## 3 Results
### Genome Sequencing and Mutation Analysis  
Whole-genome sequencing of the mutants was conducted with at least 200-fold genome coverage. Read mapping and SNP calling were performed by the Snippy script. Snippy finds SNPs between a haploid reference genome (_M. luteus_ trpE16 in this case) and the NGS sequencing reads. The obtained data were used to generate a table containing each SNP position, the original nucleotide, and the new variant present in each mutant. As expected, after using EMS as mutagen [38], almost all documented mutations were G:C\(\rightarrow\)A:T transitions. There were on average nine SNPs per genome in the M-mutants and eight SNPs per genome in the R-mutants. Besides the positions and effect of the mutations, the affected loci and their annotations were also considered. By using these data, an interactive plot was created for each set of mutants, in which each mutant along with its mutation profile and the position of each individual SNP could be inspected (Figures 2 and S5). It was possible to observe the general distribution of the mutations along the _M. luteus_ genome and to analyze how many SNPs there were per nucleotide position or if an increased SNP density was observable in specific genes or regions along the chromosome. To systematically detect sequence regions with higher SNP densities, a programmable \"sliding window\" function was implemented for data analysis in which a sequence \"window\" with adjustable length (e.g., 5000 bp) screens a defined number of positions (e.g., every 500 bp) along the genome sequence, producing a score at each step. This score is a measure for the number of SNPs within the covered area and of the distance between them. The more SNPs within the window and the closer their positions, the higher the final score. In this way, two windows with the same number of SNPs will have different scores if the distance between the SNPs is different. Sixteen different clusters, i.e., regions with increased SNP incidence, were recognized for the M-mutants (Figure 2), while for the R-mutants there was not a clear clustering of the SNPs (Figure S5). Beside the SNPs belonging to specific clusters, all SNPs affecting genes for transcription factors, proteases, or having other annotations of interest were also considered to decide if they should be included in further investigation. The most interesting clusters are listed in Tables 2 and 3. One region (cluster 13) in particular revealed a high density of SNPs (Figure 2). Almost every M-mutant was found to have an SNP in this region, and in addition, one R-mutant (R4) with a strongly repressed phenotype (Figure S6) carrying an SNP within the same area. All mutations in cluster 13 were within a region of 550 bp that encompasses two genes, Mlut_14660 encoding a DNA binding protein with a Helix-turn-Helix 17 motif domain and Mlut_14650 encoding a putative Flp pilus assembly protein RcpC/CpaB with an annotated SAF domain. The ORF for the latter protein forms an operon along with Mlut_14640 and Mlut_14670, which encodes the putative Flp pilus assembly ATPase CpaE and a two-component sensor histidine-kinase containing a PAS domain, respectively (all annotation information is from NCBI). The intergenic region between Mlut_14660 and Mlut_14650 was also affected by two different SNPs (see zoomed part in Figure 2). Among other affected genes, there is Mlut_09880 and Mlut_09900, which code for the Clp protease proteolytic and ATP-binding subunits, respectively. The only M-mutant which did not carry an SNP within cluster 13 had an SNP in Mlut_02150, which encodes a protein with the same annotation as the DNA binding protein encoded by Mlut_14660. Due to the lack of clear clustering in the R-mutants, certain mutations were selected after visual inspection of the affected genes, considering the number of SNPs affecting them. Figure 2: Distribution and clustering analysis of single nucleotide polymorphisms (SNPs) in M-mutants. All plots in the figure share the horizontal axis, which represents the reference genome of _M. luteus_ trpE16.