Methods: Genome analysis of the sequenced S. mitis strain NCTC 12261 was conducted in two-steps: (1)Identification of streptococcal Rgg homologues, and (2)search for open reading frame (ORF) coding oligopeptides in the rgg flanking regions. Real-time PCR was used to investigate whether the identified oligopeptide DIIIVGG fulfilled the criterion for autoinducing activity that characterizes pheromone systems. Transcriptome analysis using RNA sequencing was performed for a comprehensive assessment of changes in gene expression in response to the synthetic pheromone.
Results: Genome analysis revealed a non-annotated gene coding a possible novel pheromone (N-DIIIVGG), which was upstream of an Rgg transcriptional regulator. RT-PCR results showed that gene expression of the pheromone was increased by seven-fold in samples where the synthetic pheromone was added in comparison to the ones without it, indicating an autoinducing effect. Transcriptome analysis revealed that twelve genes were up-regulated at least two-fold by the new pheromone. The highest induction was observed for gene 0092 (55.9-fold) which codes an hypothetical protein with unknown function. This gene forms probably an operon, together with the 0093 gene, which codes for an ABC transporter ATP-binding protein (possibly multidrug efflux), and the pheromone gene. This last one was located in the intergenic sequence between 0093 and the rgg gene (ID0094). Transcription of the Rgg regulator was found to occur in the opposite direction of the pheromone operon.
Conclusion: We identified a novel pheromone system in S.mitis that induces expression of more than ten genes. Next efforts will be directed to elucidating which specific functions this pheromone is taking part in and how it affects S. mitis group behavior.