## 3 Results
### Mutant Screening and Phenotype Selection  
EMS-treated cells were screened for mutants with altered expression of the _comEA/EC_ promoter, which regulates _lacZ_ gene expression in our reporter strain in dependence of the growth medium (Figure 1A). For this, mutagenized cells from a glycerol stock were inoculated in full medium and incubated O/N at 30 \({}^{\circ}\)C before plating serial dilutions on different media. EMS mutations leading to an increased promoter activity under competence-repressing conditions were identified on LB\({}_{\text{Kan}}\) agar supplemented with X-gal (M-mutants), on and those repressing promoter activity under competence-promoting conditions were identified on GMM\({}_{\text{Kan}}\) agar supplemented with X-gal (R-mutants). Use of the chromogenic LacZ substrate X-gal enabled a blue/white plate assay for mutant screening instead of a MUG plate-spraying technique with fluorescence read-out [28]. When looking for M-mutants with the MUG plate-spraying assay, it was not easy to qualitatively distinguish colonies displaying upregulation of the reporter gene construct from regular colonies. The fluorescence was too homogenous when many colonies were close to each other, making it difficult to distinguish between colonies with variations in fluorescence intensity. This method was also inconvenient for the screening of the R-mutants, given its lower sensitivity. Conversely, with the use of X-gal at relatively high concentrations (80 \(\mu\)g/mL), it was possible to differentiate strongly repressed phenotypes from the background of colonies with unchanged reporter gene expression. Although the expression of _comEA/EC_ is normally inhibited in full medium, M-mutants exhibited a clear and strong expression, yielding blue-colored colonies on LB\({}_{\text{Kan}}\)/X-gal (45 \(\mu\)g/mL) plates (Figure 1B). Conversely, R-mutants were selected as white colonies among a background of blue colonies on GMM\({}_{\text{Kan}}\)/X-gal plates. Such phenotypically white colonies indicated repression of the _comEA/EC_ promoter under competence-inducing conditions. Thus, compared to the wild-type, both groups of mutants failed to show the usual response of the _comEA/EC_ promoter to nutrient availability.  
More than 100 colonies of M-mutants showing enhanced activity of the \(\beta\)-galactosidase marker under repressing conditions and around 80 colonies of R-mutants with no or reduced \(\beta\)-galactosidase activity under inducing conditions were repeatedly re-streaked in order to allow genetic segregation of the mutations and to find stable phenotypes.  
In order to assess which mutants of each group to choose for genome sequencing, a quantitative \(\beta\)-galactosidase activity assay was performed. For the M-mutants, we identified which mutants had high levels of expression of the _lacZ_ reporter gene on full medium. For the R-mutants, which were expected to have largely reduced or lacking \(\beta\)-galactosidase activeity, we selected strains that had less than half of the activity of the unmutagenized _lacZ_ reporter strain using an end-point measurement (t = 10 min). Besides that, we wanted to evaluate if there were cases of SNPs occurring within the 7 kb reporter construct inserted in the genome, meaning higher genetic linkage of the causative SNP and the promoter. Fourteen R-mutants and thirty-two M-mutants fulfilled both the reporter activity and the genetic linkage screening criteria (less than 3% of lineage; for linkage scores see Supplementary Tables S1 and S2), and from these, all the R-mutants and 22 of the M-mutants were grown to stationary phase for high quality gDNA preparation and sequencing.