Changes in gene expression, cell physiology and toxicity of the harmful cyanobacterium Microcystis aeruginosa at elevated CO2

Abstract

Rising CO2 concentrations may have large effects on aquatic microorganisms. In this study, we investigated how elevated pCO2 affects the harmful freshwater cyanobacterium Microcystis aeruginosa. This species is capable of producing dense blooms and hepatotoxins called microcystins. Strain PCC 7806 was cultured in chemostats that were shifted from low to high pCO2 conditions. This resulted in a transition from a C-limited to a light-limited steady state, with a ~2.7-fold increase of the cyanobacterial biomass and ~2.5-fold more microcystin per cell. Cells increased their chlorophyll a and phycocyanin content, and raised their PSI/PSII ratio at high pCO2. Surprisingly, cells had a lower dry weight and contained less carbohydrates, which might be an adaptation to improve the buoyancy of Microcystis when light becomes more limiting at high pCO2. Only 234 of the 4691 genes responded to elevated pCO2. For instance, expression of the carboxysome, RuBisCO, photosystem and C metabolism genes did not change significantly, and only a few N assimilation genes were expressed differently. The lack of large-scale changes in the transcriptome could suit a buoyant species that lives in eutrophic lakes with strong CO2 fluctuations very well. However, we found major responses in inorganic carbon uptake. At low pCO2, cells were mainly dependent on bicarbonate uptake, whereas at high pCO2 gene expression of the bicarbonate uptake systems was down-regulated and cells shifted to CO2 and low-affinity bicarbonate uptake. These results show that the need for high-affinity bicarbonate uptake systems ceases at elevated CO2. Moreover, the combination of an increased cyanobacterial abundance, improved buoyancy, and higher toxin content per cell indicates that rising atmospheric CO2 levels may increase the problems associated with the harmful cyanobacterium Microcystis in eutrophic lakes.

Keywords: CO2-concentrating mechanisms; bicarbonate uptake; climate change; harmful algal blooms; inorganic carbon uptake; microarrays; microcystins; phytoplankton.

Figure 1

Changes in cyanobacterial abundance, light, dissolved inorganic carbon (DIC) and pH during a shift from low pCO₂ (200 ppm, white area) to high pCO₂ (1450 ppm, shaded area). (A) Cyanobacterial abundance (expressed as biovolume) and light intensity penetrating through the chemostat (I_out). (B) Dissolved CO₂ (dCO₂), bicarbonate (HCO⁻₃) and carbonate (CO²⁻₃) concentrations (logarithmic scale). (C) Dissolved inorganic carbon (DIC) and pH. Error bars indicate standard deviations (n = 4).

Figure 2

Changes in cell properties during a shift from low pCO₂ (200 ppm, white area) to high pCO₂ (1450 ppm, shaded area). (A) Average cell volume and cell weight. (B) Cellular elemental C and N content. (C) Molar C/N ratio of the cells. (D) Dry weight composition of cells from the steady states at 200 and 1450 ppm pCO₂. Error bars indicate standard deviations (n = 4).

Figure 3

Light absorption and emission spectra of cells grown at low pCO₂ (200 ppm) and high pCO₂ (1450 ppm). (A) Light absorption spectra normalized at 750 nm, with peaks of chlorophyll a (436 and 678 nm), β-carotene (shoulder at 490 nm) and phycocyanin (626 nm). (B) 77 K fluorescence emission spectra normalized based on the mean emission at 600–660 nm, with peaks of PSI (720 nm) and PSII (695 nm). The spectra are the average of four biological replicates.

Figure 4

Changes in microcystin concentration and gene expression of secondary metabolite genes. (A) Total microcystin concentration and microcystin content per cell, during the shift from low pCO₂ (200 ppm, white area) to high pCO₂ (1450 ppm, shaded area). Error bars indicate standard deviations (n = 4). (B) Changes in expression of the secondary metabolite genes after the increase of pCO₂ to 1450 ppm. Expression changes are quantified as log2 values. Red indicates significant upregulation and green significant downregulation; non-significant changes are in black. Hierarchical clustering was used to order the genes. The underlying data are presented in Supplementary Table 4.

Figure 5

Changes in expression of the CO₂-concentrating mechanism (CCM) genes after the increase of pCO₂ to 1450 ppm. Expression changes are quantified as log2 values. Red indicates significant upregulation and green significant downregulation; non-significant changes are in black. Hierarchical clustering was used to order the genes. The underlying data are presented in Supplementary Table 4.

Figure 6

Inorganic carbon uptake kinetics of cells grown at low pCO₂ (200 ppm) and high pCO₂ (1450 ppm). (A) Bicarbonate response curves (O₂ evolution rates) of cells grown at low vs. high pCO₂ after addition of different concentrations of NaHCO₃ at pH 9.8. (B) O₂ evolution rates in the presence and absence of LiCl, at pH 7.8 and pH 9.8. The cells were provided with 200 μmol L⁻¹ KHCO₃. Lithium ions block sodium-dependent bicarbonate uptake, while differences in pH produce different concentrations of dCO₂ and bicarbonate. Error bars indicate standard deviations (n = 4). Bars with different letters were significantly different, as tested by a one-way analysis of variance with post-hoc comparison of the means (α = 0.05).