A Multimodal Strategy Used by a Large c-di-GMP Network

Abstract

The genome encodes more than 50 proteins predicted to be involved in c-di-GMP signaling. Here, we demonstrated that, tested across 188 nutrients, these enzymes and effectors appeared capable of impacting biofilm formation. Transcriptional analysis of network members across ∼50 nutrient conditions indicates that altered gene expression can explain a subset of but not all biofilm formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing ∼2,000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy using combinations of ligand-mediated signals, protein-protein interaction, and/or transcriptional regulation to fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria. Cyclic diguanylate (c-di-GMP) is a key signaling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break, or bind this dinucleotide. A major open issue in the field is how signaling specificity is conferred in the unpartitioned space of a bacterial cell. Here, we took a systems approach, using mutational analysis, transcriptional studies, and bacterial two-hybrid analysis to interrogate this network. We found that a majority of enzymes are capable of impacting biofilm formation in a context-dependent manner, and we revealed examples of two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) being utilized to generate an observable impact on biofilm formation.

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

“…Next, we selected the CACHE domain-containing Pfl01_2295 and Pfl01_2297 proteins (Fig. 1A), both of which interact with LapD (20), to serve as controls to determine if other CACHE domains also respond to citrate to promote biofilm formation. For these studies, we used a P. fluorescens PfO-1 strain lacking four DGCs, referred to as Δ4DGC, which does not form a biofilm under our laboratory conditions; this low c-di-GMP producing strain was previously used to investigate c-di-GMP production by other P. fluorescens DGCs (21).…”

“…actinidiae , the CACHE domain PscD (PDB ID: 5G4Z) binds glycolate, acetate, propionate, and pyruvate (22), indicating that CACHE domains allow for some promiscuity in their binding of ligands. GcbC has been shown to be part of a large signaling network, interacting with a phosphodiesterase and multiple dual domain containing proteins (20), thus also opening the possibility that different ligands sensed via the CACHE domain of GcbC could dictate which proteins interact with this DGC.…”

A novel bacterial l‐arginine sensor controlling c‐di‐GMP levels in Pseudomonas aeruginosa

“…[2][3][4] To further complicate the scenario, genetic mutants in putative DGCs and/or PDEs showed unexpected phenotypes in terms of biofilm formation/dispersal. 5,6 A recent analysis on the regulation of the c-di-GMP network in Pseudomonas fluorescens Pf01 clearly showed that multifaceted regulatory strategies can take place to achieve the final phenotype, including combinations of ligand-mediated signals and/or transcriptional regulation. 5 Therefore, it appears that multiple signaling pathways could act together to obtain a unique c-di-GMP-dependent phenotype, and that different pathways could lead to the same output.…”

“…5,6 A recent analysis on the regulation of the c-di-GMP network in Pseudomonas fluorescens Pf01 clearly showed that multifaceted regulatory strategies can take place to achieve the final phenotype, including combinations of ligand-mediated signals and/or transcriptional regulation. 5 Therefore, it appears that multiple signaling pathways could act together to obtain a unique c-di-GMP-dependent phenotype, and that different pathways could lead to the same output. In this regard, the activity of DGCs and PDEs is often allosterically regulated by environmental/metabolic signals acting on upstream sensory domains.…”

Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

“…In P. fluorescens SBW25, screens for wrinkly spreader biofilm phenotypes, which are associated with enhanced formation of cellulose-based matrix at the air-liquid (AL) interface, identified many DCGs and associated regulators involved in c-di-GMP homeostasis [47][48][49][50][51] . In P. fluorescens Pf0-1, a systematic survey of knockout mutants revealed that about a third of c-di-GMP-associated proteins exhibit strong biofilm phenotypes across many growth conditions whereas the remaining mutants exhibited weak phenotypes in only a small number of conditions 52 . However, the biological roles of most the c-di-GMPbinding proteins still remain to be characterized.…”