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1. Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme

   https://doi.org/10.1073/pnas.2100657118  

   Patterson, Dayna C.; Ruiz, Myrrh Perez; Yoon, Hyerin; Walker, Johnnie A.; Armache, Jean-Paul; Yennawar, Neela H.; Weinert, Emily E. (September 2021, Proceedings of the National Academy of Sciences)

   Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) fromPaenibacillus dendritiformis(DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O₂state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation ofP. dendritiformisin response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities. 

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2. A Conserved Regulatory Circuit Controls Large Adhesins in Vibrio cholerae

   https://doi.org/10.1128/mBio.02822-19  

   Kitts, Giordan; Giglio, Krista M.; Zamorano-Sánchez, David; Park, Jin Hwan; Townsley, Loni; Cooley, Richard B.; Wucher, Benjamin R.; Klose, Karl E.; Nadell, Carey D.; Yildiz, Fitnat H.; et al (December 2019, mBio)

   ABSTRACT The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae , where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. We further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations. IMPORTANCE Vibrio cholerae , the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae ; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae , which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments. 

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3. Quantitative input–output dynamics of a c-di-GMP signal transduction cascade in Vibrio cholerae

   https://doi.org/10.1371/journal.pbio.3001585  

   Bridges, Andrew A.; Prentice, Jojo A.; Fei, Chenyi; Wingreen, Ned S.; Bassler, Bonnie L. (March 2022, PLOS Biology)

   Waldor, Matthew K. (Ed.)

   Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae . Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which, in turn, drives alterations specifically to biofilm gene expression. A long-standing question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V . cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input–output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies. 

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4. Regulation of Biofilm Exopolysaccharide Production by Cyclic Di-Guanosine Monophosphate

   https://doi.org/10.3389/fmicb.2021.730980  

   Poulin, Myles B.; Kuperman, Laura L. (September 2021, Frontiers in Microbiology)

   Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly- N -acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c - di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP. 

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5. Vibrio cholerae adapts to sessile and motile lifestyles by cyclic di-GMP regulation of cell shape

   https://doi.org/10.1073/pnas.2010199117  

   Fernandez, Nicolas L.; Hsueh, Brian Y.; Nhu, Nguyen T. Q.; Franklin, Joshua L.; Dufour, Yann S.; Waters, Christopher M. (November 2020, Proceedings of the National Academy of Sciences)

   The cell morphology of rod-shaped bacteria is determined by the rigid net of peptidoglycan forming the cell wall. Alterations to the rod shape, such as the curved rod, occur through manipulating the process of cell wall synthesis. The human pathogenVibrio choleraetypically exists as a curved rod, but straight rods have been observed under certain conditions. While this appears to be a regulated process, the regulatory pathways controlling cell shape transitions inV. choleraeand the benefits of switching between rod and curved shape have not been determined. We demonstrate that cell shape inV. choleraeis regulated by the bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) by posttranscriptionally repressing expression ofcrvA, a gene encoding an intermediate filament-like protein necessary for curvature formation inV. cholerae.This regulation is mediated by the transcriptional cascade that also induces production of biofilm matrix components, indicating that cell shape is coregulated withV. cholerae’s induction of sessility. During microcolony formation, wild-typeV. choleraecells tended to exist as straight rods, while genetically engineering cells to maintain high curvature reduced microcolony formation and biofilm density. Conversely, straightV. choleraemutants have reduced swimming speed when using flagellar motility in liquid. Our results demonstrate regulation of cell shape in bacteria is a mechanism to increase fitness in planktonic and biofilm lifestyles. 

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