Dover, C.; Tamrakar, K.; Dwivedi, B.; Roberts, E. R.; Chudal, S.; King, S.; Chavez, E. S.; de Crecy-Lagard, V.; Shields, R. C.

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans. However, particularly for genes annotated as "hypothetical" or uncharacterized, translating these findings into mechanistic biological functions remains a significant bottleneck. In this study, we developed an integrated functional genomics pipeline combining predictive bioinformatics, tunable CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to characterize nine legacy hypothetical essential genes in S. mutans. Comparative transcriptomics and proteomics revealed a conserved baseline stress signature across diverse essential pathways, marked by the coordinated downregulation of the citZ-citB-idh metabolic locus and insoluble matrix synthesis enzymes (gtfBC), paired with the robust activation of the integrative and conjugative element TnSmu1. Against this backdrop of systemic stress, we successfully resolved the function of SMU_393, defining it as a functional equivalent of the pneumococcal regulator of chromosome segregation, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. Remarkably, these phenotypes were bypassed by a spontaneous surface-exposed missense mutation (dnaAQ197E) within the AAA+ ATPase domain of the replication initiator. Together, this work uncovers an important cell cycle regulator and provides a framework for exploring uncharacterized essential genes of the oral microbiome.