## 4 Discussion
### M-Mutations  
Cluster 3 of the M-mutants encompasses six mutants with two positions affected within the same gene, Mlut_03135, which codes for a N6-adenine DNA methyltransferase of unknown recognition sequence. These type of enzymes are widely spread in prokaryotes, and besides their role in restriction-modification systems [52], they are an important method of regulation of gene expression. Changes in promoter methylation status has been shown to control several genes in different model organisms such as _E. coli_ and _Caulobacter sp_. A transcriptomic study conducted in _E. coli_ reported many deregulated genes in DNA adenosine methyltransferase mutants, including global regulators such as catabolite activator protein or fumarate nitrate reductase [53]. However, the single-nucleotide mutant derived from M43, 3-M43, and the corresponding gene deletion mutant trpE16 \(\Delta\)Mlut_03135 did not show increased activity of the _comEA/EC_ promoter, suggesting that promoter adenine methylation by the putative methylase encoded by Mlut_03135 probably does not play a role in regulation. Cluster 7 of the M-mutants is composed of three positions affected in six different mutants. The affected loci, Mlut_09880 and Mlut_09900, encode the AAA+ ATP-dependent Clp protease proteolytic subunit ClpP and the ATP-binding subunit ClpX, respectively. ClpX recognizes unstructured peptide tag sequences in protein substrates, proceeds to unfold stable tertiary structure in the protein, and then spools or translocates the unfolded polypeptide chain into a sequestered proteolytic compartment in ClpP for degradation into small peptide fragments [54]. The levels of the main transcriptional regulator ComK of _B. subtilis_ are controlled by this type of proteolytic system, composed of the adapter protein MecA (see Introduction) and the ATP-binding subunit ClpC (formerly known as MecB) [27,Mlut_0749042]. In _B. subtilis_, mutants in _mecA_, ClpC, and ClpP lead to the overexpression of _com_ genes and other genes activated by ComK. This scenario may be similar to what was observed in the M-mutants carrying changes in the region of cluster 7. However, none of the mutations from cluster 7 (i.e., strains 7-M28, 7-M89, and 7-M93) restored the screened EMS mutants' _comEA/EC_ promoter activity when introduced into the _M. luteus_ chromosome as single-SNP mutations (Figure 3). This means that at least the effect of those single mutations on the Clp proteases are not enough to affect _comEA/EC_ promoter expression rates. None of the aforementioned single-SNP M-mutants or gene knockout mutants had significant changes in their transformation frequencies in comparison to the trpE16 wild-type strain in late exponential phase GMM broth cultures. It is possible that the resulting phenotypes for certain M- or R-mutants depend on the combined effect of more than one mutation. Further genome editing experiments are needed to see if the combination of certain single-SNP mutations, such as those from strains 7-M89 or 13-M89 which have an unaltered _comEA/EC_ expression phenotype compared with the non-mutagenized \(\Delta\)_comEA/EC:lacZ_ control strain (Figure 3), resulting in the same LacZ reporter phenotype as their parental strain M89. Cluster 13 encompasses six different positions, with one of them detected in almost every M-mutant, the only exception being M33. Thus, the highest density of SNPs identified by our phenotypic screening (see Figure 2) was in this region of the genome. ORFs of two apparently divergently overlapping transcriptional units were affected: G1606810A/V31I (13-M27) lies within the Mlut_14650 ORF, which codes for a SAF-domain containing protein with similarity to RcpC/CpaB Flp pilus assembly proteins, while G1607081A (13-M89)and G1607117A (13-M1090) putatively affect the 5'UTR of Mlut_14660, and mutations C1607204T/S27L (13-M96), C1607261T/S46F (13-M9) and C1607314T/R64W (13-M93) affect the Mlut_14660 ORF. The only R-mutation identified in this cluster was 13-R4, which has the mutation G1606821A/G27D in the Mlut_14650 ORF.