## 3 Results
### Transformation Frequency Evaluation  
An important question was if the selected and individually reinserted single-SNP mutations had a direct impact on _M. luteus_ transformability. To answer this question,  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Cluster** & **SNP** & **Mutant/s** & **Locus** & **Orientation** & **Annotated Product** \\ \hline
1 & C9672T & R42 & Mlut\_00060 & \(>\) & DNA gyrase subunit A \\
1 & C10325A & RE & Mlut\_00060 & \(>\) & DNA gyrase subunit A \\
2 & C810889A & R33 & Mlut\_07490 & \(<\) & DNA segregation ATPase, FtsK/SpoIIIE family \\
2 & C81276A & R13 & Mlut\_07500 & \(>\) & Flp pilus assembly protein, ATPase CpaF \\
2 & C812940T & R16 & Mlut\_07500 & \(>\) & Flp pilus assembly protein, ATPase CpaF \\
2 & C816694A & R3 & Mlut\_07560 & \(>\) & hypothetical protein \\
3 & C934586T & R21 & Mlut\_08640 & \(>\) & glutamyl-tRNA synthetase \\
3 & C935940A & R16 & Mlut\_08660 & \(>\) & tRNA-Ga \\
4 & G1011921A & R4 & Mlut\_09340 & \(<\) & 2-oxo-acid dehydrogenase E1 component, \\  & & & & & homodimeric type \\
4 & C1014740T & R13 & Mlut\_09380 & \(>\) & 4-azaleucine resistance probable transporter \\
5 & G1029395A & R7 & Mlut\_09550 & \(<\) & peptide deformylase \\
5 & G1031740A & R13 & Mlut\_09580 & \(>\) & translocating P-type ATPase, \\  & & & & & Cd/Co/Hg/Pb/Zn-transporting \\
6 & G1249315A & R42 & Mlut\_11600 & \(<\) & phosphoserine phosphatase SerB \\
6 & C1250564T & R42 & Mlut\_11610 & \(>\) & ABC-type molybdenum transport system \\
7 & G1460599A & RE & Mlut\_13330 & \(>\) & 2-oxoglutarate dehydrogenase E2 component \\
7 & C1462336 & R4 & Mlut\_13350 & \(<\) & protein kinase family protein \\
8 & C1541663A & R34 & Mlut\_14110 & \(>\) & response regulator citrate/malate metabolism \\
8 & C1542830T & R41 & Mlut\_14120 & \(<\) & acyl-CoA synthetase/AMP-acid ligase \\
9 & G1814683A & R3, R30 & - & Intergenic sequence, Mlut\_16600 promoter \\
10 & C1962653T & R4 & Mlut\_18140 & \(>\) & helicase family protein with metal-binding \\  & & & & & \\  & & & & & \\
11 & C2027721T & R3, R30 & Mlut\_18760 & \(>\) & tRNA (guanine-N(?)-)methyltransferase \\
11 & C2028876T & R34 & Mlut\_18770 & \(<\) & \(\alpha/\beta\) hydrolase of unknown function (DUF1023) \\
12 & G2427638A & R3, R30 & Mlut\_22740 & \(<\) & hypothetical protein \\
13 & G1606821A & R4 & Mlut\_14650 & \(<\) & SAF domain-containing protein \\ \hline \hline \end{tabular}
\end{table}
Table 3: R-Mutants Main Mutation Clusters, SNPs Features and Affected Genes.  
transformation frequency assays were conducted. _M. luteus_ trpE16-derived strains with edited single-SNP chromosomes or with clean gene deletions (see above) were cultured in GMM broth before cells were harvested and transformed with pJET-_trpE_ plasmid to assess the frequency of conversion to Trp prototrophy. The mutant strain 13-M89 showed a 100-fold decrease in transformability compared to the parental strain _M. luteus_ trpE16. Mutants _M. luteus_ trpE16 2-R33 and _M. luteus_ trpE16 8-R34 had lower frequencies of transformation, but not as low as the knockout _M. luteus_ trpE16 \(\Delta\)14650 or _M. luteus_ trpE16 13-R4 (Figure 4). This last mutant is the only mutant that had a transformation frequency well below the detection limit of the assay. Consequently, it was not feasible to construct a \(\Delta\)_comEA/EC:lacZ-Kan_ reporter strain after introducing the corresponding point mutation (G106821A) into _M. luteus_ trpE16. The transformation frequencies of the _M. luteus_ trpE16 mutants 13-M93, 13-M1090, 13-M27, \(\Delta\)02150, and \(\Delta\)03135 did not differ significantly from the reference strain _M. luteus_ trpE16.