## 5 Conclusions  
In conclusion, random EMS mutagenesis followed by introduction of specific mutations into the _M. luteus_ genome as single-SNP mutations and markerless gene deletions  
Figure 7: Transcriptional regulation model proposed for Mlut_14660 and Mlut_14650/40/30. As a working model, transcriptional interference is proposed for the divergently overlapping transcriptional units shown above. Grey dashed lines represent presumed but yet unproven effects. (**A**) Under non-competence-inducing conditions such as high nutrient concentrations, Mlut_14660 is expressed, impeding the expression of the Mlut_14650/40/30 operon which could play a role in pili structure building. The Mlut_14660 expression product, a putative DNA-binding protein with HTH motif, may cause the repression of essential _com_ genes. (**B**) Opposite to the first scenario, under competence-inducing conditions such as low nutrient availability, the Mlut_14650/40/30 operon is expressed while simultaneously Mlut_14660 is not transcribed. This enhances _com_ gene expression, resulting in higher abundance of transformation proteins and pili structure both necessary for DNA uptake. This model is based on results of this work and previous work but has not been verified in detail.  
has allowed the identification of new genes previously not reported to be involved in competence development and natural transformation. While certain constructed single-SNP mutations did not affect _com_ gene expression and natural transformation on their own and possibly only exert an effect when combined with other SNPs, single-SNP mutations in Mlut_14660 and in the 5\({}^{\prime}\) regions of Mlut_14660 and Mlut_14650/40/30 clearly affected _com_ gene expression and natural transformation. Further work is planned to confirm the hypothetical regulation model for these genes presented here and to unravel the molecular basis of competence regulation in the Actinobacteria model organism _M. luteus_.  
The following are available online at [[https://www.mdpi.com/article/10.3390/genes12091307/s1](https://www.mdpi.com/article/10.3390/genes12091307/s1)]([https://www.mdpi.com/article/10.3390/genes12091307/s1](https://www.mdpi.com/article/10.3390/genes12091307/s1)), Table S1: M-mutants linkage assay results, Table S2: R-mutants linkage assay results, Figure S1: Chromosomal situation at _comEA/EC_ locus in _M. luteus_ trpE16 wild-type and in the _\(\Delta\)comEA/EC:lacZ-Kan_ reporter strain, Figure S2: Vector map of pKOS6 bMarget gene used for clean deletions and genome editing via double crossover recombination, Figure S3: Log10 transformed number of rifampicin resistant colonies obtained with and without EMS treatment, Figure S4: Frequency of rifampicin resistance appearance, Figure S5: Distribution and clustering analysis of SNPs in R-mutants, Figure S6: \(\beta\)-galactosidaseastive assay based on the fluorescent substrate 4-methylumbellifery \(\beta\)-D-galactopyranoside, Figure S7: Translation initiation rates of _M. luteus_ trpE16 and the mutant 13-M1090. R-script for SNPs mapping and clusters determination. Sliding window function R-script.  
[PERSON], [PERSON] and [PERSON] designed the research and interpreted results, with important input by [PERSON] and [PERSON]. [PERSON] constructed and analyzed the mutants with help by [PERSON] [PERSON] drafted the manuscript, which was revised by [PERSON], [PERSON] and the other authors. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
The authors declare no conflict of interest.