
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.01.610008
preprint
1
Article
Negative feedback of cyclic di-GMP levels optimizes switching between sessile and motile lifestyles in Vibrio cholerae
Rangarajan Aathmaja Anandhi http://orcid.org/0000-0003-3471-0257

Schroeder Jeremy W. http://orcid.org/0000-0001-8829-8494

Hurto Rebecca L.
Severin Geoffrey B. http://orcid.org/0000-0003-2441-5916

Pell Macy E. http://orcid.org/0000-0002-1846-5063

Hsieh Meng-Lun http://orcid.org/0000-0002-6360-005X

Waters Christopher M. http://orcid.org/0000-0003-2336-7836

Freddolino Lydia http://orcid.org/0000-0002-5821-4226

01 9 2024
2024.09.01.610008https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.01.610008
nihpp-2024.09.01.610008.pdf
Abstract

The signaling molecule cyclic di-GMP (cdG) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of cdG have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of cdG are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize cdG to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyse cdG to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing cdG levels, we followed a timecourse of overexpression of either the V. harveyi diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases cdG levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on cdG levels plateaus at 30 minutes, indicating tight adaptive control of cdG levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to cdG, leads to higher baseline levels of cdG and continuously increasing cdG through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on cdG levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC , is activated by VpsR at high cdG concentrations, mediating this negative feedback on cdG levels. We further identify a transcript encoded within, and antisense to, the cdgC open reading frame which we name s RNA n egative r egulator of C dgC (SnrC). RNA polymerase ChIP-seq and RNA-seq demonstrate SnrC to be expressed specifically under conditions of high cdG in the absence of VpsR. Ectopic SnrC expression increases cdG levels in a manner dependent on CdgC, demonstrating that its effect on cdG levels is likely through interference with CdgC production. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of cdG to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.
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pmc
