Dover, C. | bioRxiv Science

bioRxiv2026

Deciphering the Streptococcus mutans essentialome: multi-omic resolution of hypothetical genes and identification of a functional RocS equivalent

Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans. In this study, we combined predictive bioinformatics, conditional CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to investigate nine poorly characterized essential genes in S. mutans. From this screen, phenotypic and genetic analyses identified SMU_393 as a functional homolog of the pneumococcal chromosome segregation factor, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. These phenotypes were bypassed by a spontaneous surface-exposed missense mutation (dnaAQ197E) within the AAA+ ATPase domain of the replication initiator. Together, this study refines annotations within the S. mutans essential genome and provides genetic insights into streptococcal chromosome segregation and cell cycle control.

microbiology↗

bioRxiv2025

Post-translational modifications via serine/threonine phosphorylation and GpsB in Streptococcus mutans

Post-translational modifications (PTMs), such as protein phosphorylation, are critical regulators of bacterial physiology. Here, we present the first comprehensive phosphoproteomic analysis of Streptococcus mutans, revealing extensive O-phosphorylation under non-stressed conditions. Using tandem mass tag (TMT)-based mass spectrometry and phosphopeptide enrichment, we identified 231 high-confidence phosphosites on 131 proteins, representing approximately 6.7% of the detected proteome. These phosphorylated proteins were enriched in pathways related to translation, carbohydrate metabolism, and the cell cycle, suggesting a broad role for O-phosphorylation in core cellular functions. To define the functional roles of the sole serine/threonine protein kinase (PknB) and phosphatase (PppL) encoded by S. mutans, we analyzed phosphoproteomic and proteomic changes in {Delta}pknB and {Delta}pppL mutants. These mutants exhibited widespread alterations in protein abundance and phosphorylation, revealing overlapping but distinct sets of putative kinase and phosphatase substrates, including DivIVA, MapZ, MltG, and ribosomal proteins. Notably, we discovered that repression of gpsB, a predicted PknB binding partner, causes lethal defects that can be rescued by a suppressor mutation (G98R) in pppL. This mutation restores phosphorylation of DivIVA, suggesting that GpsB regulates the PknB/PppL signaling axis to maintain appropriate phosphorylation of essential targets. This work highlights conserved and unique features of bacterial phospho-signaling and provides a foundation for future studies on PTM-mediated regulation in S. mutans.

microbiology↗