Effect of Zinc on Microcystis aeruginosa UTEX LB 2385 and Its Toxin Production

Abstract

Cyanobacteria harmful algal blooms (CHABs) are primarily caused by man-made eutrophication and increasing climate-change conditions. The presence of heavy metal runoff in affected water systems may result in CHABs alteration to their ecological interactions. Certain CHABs produce by-products, such as microcystin (MC) cyanotoxins, that have detrimentally affected humans through contact via recreation activities within implicated water bodies, directly drinking contaminated water, ingesting biomagnified cyanotoxins in seafood, and/or contact through miscellaneous water treatment. Metallothionein (MT) is a small, metal-sequestration cysteine rich protein often upregulated within the stress response mechanism. This study focused on zinc metal resistance and stress response in a toxigenic cyanobacterium, Microcystis aeruginosa UTEX LB 2385, by monitoring cells with (0, 0.1, 0.25, and 0.5 mg/L) ZnCl₂ treatment. Flow cytometry and phase contrast microscopy were used to evaluate physiological responses in cultures. Molecular assays and an immunosorbent assay were used to characterize the expression of MT and MC under zinc stress. The results showed that the half maximal inhibitory concentration (IC₅₀) was 0.25 mg/L ZnCl₂. Flow cytometry and phase contrast microscopy showed morphological changes occurred in cultures exposed to 0.25 and 0.5 mg/L ZnCl₂. Quantitative PCR (qPCR) analysis of selected cDNA samples showed significant upregulation of Mmt through all time points, significant upregulation of mcyC at a later time point. ELISA MC-LR analysis showed extracellular MC-LR (µg/L) and intracellular MC-LR (µg/cell) quota measurements persisted through 15 days, although 0.25 mg/L ZnCl₂ treatment produced half the normal cell biomass and 0.5 mg/L treatment largely inhibited growth. The 0.25 and 0.5 mg/L ZnCl₂ treated cells demonstrated a ~40% and 33% increase of extracellular MC-LR(µg/L) equivalents, respectively, as early as Day 5 compared to control cells. The 0.5 mg/L ZnCl₂ treated cells showed higher total MC-LR (µg/cell) quota yield by Day 8 than both 0 mg/L ZnCl₂ control cells and 0.1 mg/L ZnCl₂ treated cells, indicating release of MCs upon cell lysis. This study showed this Microcystis aeruginosa strain is able to survive in 0.25 mg/L ZnCl₂ concentration. Certain morphological zinc stress responses and the upregulation of mt and mcy genes, as well as periodical increased extracellular MC-LR concentration with ZnCl₂ treatment were observed.

Keywords: Microcystis aeruginosa; cyanobacteria; cyanotoxins; metal; microcystin; zinc.

Figure 1

Growth curves of Microcystis aeruginosa UTEX LB 2385 treated with 0, 0.1, 0.25, and 0.5 mg/L ZnCl₂. (a) Direct count of cell number was made via hemocytometer through 15 days. (b) Turbidity was evaluated via optical density (OD_{750 nm}) readings through 15 days. Data is presented as mean ± SD of three replicates.

Figure 2

Phase-contrast microscopy of Microcystis aeruginosa UTEX LB 2385 treated with 0, 0.1, 0.25, and 0.5 mg/L ZnCl₂ at Days 1, 5, and 8, 1000× total magnification. Although greatly reduced in cell number, M. aeruginosa UTEX LB 2385 cells treated with 0.5 mg/L ZnCl₂ presented morphology similar to 0 mg/L cells through Day 8, as indicated by blue arrows. The red arrow indicates a small cluster of multi-cell aggregation at Day 5 for 0.5 mg/L ZnCl₂ cells. Scale bar: 5 μm.

Figure 3

Flow cytometry analysis of population size (forward scatter: FSC-A) histograms within Microcystis aeruginosa UTEX LB 2385 treated with 0, 0.25, and 0.5 mg/L ZnCl₂. (a) 0 mg/L ZnCl₂ cells showed relative uniform FSC-A size through eight days. (b) 0.25 mg/L ZnCl₂ cells and (c) 0.5 mg/L ZnCl₂ cells showed increased FSC-A population size at Day 8 compared to control cells. Histograms were generated via FlowJo software analysis. (D represent days).

Figure 4

qPCR analysis after cDNA synthesis, of three genes: Mmt, mcyC, and mcyE as potential zinc metal response genes in M. aeruginosa UTEX LB 2385. Mmt (top row) overall expression was shown to be highest in 0.25 mg/L ZnCl₂ treated cells, with significant (Day 1) upregulation in comparison to 0 and 0.1 mg/L ZnCl₂ treatment. mcyC and mcyE (middle and bottom rows, respectively) were distinct in expression patterns, with mcyC gene significantly upregulating at Day 12, showing a fold-increase >2.5 in ZnCl₂ treated cells. For 0.1 and 0.25 mg/L ZnCl₂ treated cells, Day 12 showed gene expression increase for Mmt and mcyC genes. (D represent days; 0, 0.1, 0.25 mg/L represent ZnCl₂ concentrations; color scale of yellow refers to no change, green-downregulation, orange-red-upregulation).

Figure 5

ELISA quantitative analysis of intracellular and extracellular MC-LR (µg/L) equivalents within Microcystis aeruginosa UTEX LB 2385 treated with 0, 0.1, 0.25, and 0.5 mg/L ZnCl₂ at Days: One, 5, 8, 12, 15. Extracellular MC-LR equivalents (µg/L) of 0.1 mg/L ZnCl₂ cells showed a 18% and 23% increase for Days 8 and 12 compared to control; 0.25 and 0.5 mg/L ZnCl₂ treated cells showed a ~40% and 33% increase of extracellular MC-LR, respectively, as early as day 5. The 0.25 mg/L ZnCl₂ extracellular MC-LR (µg/L) equivalents decreased and increased from Days 8 to 15, and 0.5 mg/L ZnCl₂ intracellular MC-LR (µg/L) intracellular measurements decreased and increased from this same time point. All samples were performed as duplicate replicates.