PROJECT SUMMARY Dental plaque and associated periodontal diseases represent a common infectious disease afflicting nearly half of American adults (CDC). The complex intra- and interspecies interactions regulate the development of multispecies oral microbial communities called dental plaque. The dental plaque is made of an organized, highly complex microbial social community and the Gram-negative anaerobe F.nucleatum is a key organism of this microbiome by virtue of its unique capability to physically aggregate with many early and late colonizers. Besides its role in periodontitis, F. nucleatum has been linked to several extra-oral diseases including preterm birth and colorectal cancer. Although the fusobacterial interactions with other bacteria have been widely studied and four coaggregation adhesins identified, little is known about mechanisms that regulate Fusobacterium-mediated coaggregation, mainly due to the lack of a robust genetic toolkit for manipulation of F. nucleatum. To overcome this, we recently developed a convenient gene deletion system for F. nucleatum and generated a large library of random transposon mutants with ~10-fold genome coverage. Screening of this library uncovered several coaggregation factors, which include a unique two-component system termed CarS- CarR and a nine-gene-operon that encodes a lysine-degrading pathway (LDP) that controls the amount and activity of RadD, respectively. RadD, a type IV autotransporter, is a versatile adhesin that mediates fusobacterial adhesion with many early and some late colonizers. Based on these findings, we plan to characterize the two regulatory factors in-depth. We will be the first time to show an oral bacterial two component signaling component regulates expression of cell-cell adhesin in a cell density-dependent manner and characterize a lysine riboswitch related to lysine catabolism in bacteria. Upon the successful completion of this research, we expect to have significantly contributed to the understanding of how the versatile adhesin RadD is regulated by identifying and deciphering factors and mechanism involved. Because RadD requires F. nucleatum to incorporate into an established community made of initial commensal colonizers, such as streptococci and actinomyces, our discoveries will have a significant impact on the understanding of fusobacterium-mediated coaggregation role in development of dental plaque, will provide new insights into the development of potent therapeutic strategies against this important pathogen.