Identification of Vibrio cholerae proteins that bind 3′,3′-cyclic guanosine adenine dinucleotide

ABSTRACT Cyclic dinucleotides are used as signaling molecules in both antiphage defense and normal cellular functions. Horizontal gene transfer can introduce antiphage defense systems that encode specific signaling molecules into a new host. Whether these new signaling molecules interact with existing host signaling proteins and whether such interactions have evolutionary implications remain unknown. Vibrio cholerae represents an organism in which a new cyclic dinucleotide signaling molecule, 3′,3′-cyclic guanosine adenine dinucleotide (3′,3′-cGAMP) produced by DncV, is introduced by horizontal gene transfer of an antiphage system. 3′,3′-cGAMP activates CapV phospholipase activity, leading to reduced growth and swim motility. To identify additional 3′,3′-cGAMP-binding proteins, we performed a 3′,3′-cGAMP-binding screen of the V. cholerae El Tor ORFeome. In addition to the expected 3′,3′-cGAMP-binding proteins CapV and three V-cGAP proteins, this screen identified seven additional 3′,3′-cGAMP-binding proteins that were previously found to bind c-di-GMP. These seven all bind to c-di-GMP with greater affinity, suggesting that these proteins do not bind 3′,3′-cGAMP specifically. We assessed the function of 3′,3′-cGAMP-binding proteins by testing the expression of DncV, CapV, or both in V. cholerae . Expression of DncV alone did not affect growth or swimming motility, in contrast to co-expression with CapV. In Escherichia coli, which lacks native 3′,3′-cGAMP-signaling pathways, co-expression of CapV and DncV also led to reduced growth and swimming motility. In contrast, Pseudomonas aeruginosa, which also lacks endogenous 3′,3′-cGAMP, is less susceptible to CapV and DncV co-expression. Signaling nucleotides from antiphage systems primarily function in phage defense but have the potential to regulate other processes when the phage pressure subsides. IMPORTANCE In this study, we systematically identified 3′,3′-cGAMP-binding proteins in V. cholerae , which also uses the similar cyclic dinucleotide c-di-GMP for signaling. In addition to previously observed 3′,3′-cGAMP-binding proteins, including 3′,3′-cGAMP-activated phospholipase CapV and V-cGAP proteins, this screen identified seven new 3′,3′-cGAMP-binding proteins that bound c-di-GMP as well. We found that both 3′,3′-cGAMP production and CapV phospholipase activity were necessary and sufficient to inhibit growth and swimming motility as previously observed. In heterologous bacteria that use c-di-GMP but lack 3′,3′-cGAMP-signaling pathways, 3′,3′-cGAMP activation of CapV phospholipase was similarly required to inhibit growth and swimming motility as observed in V. cholerae . These results suggest that the acquisition of signaling nucleotides from antiphage systems may evolve to regulate cellular processes.