## 2 Materials and Methods
### \(\upbeta\)-Galactosidase Activity Assays  
Precultures of all the evaluated strains containing the transcriptional reporter _\(\Delta\)comEA/EC:lacZ_ were grown O/N in LB. Adequate aliquots of the cells were harvested and washed with PBS by centrifugation at 14,000\(\times\)\(g\) for 10 min. All samples were normalized such that they would have the same OD\({}_{600\text{nm}}\) of 1.5. All suspensions were treated  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Name** & **Genotype and Relevant Phenotype** & **Source** \\ \hline trpE16 & _trpE16_, mutagenesis derivative of ATCC 27141, Trp\({}^{-}\) & [29] \\ \hline _\(\Delta\)comEA/EC:lacZ_ & _trpE16_\(\Delta\)comEA/EClacZ-KanR_, LacZ expression & [26] \\ \hline \(\Delta\)14650 & _trpE16_\(\Delta\)Mlut\_14650 & This study \\ \(\Delta\)02150 & _trpE16_\(\Delta\)Mlut\_02150 & This study \\ \(\Delta\)03135 & _trpE16_\(\Delta\)Mlut\_03135 & This study \\ 3-M43 & _trpE16_ G341241A (Mlut\_03135) & This study \\ 7-M89 & _trpE16_ C1061676T (Mlut\_09880) & This study \\ 7-M93 & _trpE16_ C1063997 (Mlut\_0990) & This study \\ 7-M28 & _trpE16_ G1064341A (Mlut\_09900)  
with 0.1 mg/mL of lysozyme for 10 min at 37 \({}^{\circ}\)C, followed by transfer of 200 \(\upmu\)L aliquots to a 96-well microtiter plate. For the measurement of LacZ activity, the \(\beta\)-galactosidase substrate 4-methylumbelliferyl \(\beta\)-D-galactopyranoside (MUG) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well at a final concentration of 250 \(\upmu\)g/mL. Kinetic measurements were performed by incubating the cells in a FLUOstar Omega (BMG LABTECH, Ortenberg, Germany) microplate reader at 30 \({}^{\circ}\)C for 10 h and measuring fluorescence caused by MUG cleavage every 10 min at 460 nm. The wildtype strain of _M. luteus_ was incubated with and without a substrate to establish the background fluorescence. There were at least 3 biological replicates and 3 technical replicates for every strain assayed. Controls without cells and without substrate were created to evaluate background noise. To determine the relative activity of the promoter of _comEA/EC_, a four parameter logistic function was fitted to the measured kinetic curves. The linear range of the kinetic curves prior to substrate depletion was used for evaluation. This was accomplished by calculating the steepest slope within the linear range of the curves which corresponds to the maximal velocity of the enzymatic reaction and is directly proportional to the enzyme concentration [26].  
Figure 1: _M. luteus comEA/EC_ promoter activity and screening for EMS mutants with dysregulated activity on rich and minimal medium. (**A**) End-point measurement (t = 30 min) of _comEA/EC_ promoter transcriptional levels after growth of the reporter strains _M. luteus_ trpE16 \(\Delta\)_comEA/EC:lacZ_ under different nutritional conditions (lysogeny broth (LB) and glutamate minimal medium (GMM)) using the \(\beta\)-galactosidase substrate 4-methylumbelliferyl \(\beta\)-D-galactopyranoside (MUG). (**B**) Screening of EMS mutants with deregulated _comEA/EC_ promoter. On the left panel, mutants (blue) show higher enzymatic activity, indicating an increase in the _comEA/EC_ promoter activity under usually competence-repressing conditions (LB\({}^{\text{Kan}}\) full medium with 45 \(\upmu\)g/mL of 5-bromo-4-chloro-3-indolyl-\(\beta\)-D-galactopyranoside (X-gal)). The mutants with this phenotype were called M-mutants. The right panel shows colonies derived from EMS-mutagenized cells plated on competence-inducing minimal media (GMM\({}^{\text{Kan}}\) with 80 \(\upmu\)g/mL of X-gal), including mutants with no enzymatic activity (yellow due to natural color of _M. luteus_ cells), which were designated as R-mutants. Arrow: Blue: _comEA/EC_ promoter sequence; Red: _lacZ_ gene sequence. Normal arrow indicates promoter induction; Blunt arrow indicates promoter repression.