A ComX-based strategy for artificially inducing competence in naturally non-transformable Streptococcus parasanguinis

Many streptococci, including strains of Streptococcus salivarius and Streptococcus parasanguinis, harbor com genes but do not exhibit natural competence. In S. salivarius YS18, a chromosomal rearrangement in the 5' flanking region of comX, encoding σ^X, the alternative sigma factor responsible for the late com gene expression, was found to confer competence, whereas its parental strain 57.I was not competent. This genetic arrangement led to constitutive, high-level comX expression, with ComX production independent of quorum-sensing regulation. By establishing YS18 comX on the Streptococcus-Escherichia coli shuttle vector pDL278 in non-competent S. parasanguinis FW213, transient competence could be established, enabling mutant construction via ligation mutagenesis; thereby, bypassing the need to generate plasmids carrying mutated loci that would be introduced via electroporation. Plasmids in the mutant isolates were successfully cured by cultivation in antibiotic-free media post-mutagenesis. In conclusion, the lack of competence in com gene-containing streptococci may likely be due to inadequate ComX production. Given the structural conservation of ComX, constitutive comX expression provides a practical strategy to establish competence in non-competent streptococcal hosts.IMPORTANCEMany streptococci encode com genes but remain non-competent, limiting genetic manipulation. This study found that insufficient ComX production was the key barrier to competence development in these strains. However, variations in competence-stimulating peptides and σ^X-inducing peptides make optimizing comX expression with synthetic peptides impractical. Instead, by introducing a constitutively expressed comX from Streptococcus salivarius, a transient competence stage can be established in non-competent Streptococcus parasanguinis, enabling efficient mutant construction via ligation mutagenesis. This approach provides a generalizable strategy to simplify mutagenesis workflows and expands the genetic toolkit for functional studies in otherwise intractable streptococcal strains.