Lai, XuanXuanLaiYu, MandaMandaYuCHIU-PING CHENGLai, Erh-MinErh-MinLai2025-06-162025-06-162025-04-17https://www.scopus.com/record/display.uri?eid=2-s2.0-105005558146&origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/730007Diguanylate cyclases (DGCs) synthesize bis-(3′,5′)-cyclic diguanylic acid (c-di-GMP), a critical bacterial second messenger that coordinates diverse biological processes. Agrobacterium tumefaciens, a plant pathogen causing crown gall disease, relies on type IV secretion system for pathogenesis and type VI secretion system (T6SS) for interbacterial competition. Our study identified two putative DGCs, named diguanylate cyclase domain proteins regulating virulences A and B (DcvA and DcvB), that negatively regulate virulence through distinct mechanisms. DcvA suppresses virulence by targeting the VirA/VirG two-component system downstream of VirA. This inhibition is independent of c-di-GMP levels. DcvB positively regulates biofilm formation, inhibits T6SS-mediated interbacterial competition, and suppresses virulence via the ChvG/ChvI two-component system downstream of ChvG. These effects are dependent on its cyclase activity and the associated increase in intracellular c-di-GMP levels. These findings suggest that DcvA and DcvB control virulence and interbacterial competition using different mechanisms in Agrobacterium. DcvA suppresses virulence, independent of c-di-GMP, and DcvB enhances global c-di-GMP concentration to promote biofilm formation and inhibits virulence and T6SS antibacterial activity. The findings provide understanding of how DGC domain proteins orchestrate complex regulatory networks to balance virulence, biofilm formation, and interbacterial competition, enabling them to adapt to changing environments.enAgrobacterium tumefaciensc-di-GMPdiguanylate cyclasesinterbacterial competitionvirulence regulation[SDGs]SDG2[SDGs]SDG3journal article10.1128/mbio.00039-25