## 4 Discussion  
### Mutant Selection and Clustering of Mutations  
The most fundamental and conserved proteins involved in natural transformation are _ComEC_, a polytopic integral membrane protein that forms the DNA uptake channel, and the extracytoplasmic DNA receptor _ComEA_. Their importance is also true for the actinobacterial model organism _Microococcus luteus_[4; 15]. We aimed to find out more about how these co-transcribed genes are regulated. Information about the genes involved in competence development for natural transformation and its regulation in the _Actinobacteria_ is scarce. It was only recently shown that the pilus-like structure, which is commonly found to be necessary for natural transformation in different bacterial lineages (usually type IV pili or pseudo-pili, and in certain cases, type IV secretion system), is a Flp pilus structure in _M. luteus_ and is thus different from other bacteria studied previously. Orthologs of most other genes implicated in competence development and natural transformation in the best-studied low-GC-Gram-positive and Gram-negative model bacteria were not found in _M. luteus_[15; 26]. Therefore, we decided to take a classical random mutagenesis approach for the search for gene candidates involved in this bacterium. In the _Firmicutes_ model bacterium _Bacillus subtilis_, [PERSON] and others in the 1980s isolated 28 _Tn917-lacZ_ mutants that were deficient in competence as well as mapped the mutations and characterized the mutants with respect to DNA binding and uptake [39; 40]. The expression of \(\beta\)-galactosidase under the control of the _com_ promoter was studied as a function of growth stage, medium, and genetic background [41]. Later, [PERSON] and [PERSON] [27] used EMS-induced chemical mutagenesis to generate mutants that would permit the overexpression of _com_ genes under normally repressing conditions, such as in full medium. The _mec_ (for medium-independent expression of competence) mutations found by this approach mainly affected two genes, _mecA_ and _mecB_. Later, it was determined how they affected the regulation of medium-dependent expression of competence [42]. Our group previously constructed a _lacZ_ reporter strain to study expression of the late _com_ genes _comEA/EC_ in _M. luteus_[26]. We chose EMS random chemical mutagenesis as an efficient method for introducing random mutations into _M. luteus_ trpE16 _\(\Delta\)comEA/EC:lacZ-Kan_. On average, we obtained one stable transition every \(3\times 10^{5}\) bp. If lower, the frequency of appearance of a desired phenotype would make the screening for its detection laborious, whereas if it were higher, it would be difficult to pinpoint the causative mutation for a specific trait among numerous other point mutations in the genome. Regarding the mutagenesis efficiency, our results are within the same order of magnitude as described in the study on _B. subtilis_ by [PERSON] and [PERSON] (1990) [27]. It was crucial to filter out EMS mutants that exhibited the medium-dependent overexpression or repression phenotype of interest but were genetically closely linked to the _comEA/EC_ locus or the _lacZ_ reporter gene because it was our goal to identify new loci involved in competence development that are different from _comEA/EC_. By natural transformation of the wild-type strain with gDNA isolated from the M or R mutants, it was possible to analyze the degree of linkage between the phenotype-causing SNPs and the seven kbp \(\Delta\)_comEA/EC:lacZ-Kan_ reporter construct region. A linkage analysis similar to the LOD score [43] was conducted to study _cis_-acting mutations in _comG_ of _B. subtilis_[28]. In our study, the linkage analysis was successfully used to identify and focus on _M. luteus_ trpE16 EMS mutants that were genetically unlinked to the _comEA/EC_ locus.