
Intracellular-extracellular MC concentrations were compared to mcy genes upregulation. MC production and localization was evaluated in ZnCl2 treated M. aeruginosa UTEX LB 2385 cultures by ELISA MC-LR analysis. M. aeruginosa UTEX LB 2385 treated with 0.25 and 0.5 mg/L ZnCl2 demonstrated extracellular MC-LR (μg/L) increase by day 5 vs. control cells. Total MC-LR (μg/cell) quota was significantly higher for 0.5 mg/L ZnCl2 treated cells vs. control cells by day 8, indicating release of microcystins into the extracellular space upon cell lysis.
These studies showed M. aeruginosa UTEX LB 2385 and M. aeruginosa UTEX LB 2386 strains survived 0.25 mg/L ZnCl2 concentration. Certain morphological and photosynthetic responses were observed in cultures treated with 0.25 and 0.5 mg/L ZnCl2. Upregulation of mt and mcy genes in ZnCl2 treated M. aeruginosa UTEX LB 2385 cultures was shown, with 0.25 and 0.5 mg/L ZnCl2 treatment yielding greater extracellular MC-LR (μg/L) concentrations compared to control cells by day 5. Taken together, these studies demonstrated that M. aeruginosa UTEX LB 2385 and M. aeruginosa UTEX LB 2386 respond to zinc heavy metal stress and activate certain coping, survival mechanisms.

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