Genetic and functional characterization of the natural transformation system in Streptococcus constellatus

Streptococcus constellatus is an opportunistic pathogen frequently associated with abscess formation in various body sites. While the species has been shown to acquire exogenous DNA through natural transformation, functional analyses of its underlying mechanisms and optimized genetic editing protocols remain limited. Thus, our aim was to characterize the natural transformation system in S. constellatus and investigate environmental factors coordinating its activation. In addition, we sought to develop an optimized protocol for genome editing. Genomic analysis revealed that 73% of analyzed strains possess orthologs for essential competence regulon genes, with 58% harboring both a complete ComCDE-based operon and the putative transformation machinery required for natural competence. While all complete genomes harbored three copies of the master regulator sigX , the accessory regulator comW was seemingly absent. Lacking the peptide exporter comAB , we demonstrated that S. constellatus utilizes the bacteriocin transporter silED for competence-stimulating peptide export. Gene expression assays indicated system activation at peptide concentrations as low as 4 nM, with peak sigX expression obtained over 60 nM. With the goal of optimizing gene editing strategies, we developed a protocol utilizing rich media supplemented with BSA and calcium chloride, significantly increasing transformation frequencies. Furthermore, we observed that environmental stressors can upregulate the system, including hydrogen peroxide and subinhibitory concentrations of the antibiotics erythromycin, chloramphenicol and ampicillin. Given the increasing clinical relevance of the anginosus group, elucidating horizontal gene transfer mechanisms can provide critical insights into the evolutionary process and pathogenic potential of these species.