The second messenger c-di-GMP is a key regulator of bacterial physiology. The V.?vulnificus genome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation in V.?vulnificus . Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers brp -mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen. ABSTRACT Poor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogen Vibrio vulnificus . V.?vulnificus , as well as the bivalves (oysters) it naturally colonizes, is indigenous to estuaries and human-inhabited coastal regions and must endure constantly changing environmental conditions as freshwater and seawater enter, mix, and exit the water column. Elevated cellular c-di-GMP levels trigger biofilm formation, but relatively little is known regarding the environmental signals that initiate this response. Here, we show that calcium is a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers expression of the brp extracellular polysaccharide that enhances biofilm formation. A transposon screen for the loss of calcium-induced P _( brpA )expression revealed CysD, an enzyme in the sulfate assimilation pathway. Targeted disruption of the pathway indicated that the production of a specific metabolic intermediate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), was required for calcium-induced P _( brpA )expression and that PAPS was separately required for development of the physiologically distinct rugose phenotype. Thus, PAPS behaves as a second messenger in V.?vulnificus . Moreover, c-di-GMP and BrpT (the activator of brp expression) acted in concert to bias expression of the sulfate assimilation pathway toward PAPS and c-di-GMP accumulation, establishing a feed-forward regulatory loop to boost brp expression. Thus, this signaling network links extracellular calcium and sulfur availability to the intracellular second messengers PAPS and c-di-GMP in the regulation of V.?vulnificus biofilm formation and rugosity, survival phenotypes underpinning its evolution as a resilient environmental organism.
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