## 3 Results
### Genomic Syntenty of Cluster 13-Associated Genes  
As cluster 13 mutations showed a strong influence on the _comEA/EC_ promoter, and certain mutations from this cluster affected the transformability of _M. luteus_, we evaluated the presence, distribution, and genomic context of the genes in this cluster and flanking genes in other members of _Actinobacteria_ using the microbial genomic context viewer. BLAST was used to obtain the E-values of the homologs with _M. luteus_ gene sequences as queries. Similar gene arrangements as at cluster 13 of _M. luteus_ were found in species of the genera _Geodermatophilus_, _Rothia_, _Jonesia_, and _Cellomonas_. In _Nocardioides_, the genes were also present, although they had a different orientation relative to each other. For _Gordonia_ and _Kytococcus_ species, the analysis revealed that homologues of the genes are present, but in a different organization (Figure 5).  
Figure 3: Relative initial velocities of \(\beta\)-galactosidase activity of _M. luteus_ trpE16, _the \(\Delta\)comEA/EC:lacZ_ reporter strain and its derivative mutants. _ComEA/EC_ promoter transcriptional activities for _M. luteus_ trpE16, the \(\Delta\)_comEA/EC:lacZ_ reporter strain (orange), and its derivative mutants under normally competence-repressing conditions (LB full medium). All cultures were normalized to cell density prior to lysozyme treatment. LacZ substrate MUG was added and kinetic data for the promoter activity was recorded over 8 hours by measuring the fluorescence generated from the substrate cleavage. A four parameter logistic model was fitted to the kinetic curves (\(n\geq 3\)). The slope of the logistic curve served as a measure for the relative promoter activity. Expression levels of the M-mutants are shown in blue, with the single SNP mutants in dark blue and the gene knockouts in turquoise. The mean values of \(\geq\)3 independent experiments and standard deviations of all experiments are presented. An unpaired, two-tailed Student’s t-test showed no difference between M27, M93, and M1090 and 13-M27, 13-M93, and 13-M1090, respectively. *, indicates statistically significant difference.  
Figure 4: Effect of specific genome modifications on the transformability of _M. luteus_. Transformation frequencies of clean deletion mutants are shown in turquois, while single-SNP mutants with SNPs from the M-mutants and from the R-mutants are shown in dark blue and brown, respectively. Each dot represents the log\({}_{10}\)-transformed mean transformation frequency relative to the mean of the trpE16 strain. All strains were grown in GMM for 20 h until OD\({}_{600\text{nm}}\) reached 1. The error bars indicate the SD of the biological replicates. An ordinary one-way ANOVA test was performed to compare all means with each other (\(p<0.005\), \(n\geq 3\)).  
Figure 5: Physical maps of the Mlut_14660 and Mlut_14650/40/30 genes and their genomic surroundings on the chromosomes of a selection of actinobacterial (high-GC Gram-positive) representatives and from model organism _M. luteus_. ORFs with the same color encode proteins with significant similarity (BLAST E \(<10^{-4}\)) to the respective gene products of _M. luteus_. The predicted gene products are shown and the numbers in the arrows representing homologs are E-values of a BLAST search using the full-length _M. luteus_ genes as queries. Pink: Mlut_14660; light yellow: Mlut_14650; light brown: Mlut_14640; green: Mlut_14630; blue: Mlut_14670. All representatives harbor the genes comEA and comEC in the chromosome.