Synergy between c-di-GMP and Quorum-Sensing Signaling in Vibrio cholerae Biofilm Morphogenesis

Transitions between individual and communal lifestyles allow bacteria to adapt to changing environments. Bacteria must integrate information encoded in multiple sensory cues to appropriately undertake these transitions. Here, we investigate how two prevalent sensory inputs converge on biofilm morphogenesis: quorum sensing, which endows bacteria with the ability to communicate and coordinate group behaviors, and second messenger c-di-GMP signaling, which allows bacteria to detect and respond to environmental stimuli. We use Vibrio cholerae as our model system, the autoinducer AI-2 to modulate quorum sensing, and the polyamine norspermidine to modulate NspS-MbaA-mediated c-di-GMP production. Individually, AI-2 and norspermidine drive opposing biofilm phenotypes, with AI-2 repressing and norspermidine inducing biofilm formation. Surprisingly, however, when AI-2 and norspermidine are simultaneously detected, they act synergistically to increase biofilm biomass and biofilm cell density. We show that this effect is caused by quorum-sensing-mediated activation of nspS-mbaA expression, which increases the levels of NspS and MbaA, and in turn, c-di-GMP biosynthesis, in response to norspermidine. Increased MbaA-synthesized c-di-GMP activates the VpsR transcription factor, driving elevated expression of genes encoding key biofilm matrix components. Thus, in the context of biofilm morphogenesis in V. cholerae, quorum-sensing regulation of c-di-GMP-metabolizing receptor levels connects changes in cell population density to detection of environmental stimuli. IMPORTANCE The development of multicellular communities, known as biofilms, facilitates beneficial functions of gut microbiome bacteria and makes bacterial pathogens recalcitrant to treatment. Understanding how bacteria regulate the biofilm life cycle is fundamental to biofilm control in industrial processes and in medicine. Here, we demonstrate how two major sensory inputs-quorum-sensing communication and second messenger c-di-GMP signaling-jointly regulate biofilm morphogenesis in the global pathogen Vibrio cholerae. We characterize the mechanism underlying a surprising synergy between quorum-sensing and c-di-GMP signaling in controlling biofilm development. Thus, the work connects changes in cell population density to detection of environmental stimuli in a pathogen of clinical significance.