A cyclic di-GMP phosphodiesterase in the VSP-2 island of <i>Vibrio cholerae</i> is regulated by zinc and quorum sensing

The second messenger cyclic di-GMP (cdG) is indispensable for the regulation of biofilm formation, motility, and a variety of other important bacterial behaviors in the majority of bacteria. The human pathogen <i>Vibrio cholerae</i> has a diverse repertoire of diguanylate cyclase and phosphodiesterase (PDE) enzymes that control the intracellular cdG concentration depending on local environmental cues and its physiological state. Determining the transcriptional regulation of these enzymes and the respective environmental signals that control their activity is important to understand how and when <i>V. cholerae</i> switches between motile and sessile lifestyles in different environments. In some strains of the current <i>V. cholerae</i> seventh pandemic El Tor biotype, the horizontally acquired <i>Vibrio</i> seventh pandemic 2 (VSP-2) island encodes an uncharacterized PDE at the gene locus <i>vc0515</i>, which we named <i>zpdA</i> (<u>z</u>inc-inhibited <u>p</u>hospho<u>d</u>iesterase <u>A</u>). We show, here, that <i>zpdA</i> transcription is repressed by Zur when Zn²⁺ is abundant, as well as by the quorum sensing regulator HapR when cells grow to high density. Furthermore, we find that the PDE activity of the purified ZpdA protein is inhibited by Zn²⁺ but is dependent on alternative divalent cations such as Mn²⁺, which we find are elevated in <i>V. cholerae</i> cells grown under zinc-limiting conditions. We conclude that ZpdA is an active metal-dependent PDE that is regulated by Zn²⁺ availability at both the level of transcriptional and post-translational leading to elevated cdG levels when Zn²⁺ is abundant. Our results demonstrate the important role of metal availability in modulating cdG signaling in bacteria.IMPORTANCE<i>Vibrio cholerae</i> colonizes estuarine environments and human hosts, where it transitions between motile and sessile states using the second messenger cyclic di-GMP (cdG). cdG levels change in response to a variety of signals and are controlled by the activity of diguanylate cyclases and phosphodiesterase (PDEs), enzymes that make and degrade cdG. In this work, we show that Zn²⁺ and the cell density regulator HapR repress ZpdA, the PDE present in the <i>Vibrio</i> seventh pandemic 2 island, at the level of transcription, and that Zn²⁺ unexpectedly alters the PDE activity of ZpdA protein itself. Our study highlights the role of metal availability as an important signaling cue that controls <i>V. cholerae</i> biology.