Abstract
Historically, Micrococcus luteus was one of the first organisms used to study natural transformation, one of the main routes of horizontal gene transfer among prokaryotes. However, little is known about the molecular basis of competence development in M. luteus or any other representative of the phylum of high-GC Gram-positive bacteria (Actinobacteria), while this means of genetic exchange has been studied in great detail in Gram-negative and low-GC Gram-positive bacteria (Firmicutes). In order to identify new genetic elements involved in regulation of the comEA-comEC competence operon in M. luteus, we conducted random chemical mutagenesis of a reporter strain expressing lacZ under the control of the comEA-comEC promoter, followed by the screening of dysregulated mutants. Mutants with (i) upregulated com promoter under competence-repressing conditions and (ii) mutants with a repressed com promoter under competence-inducing conditions were isolated. After genotype and phenotype screening, the genomes of several mutant strains were sequenced. A selection of putative com-influencing mutations was reinserted into the genome of the M. luteus reporter strain as markerless single-nucleotide mutations to confirm their effect on com gene expression. This strategy revealed mutations affecting com gene expression at genetic loci different from previously known genes involved in natural transformation. Several of these mutations decreased transformation frequencies by several orders of magnitude, thus indicating significant roles in competence development or DNA acquisition in M. luteus. Among the identified loci, there was a new locus containing genes with similarity to genes of the tad clusters of M. luteus and other bacteria.