Ammonium toxicity and potassium limitation in yeast

Abstract

DNA microarray analysis of gene expression in steady-state chemostat cultures limited for potassium revealed a surprising connection between potassium and ammonium: potassium limits growth only when ammonium is the nitrogen source. Under potassium limitation, ammonium appears to be toxic for Saccharomyces cerevisiae. This ammonium toxicity, which appears to occur by leakage of ammonium through potassium channels, is recapitulated under high-potassium conditions by over-expression of ammonium transporters. Although ammonium toxicity is well established in metazoans, it has never been reported for yeast. To characterize the response to ammonium toxicity, we examined the filtrates of these cultures for compounds whose excretion might serve to detoxify the ammonium (such as urea in mammals). Using liquid chromatography-tandem mass spectrometry to assay for a wide array of metabolites, we detected excreted amino acids. The amounts of amino acids excreted increased in relation to the severity of growth impairment by ammonium, suggesting that amino acid excretion is used by yeast for ammonium detoxification.

Figure 1. Potassium Limitation in the Chemostat

Steady-state cell number (green diamonds, left axis) was measured over a potassium concentration of 0.65 mM to 13 mM. Additionally, residual phosphate at steady state (blue circles, right axis) was measured from filtrates of these chemostats.

Figure 2. Expression Arrays Reveal Changes in Nitrogen Transporters

(A) Ranked list of expression data from steady-state chemostats with different potassium concentrations (the same chemostats shown in Figure 1). The arrays use 13 mM potassium (far left) as a reference and are ranked from most induced in 1.3 mM potassium to most repressed in 1.3 mM potassium. Each row represents a single gene and each column is a single array. Red values indicate a higher expression compared to the reference, whereas green values indicate a lower expression. The intensity of the color represents the strength of the fold change and corresponds to the legend in the bottom right. (B) Genes that were 3-fold induced (top) or 3-fold repressed (bottom) in 1.3 mM potassium are shown with their gene name.

Figure 3. Ammonium Is Toxic to Yeast in Low Potassium

Steady-state cell number for 1.3 mM potassium (blue diamonds) or 13 mM potassium (black circles) medium was measured over an ammonium concentration of 19 mM to 606 mM.

Figure 4. Toxicity Is Specific to Ammonium as a Nitrogen Source

Chemostats were run in high (13 mM, black bars) or low (1.3 mM, blue bars) potassium with either ammonium (left) or asparagine (right) as the sole nitrogen source. As demonstrated before, steady-state cell mass (measured here by Klett Colorimeter) decreased with lower potassium when ammonium was the nitrogen source. However, if asparagine was used as the nitrogen source, then reduced potassium had no effect on steady-state cell mass.

Figure 5. Ammonium Toxicity Induces Amino Acid Excretion

LC-MS/MS was used to analyze filtrates from steady-state chemostats. The detected amino acids were expressed in nmoles based on standards (Materials and Methods). The detected amino acids were scaled for number of excreted nitrogen molecules per amino acid (i.e., one for alanine and two for glutamine) and expressed in nmoles excreted nitrogen per million cells. (A) Excreted amino acids for a range of potassium (same as the chemostats shown in Figure 1). (B) Excreted amino acids were measured for a range of ammonium in 1.3 mM potassium (same as the chemostats shown in Figure 3).

Figure 6. Ammonium Toxicity Is Correlated with Increased Glucose Utilization and Ethanol Production

Specific glucose utilization and ethanol production were measured from steady-state chemostat cultures. Specific glucose utilization (blue points) is expressed in nmoles consumed per million cells, and specific ethanol production (green points) is expressed in nmoles produced per million cells. (A) Glucose utilization and ethanol production from steady-state chemostats with different concentrations of potassium (same as the chemostats shown in Figure 1). (B) Glucose utilization and ethanol production were measured from steady-state chemostats with different concentrations of ammonium at 1.3 mM potassium (same as the chemostats shown in Figure 3).

Figure 7. Ammonium Toxicity and Excretion of Amino Acids Is Observed across a Variety of Saccharomyces Strains

(A) Chemostats were run in high (13 mM, black bars) or low (1.3 mM, blue bars) potassium with ammonium as a nitrogen source for the indicated Saccharomyces strains. As observed in Figure 1 and Figure 4 for S228C, all strains tested showed decreased steady-state biomass in low potassium. The Sigma strain washed out of the chemostat in 1.3 mM potassium, indicating that it could not maintain a growth rate equal to the dilution rate. (B) LC-MS/MS was used to analyze filtrates as described in Figure 5. Filtrates from steady-state chemostats from (A) were analyzed, and increased amino acid excretion was observed for all low-potassium chemostats. The Sigma strain was not analyzed because it washed out in low potassium.

Figure 8. Over-Expression of High-Flux Ammonium Channels Causes Lethality on Ammonium

Growth was assessed by frogging a dilution series (from 10⁷ cells/ml to 10³ cells/ml) onto SC plates with either 2% glucose or 2% galactose as the carbon source. Nitrogen sources were as follows: 5-g/l (NH₄)₂SO₄ (NH₄), 5-g/l asparagine (ASN), 2.5-g/l citrulline (CIT), 10-g/l proline (PRO), or 10-g/l leucine (LEU). The most informative dilutions are displayed for each medium (10⁷–10⁵ cells/ml for [A] and 10⁵–10⁴ cells/ml for [B]). Plates were incubated for 3 d at 30 °C. (A) Strains frogged on plates with ammonium as a nitrogen source with either glucose (left) or galactose (right) as the carbon source. (B) Strains frogged on plates with galactose as the carbon source and a series of nitrogen sources. gal pr, galactose promoter.

Figure 9. Amino Acid Excretion Is Induced at Steady State when Ammonium Channels Are Over-Expressed

(A) Each of the MEP over-expressing strains was tested in the chemostat at high (13 mM) potassium under a range of ammonium concentrations (6 mM to 76 mM) with galactose as the carbon source. Steady-state biomass is reported as percentage of the biomass in standard phosphate-limited medium with glucose as the carbon source. MEP1 (blue) and MEP3 (pink) only maintained steady state at the lowest concentrations of ammonium with galactose as the carbon source. (B) Amino acid excretion was measured as before (Figure 5) in 6 mM ammonium medium with galactose as the carbon source.

Figure 10. Model for Ammonium Toxicity and Detoxification in Yeast

We believe the results of this paper support the following model. If ammonium is present in high concentrations in the environment, then ammonium ions can enter the cell unregulated via potassium channels. Although most of the ammonium can be taken up into new biomass (if excess carbon and other nutrients are available), the unregulated flux creates an excess of internal ammonium that becomes toxic. To reduce internal ammonium levels, amino acids are excreted (most likely through the SPS amino acid transporters). The nitrogen affixed to amino acids will not be taken up through the potassium channels and is thus detoxified with respect to the cell.