## 4 Discussion
### R-Mutations  
Four positions were affected within cluster 2 of the R-mutants (Table 3). Two missense transitions within Mlut_07500 encoding the Flp pilus assembly protein/NTPase TadA1/CpaF and another one in Mlut_07560, a hypothetical Flp pilus assembly TadG-like protein. Both proteins belong to the previously described _tad-1_ cluster and have been shown to be essential for transformation/pilus assembly [15].  
Three R-mutations that were selected for re-insertion as single-nucleotide exchange mutations gave rise to strains (2-R33, 8-R34, 13-R4) with at least 1000-fold lower transformability than the wild-type in late-exponential phase grown in GMM (Figure 4). Mutation G1541663A/D149N (8-R34) affected Mlut_14110, an ORF encoding a putative response regulator that forms an operon along with the putative histidine kinase ORF Mlut_14100, together probably composing a two-component signal transduction cascade similar to _sivS/R_ of _Streptococci__iinae_, according to a BLAST search [45]. Mutation G1606821/G27D (13-R4) affects ORF Mlut_14650 and is merely 10 bp upstream of mutation G1606810A/V31I (13-M27) from cluster 13 of the M-mutants, which is discussed below.  
Mutation G810889A/G327E (2-R33) affects Mlut_07490, an ORF encoding an AAA+ ATPase DNA translocase of the FtsK/SpoIIIE family, which is directly adjacent to the _tad-1_ cluster, one of two tad gene clusters already known to be essential for natural transformation in _M. luteus_[15]. Members of this protein family are involved in a wide variety of processes in many species, such as conjugation, DNA packaging, DNA segregation, and maintenance [46]. Mlut_07490 seems to share structural features with FtsK and SpoIIIE, such as a well conserved DNA translocation module and transmembrane helices (four in FtsK and SpoIIIE, but only two in Mlut_07490; sequence alignment in www.uniprot.org/align (accessed on the 18 August 2021) Identifiers: P46889, P21458, and C5C9X9). These transmembrane domains are localized at the proteins' N-termini, usually followed by a sequence of variable length that interacts with other proteins, and at the highly conserved C-terminus there is a DNA-binding module [47; 48] whose specific role in _M. luteus_ has not yet been studied. Mlut_07490 has the typical highly conserved P-Loop consensus amino acid sequence \"GTTSGKS\" (Walker A motif). The Walker B motif is usually located downstream of Walker A and has a higher variability. The only invariant features of this motif are a negatively charged residue following a stretch of bulky, hydrophobic amino acids [49], such as the \"AAAAAPAVAPWE\" present in _M. luteus_ Mlut_07490. In a study conducted on _B. subtilis_[50], a _spoIIIE_ mutation was the only one that significantly decreased the _lacZ_ reporter gene expression (in that case under control of the _spoIIIG_ promoter). This suggests that the _spoIIIE_ product is directly involved in a mechanism of differential gene expression.  
The membrane-spanning and mononucleotide-binding domains suggest that the protein may function as part of a signal transduction pathway based on phosphoryl group transfer, similar to the NtrB/NtrC system in other bacteria [51].  
When transforming the single-nucleotide exchange mutants 2-R33 and 8-R34 with the _lacZ_ reporter construct, blue colonies were obtained. This indicates that although these SNPs affect transformability, they do not directly repress _comEA/EC_ promoter expression, suggesting that they may play a role in the process at a different level or that one or more other mutations are required for _comEA/EC_ promoter repression.