Dataset Information

Interaction between the type 4 pili machinery and a diguanylate cyclase fine-tune c-di-GMP levels during early biofilm formation.

ABSTRACT: To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.

SUBMITTER: Webster SS

PROVIDER: S-EPMC8256011 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The bacterial intracellular second messenger, cyclic dimeric GMP (c-di-GMP), regulates biofilm formation for many bacteria. The binding of c-di-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 I-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 Pseudomonas 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.IMPORTANCE In many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP; however, it is unclear how undesired cross talk 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-di-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the I-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.

Project description:Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. A large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs), and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components employ various methods to avoid undesired cross talk to maintain signaling specificity. The synthesis of the antibiotic HSAF (heat-stable antifungal factor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE, LchP, and a c-di-GMP effector, Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, that participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP display local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. IMPORTANCE Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases [DGCs]), c-di-GMP-degrading enzymes (phosphodiesterases [PDEs]), and c-di-GMP effectors. These components deploy various methods to avoid undesired cross talk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes. We provide direct evidence to show that the DGC and PDE form a complex and also indirect evidence to argue that they may balance a local c-di-GMP pool to control antibiotic production. These results represent an important finding regarding the mechanism of a DGC and PDE pair to control the expression of specific c-di-GMP signaling pathways.

Dataset Information

Interaction between the type 4 pili machinery and a diguanylate cyclase fine-tune c-di-GMP levels during early biofilm formation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets