Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such “local signaling” is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network.

Cyclic diguanylate (c-di-GMP) is a near universal signaling molecule produced by diguanylate cyclases that can direct a variety of bacterial behaviors. A major area of research over the last several years has been aimed at understanding how a cell with dozens of diguanylate cyclases can deploy a given subset of them to produce a desired phenotypic outcome without undesired cross talk between c-di-GMP-dependent systems. Several models have been put forward to address this question, including specificity of cyclase activation, tuned binding constants of effector proteins, and physical interaction between cyclases and effectors. Additionally, recent evidence has suggested that there may be a link between the catalytic state of a cyclase and its physical contact with an effector. This review highlights several key studies, examines the proposed global and local models of c-di-GMP signaling specificity in bacteria, and attempts to identify the most fruitful steps that can be taken to better understand how dynamic networks of sibling cyclases and effector proteins result in sensible outputs that govern cellular behavior.

Many bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an autoinhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus controlling the amount of c-di-GMP available to bind to target proteins in the cell. Many DGCs studied to date have also been found to signal for a specific c-di-GMP-related process, and although recent studies have suggested that physical interaction between DGCs and target proteins may provide this signaling fidelity, the importance of the I-site has not yet been incorporated into this model. Our results from Pseudomonas fluorescens indicate that mutation of residues at the I-site of a DGC disrupts the interaction with its target receptor. By creating various substitutions to a DGC's I-site, we show that signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as participating in protein-protein interactions suggests caution in interpreting the function of the I-site as only a means to negatively regulate a cyclase. These results implicate the I-site as an important positive and negative regulatory element of DGCs that may contribute to signaling specificity. IMPORTANCESome bacteria contain several dozen diguanylate cyclases (DGCs), nearly all of which signal to specific receptors using the same small molecule, c-di-GMP. Signaling specificity in these networks may be partially driven by physical interactions between DGCs and their receptors, in addition to the autoinhibitory site of DGCs preventing the overproduction of c-di-GMP. In this study, we show that disruption of the autoinhibitory site of a DGC in Pseudomonas fluorescens can result in the loss of interactions with its target receptor and reduced biofilm formation, despite increased production of c-di-GMP. Our findings implicate the autoinhibitory site as both an important feature for signaling specificity through the regulation of c-di-GMP production and a necessary element for the physical interaction between a diguanylate cyclase and its receptor.A major method of intracellular signaling in bacteria revolves around the second messenger cyclic diguanylate (c-di-GMP). c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs) in networks that may range from one or a few such proteins to several dozen (1). Effector proteins then sense c-di-GMP by binding it and promote some specific cellular action as a result. Importantly, many DGCs studied to date promote only one or a small number of such processes (2-6), indicating that there must be mechanisms in place that allow for specificity in signaling among the many members of a c-di-GMP network.A key regulatory element in signalin...

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.

No abstract

The role of microbes in sustaining agricultural plant growth has great potential consequences for human prosperity. Yet we have an incomplete understanding of the basic function of rhizosphere microbial communities and how they may change under future stresses, let alone how these processes might be harnessed to sustain or improve crop yields. A reductionist approach may aid the generation and testing of hypotheses that can ultimately be translated to agricultural practices. With this in mind, we ask whether some rhizosphere microbial communities might be governed by "keystone metabolites", envisioned here as microbially produced molecules that, through antibiotic and/or growth-promoting properties, may play an outsized role in shaping the development of the community spatiotemporally. To illustrate this point, we use the example of redox-active metabolites, and in particular phenazines, which are produced by many bacteria found in agricultural soils and have well-understood catalytic properties. Phenazines can act as potent antibiotics against a variety of cell types, yet they also can promote the acquisition of essential inorganic nutrients. In this essay, we suggest the ways these metabolites might affect microbial communities and ultimately agricultural productivity in two specific scenarios: i) biocontrol of beneficial and pathogenic fungi in increasingly arid crop soils and ii) promotion of phosphorus bioavailability and sustainable fertilizer use. We conclude with specific proposals for future research.

Bacteria can be motile and planktonic or, alternatively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, including Bordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation in B. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of the bdcA gene, B. bronchiseptica showed enhanced motility, strengthening the hypothesis that BdcA regulates motility in B. bronchiseptica. We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of the bdcA gene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation in B. bronchiseptica, as well as its impact on pathogenesis. IMPORTANCE Pathogenesis of Bordetella spp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. In Bordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized a B. bronchiseptica diguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability of B. bronchiseptica to colonize the murine respiratory tract. These results provide us with a better understanding of how B. bronchiseptica can infect a host.

No abstract