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emi2887-sup-0001-si.pdf602K

Fig. S1. Potassium starvation activates the expression of genes related to the metabolism of sulfur-containing amino acids.

A. BY4741 wild-type strain was transformed with reporter YEp357-based plasmids containing the entire promoters of indicated genes, transferred to K+-free medium for 2 h and processed for measurement of β-galactosidase activity as described in Experimental procedures.

B. Methionine supplementation attenuates the induction of sulfur-related genes upon potassium starvation. The BY4741 wild-type strain was transformed with indicated reporters and processed as above except that cells containing the pPHO84 reporter were collected after 90 min of potassium starvation. For this experiment the standard Translucent K+-free medium, which contains 20 mg l−1 Methionine (open bars) was made 40 mg l−1 (stripped bars) or 80 mg l−1 (closed bars). Data are mean ± SEM from three to five experiments.

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Fig. S2. Induction of sulfur-related genes is also observed in MET17 strains. Wild-type strains BY4741 (BY, met17, data from Fig. S1A), W303-1A (W303, MET17) and DBY746 (DBY, MET17) were transformed with the indicated reporters and processes as in Fig. S1A for β-galactosidase assay. Data are mean ± SEM from three to five experiments.

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Fig. S3. STRE-mediated promoter activation response to potassium starvation. Wild-type strain W303-1A and its msn2 msn4 derivative (MCY5278, a generous gift from M. Carlson, Casado et al., 2011) were transformed with plasmid pGM18/17 (Marchler et al., 1993) allowing the integration of a (7x)STRE-LacZ element at the URA3 locus. The strains were transferred for the indicated times to Translucent K+-free medium either at room temperature or at 37°C (to induce a heat-shock response) and β-galactosidase activity measured. Data are mean SEM from three independent experiments performed by triplicate.

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Fig. S4. Strain JC37-1A, which expresses a Rtg1-GFP version (Crespo et al., 2002), was grown to exponential phase in K+-free medium (without ammonium sulfate and containing 5 mM glutamine as nitrogen source) supplemented with 50 mM KCl. Cells were washed and resuspended in fresh medium either with (+) or without (−) 50 mM KCl. One millilitre of samples were taken at different times (only results after 20 min are shown). Cells were fixed with 2% formaldehyde, harvested by centrifugation, washed two times with phosphate-buffered saline (PBS) solution, stained with 0.1 mg ml−1 DAPI (4′,6-diamidino-2-phenylindole) and finally resuspended in PBS. Samples were observed by using a Nikon Eclipse E800 fluorescence microscope and a FITC filter.

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Fig. S5. Comparison of transcriptional changes in trk1 trk2 and hal4 hal5 mutants. The change in mRNA levels (log2 space) for genes related to sulfur metabolism presented in Fig. 2B in the main text, plus CIT2 and DLD3, were plotted. y-axis represents data from this work, whereas x-axis represents data from Perez-Valle and colleagues (2010). The calculated correlation coefficient was 0.768.

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Table S1. Oligonucleotides used in this work.

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Table S2. Genes induced after potassium starvation. Log(2) values of the changes are listed for each time point of the experiment.

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Table S3. Genes repressed after potassium starvation. Log(2) values of the changes are listed for each time point of the experiment.

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Table S4. Genes induced or repressed in the trk1 trk2 strain growing in 50 mM KCl. Log(2) values of the changes are listed for each time point of the experiment.

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