The CarSR Two-Component System Directly Controls radD Expression as a Global Regulator that Senses Bacterial Coaggregation in Fusobacterium nucleatum

Two-component systems (TCS) enable bacteria to sense and respond to environmental signals, facilitating rapid adaptation. Fusobacterium nucleatum, a key oral pathobiont, employs the CarSR TCS to modulate coaggregation with various Gram-positive partners by regulating the expression of radD, encoding a surface adhesion protein, as revealed by RNA-Seq analysis. However, the direct regulation of the radD-containing operon (radABCD) by the response regulator CarR, the broader CarR regulon, and the signals sensed by this system remain unclear. In this study, chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) identified approximately 161 CarR-enriched loci across the genome and a 17-bp consensus motif that likely serves as the CarR binding site. Notably, one such binding motif was found in the promoter region of the radABCD operon. The interaction of CarR with this binding motif was further validated using electrophoretic mobility shift assays (EMSA), mutagenesis, and DNase I footprinting analyses. Beyond regulating radABCD, CarR directly controls genes involved in fructose and amino acid (cysteine, glutamate, lysine) utilization, underscoring its role as a global regulator in F. nucleatum. Lastly, we discovered that RadD-mediated coaggregation enhances radD expression, and deletion of carS abolished this enhancement, suggesting that coaggregation itself serves as a signal sensed by this TCS. These findings provide new insights into the CarR regulon and the regulation of RadD, elucidating the ecological and pathogenic roles of F. nucleatum in dental plaque formation and disease processes. IMPORTANCEFusobacterium nucleatum is an essential member of oral biofilms, acting as a bridging organism that connects early and late colonizers, thus driving dental plaque formation. Its remarkable ability to aggregate with diverse bacterial partners is central to its ecological success, yet the mechanisms it senses and responds to these interactions remain poorly understood. This study identifies the CarSR two-component system as a direct regulator of RadD, the primary adhesin mediating coaggregation, and reveals its role in sensing coaggregation as a signal. These findings uncover a novel mechanism by which F. nucleatum dynamically adapts to polymicrobial environments, offering new perspectives on biofilm formation and bacterial communication in complex oral microbial ecosystems.