Figure 3. The alkaline stress response is associated with S. venezuelae exploratory behaviour.

(A) The surface area and medium pH associated with S. venezuelae explorer cells beside S. cerevisiae on YPD agar were measured and plotted every day for 14 days. (B) Schematic of the method used to identify genes required for S. venezuelae exploratory growth. S. venezuelae spores were subject to chemical mutagenesis, then screened on G- agar (no glucose, exploration-permissive without S. cerevisiae) for a lack of exploratory growth. Static colonies (beige) were grown beside S. cerevisiae (pink) on YPD medium to confirm a lack of exploratory growth. Genomic DNA was isolated from strains unable to initiate exploratory growth on G- agar, and when inoculated beside S. cerevisiae on YPD medium. Whole genome sequencing was performed to identify mutations responsible for the lack of exploratory growth. (C) Morphology of a mutant cytochrome bd oxidase S. venezuelae strain (∆cydCD) and the corresponding complemented strain grown on YPD agar for 14 days. (D) Transcript levels for alkaline stress-responsive genes in S. venezuelae explorer cells (grown beside S. cerevisiae on YPD medium), divided by levels for non-exploratory S. venezuelae cells (grown alone on YPD medium). Transcript levels were normalized and differential expression was log2-transformed. The associated sven gene numbers are shown above the bar graphs.

DOI: http://dx.doi.org/10.7554/eLife.21738.011

Figure 3—figure supplement 1. S. venezuelae grown alone raises the pH of glucose-deficient medium.

S. venezuelae was grown alone on G- (no glucose) or G+ (glucose-containing) agar medium containing the pH indicator bromothymol blue. Two replicates were inoculated on each plate, and these were grown for 14 days.

DOI: http://dx.doi.org/10.7554/eLife.21738.012

Figure 3—figure supplement 2. High pH alone does not stimulate S. venezuelae exploration.

S. venezuelae was grown alone on YPD agar medium buffered to pH 9.0 using 50, 100 or 200 mM borate. Two replicates were inoculated on each plate, and these were grown for 14 days.

DOI: http://dx.doi.org/10.7554/eLife.21738.013

Figure 3—figure supplement 3. Complementation of explorer mutant phenotypes.

Top row: EMS (ethyl methanesulfonate) mutagenesis-derived S. venezuelae explorer mutants containing point mutations in the cydABCD operon, grown beside S. cerevisiae. Bottom row: Explorer mutants complemented with a cosmid carrying the wild type cydABCD operon. Two replicates were inoculated on each plate.

DOI: http://dx.doi.org/10.7554/eLife.21738.014