Comparative Experimental Evolution of Gene Essentiality in Streptococcus sanguinis and S. mutans - Title: Comparative Experimental Evolution of Gene Essentiality in Streptococcus sanguinis and S. mutans Project Summary/Abstract Streptococcus sanguinis and S. mutans are important oral microbiota. Despite both belonging to the Streptococcal genus, these bacteria exhibit distinct roles in oral health, with S. sanguinis serving as a pioneer colonizer and commensal on teeth, while S. mutans is notorious for its major pathogenic role in dental caries development. The contrasting virulence in dental caries is underscored by the similarities and differences in their essential genomes. S. sanguinis SK36 possesses 218 essential genes (EGs), whereas S. mutans UA159 has 295. As key regulators for fitness and viability, EGs control the pathogenesis of these bacteria. However, our understanding of gene essentiality has been hindered by the traditional belief that generating EG-deletion mutants is impractical due to the indispensable nature of these genes for survival. Recognizing the quantitative characteristics of gene essentiality, we have recently developed a high-throughput method that enables the generation of dozens of EG deletion mutants in S. sanguinis SK36. We categorized the 218 EGs into three groups:186 “non-evolvable EGs”, 23 “evolvable EGs” and 9 “conditional EGs”, marking a significant advancement across the entire Streptococcus species. We hypothesized that A) the list of “evolvable EGs” and “non-evolvable EGs” and B) the profile of suppressor genes for the same “evolvable EGs” are different between S. mutans and S. sanguinis. Using an experimental evolution approach combined with whole-genome sequencing, we have identified >1000 suppressor mutation in evolved populations deled of “evolvable EGs” of S. sanguinis SK36. These suppressors were further be classified into three categories: “activation,” “inhibition,” and “unknown.” in the previous study. Our primary objective is to categorize the 295 EGs in S. mutans UA159, identify and classify corresponding suppressor mutations for its evolvable EGs. By examining essential gene mutants and their corresponding suppressors, we aim to construct a comprehensive functional network of gene essentiality. Through a comparative analysis of suppressor mutation profiles evolved in essential-gene mutants between S. sanguinis and S. mutans, our goal is to comprehend gene essentiality of these two Streptococcus species. This thorough examination will provide valuable insights into the conservation and diversity of essential gene functions within the broader context of Streptococcus biology. Ultimately, these findings will be instrumental in rational design of targeting S. mutans-specific anti-caries strategies by targeting EGs whose deletion is lethal in S. mutans, while leaving S. sanguinis unaffected or allowing for compensatory mechanisms through suppressor mutations.