Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

Abstract

The second messenger, cyclic dimeric GMP (c-di-GMP) regulates biofilm formation for many bacteria. The binding of c-d¡-GMP by the inner-membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbC’s calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present GcbC activity is significantly enhanced ~8-fold, indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the l-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of P. fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.ImportanceIn many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP, however it is unclear how undesired crosstalk is mitigated in the context of this soluble signal, and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-d¡-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the l-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a “cloud” of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling and biofilm formation.

Effects of Calcium and Signal Sensing Systems on Azorhizobium caulinodans Biofilm Formation and Host Colonization

“…First, chemotaxis toward ligands in TY medium, which might be sensed by AZC_0821 and AZC_0660, is important for the calcium-mediated biofilm. In Pseudomonas fluorescens, the ligand citrate can bind to the Cache domain of GcbC to mediate biofilm formation (Giacalone et al, 2018). Second, calcium might function as a chemotaxis ligand, and AZC_0821 and AZC_0660 might be involved in the sensing of calcium signals directly.…”

Role of SpoIVA ATPase Motifs DuringClostridioides difficileSporulation

“…The transformations were incubated on LB agar supplemented with 50 µg/mL kanamycin, 50 µg/mL carbenicillin, and 0.5 mM IPTG for 40 hrs at 30˚C. After this incubation period, β-galactosidase activity was quantified in Miller units using the protocol described by (54,55).…”

Spatial organization enhances versatility and specificity in cyclic di-GMP signaling

“…Upon binding to c-di-GMP, LapD undergoes a conformational change that sequesters the periplasmic protease LapG. This in turn allows adhesin LapA to accumulate on the cell surface, a first step in biofilm formation (Chatterjee et al 2014;Dahlstrom et al 2015;Giacalone et al 2018;Navarro et al 2011;Newell et al 2011). The DGC GcbC binds to LapD via alpha helical contacts, which is crucial for signaling efficiency and thereby increases its activity up to eight-fold (Dahlstrom et al 2015;Giacalone et al 2018).…”

“…This in turn allows adhesin LapA to accumulate on the cell surface, a first step in biofilm formation (Chatterjee et al 2014;Dahlstrom et al 2015;Giacalone et al 2018;Navarro et al 2011;Newell et al 2011). The DGC GcbC binds to LapD via alpha helical contacts, which is crucial for signaling efficiency and thereby increases its activity up to eight-fold (Dahlstrom et al 2015;Giacalone et al 2018). Also in B. subtilis, two DGCs were shown to bind to two receptor proteins having different c-di-GMP binding domains, depending on c-di-GMP binding for at least one of the pairs (Kunz et al 2020), which is further detailed below.…”