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The aquatic γ-proteobacterium Shewanella oneidensis is able to form two types of biofilms: a floating biofilm at the air-liquid interface (pellicle) and a solid surface-associated biofilm (SSA-biofilm). S. oneidensis possesses the Bpf system, which is orthologous to the Lap system first described in Pseudomonas fluorescens . In the Lap systems, the retention of a large adhesin (LapA) at the cell surface is controlled by LapD, a c-di-GMP effector protein, and LapG, a periplasmic protease targeting LapA. Here, we showed that the Bpf system is mandatory for pellicle biogenesis, but not for SSA-biofilm formation, indicating that the role of Bpf is somewhat different from that of Lap. The BpfD protein was then proved to bind c-di-GMP via its degenerated EAL domain, thus acting as a c-di-GMP effector protein like its counterpart LapD. In accordance with its key role in pellicle formation, BpfD was found to interact with two diguanylate cyclases, PdgA and PdgB, previously identified as involved in pellicle formation. Finally, BpfD was shown to interact with CheY3, the response regulator controlling both chemotaxis and biofilm formation. Altogether, these results indicate that biofilm formation in S. oneidensis is under the control of a large c-di-GMP network.

Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O 2 -dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O 2 binding modulates activity of diguanylate cyclase-containing GCS proteins., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Emily Weinert reports financial support was provided by National Science Foundation. Emily Weinert reports financial support was provided by Herman Frasch Foundation for Chemical Research. Yilin Liu reports financial support was provided by National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phenotypic heterogeneity in bacteria can result from stochastic processes or deterministic programs. The deterministic programs often involve the versatile second messenger c-di-GMP, and give rise to daughter cells with different c-di-GMP levels by deploying c-di-GMP metabolizing enzymes asymmetrically during cell division. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity is switched on during cytokinesis, resulting in a transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating phenotypically similar daughters with correct motility behaviours. Thus, our findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria can ensure the formation of dissimilar or similar daughter cells by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations., (© 2024. The Author(s).)

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger in bacteria that regulates multiple biological functions, including biofilm formation, virulence, and intercellular communication. However, c-di-GMP signaling is virtually unknown in economically important filamentous cyanobacteria, Arthrospira. In this study, we predicted 31 genes encoding GGDEF-domain proteins from A. platensis NIES39 as potential diguanylate cyclases (DGCs). Phylogenetic distribution analysis showed five genes (RS09460, RS04865, RS26155, M01840, and E02220) with highly conserved distribution across 25 Arthrospira strains. Adc1 encoded by RS09460 was further characterized as a typical DGC. By establishing the genetic transformation system of Arthrospira, we demonstrated that the overexpression of Adc1 promoted the production of extracellular polymeric substances (EPS), which in turn caused the aggregation of filaments. We also confirmed that RS04865 and RS26155 may encode active DGCs, while enzymatic activity assays showed that proteins encoded by M01840 and E02220 have phosphodiesterase (PDE) activity. Meta-analysis revealed that the expression profiles of RS09460 and RS04865 were unaffected under 31 conditions, suggesting that they may function as conserved genes in maintaining the basal level of c-di-GMP in Arthrospira. In summary, this report will provide the basis for further studies of c-di-GMP signal in Arthrospira., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heme-based sensor proteins are used by organisms to control signaling and physiological effects in response to their gaseous environment. Globin-coupled sensors (GCS) are oxygen-sensing proteins that are widely distributed in bacteria. These proteins consist of a heme globin domain linked by a middle domain to various output domains, including diguanylate cyclase domains, which are responsible for synthesizing c-di-GMP, a bacterial second messenger crucial for regulating biofilm formation. To understand the roles of heme pocket residues in controlling activity of the diguanylate cyclase domain, variants of the Pectobacterium carotovorum GCS (PccGCS) were characterized by enzyme kinetics and resonance Raman (rR) spectroscopy. Results of these studies have identified roles for hydrogen bonding and heme edge residues in modulating heme pocket conformation and flexibility. Better understanding of the ligand-dependent GCS signaling mechanism and the residues involved may allow for future development of methods to control O 2 -dependent c-di-GMP production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Prokaryotic antiviral defence systems are frequently toxic for host cells and stringent regulation is required to ensure survival and fitness. These systems must be readily available in case of infection but tightly controlled to prevent activation of an unnecessary cellular response. Here we investigate how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a type II CBASS system from Bacillus cereus into the model organism Bacillus subtilis, we show that the protein-conjugating Cap2 (CBASS associated protein 2) enzyme links the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. The cyclase-PspA conjugation is reversed by the deconjugating isopeptidase Cap3 (CBASS associated protein 3). We propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage, with conjugation released upon infection, priming the cyclase for activation., (© 2024. The Author(s).)

The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans . Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan , was identified as one of the 11 "hits," with the greatest reduction (>99%) in fluorescence at 100 µM. The sm DAC inhibitory profiles of the 11 "hits" established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC 50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits sm DAC's enzymatic activity ( K i = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin's binding profile with sm DAC ( K d = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC 50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 10 5 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to sm DAC but can also inhibit the function of sm DAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.IMPORTANCEThis study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans . The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans ' diadenylate cyclase ( sm DAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of sm DAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms., Competing Interests: The authors declare no conflict of interest.

Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimeric-guanosine monophosphate. While critical signal integration elements have been described for different phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. Here we show that dynamics-driven allostery is key to understanding signal integration on a molecular level. We generated protein variants stabilized in their far-red-absorbing Pfr state and demonstrated by analysis of conformational dynamics using hydrogen-deuterium exchange coupled to mass spectrometry that single amino acid replacements are accompanied by altered dynamics of functional elements throughout the protein. We show that the conformational dynamics correlate with the enzymatic activity of these variants, explaining also the increased activity of a non-photochromic variant. In addition, we demonstrate the functional importance of mixed Pfr/intermediate state dimers using a fast-reverting variant that still enables wild-type-like fold-changes of enzymatic stimulation by red light. This supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed Pfr/intermediate-state spectra observed for many phytochrome systems. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. This will eventually impact design strategies for the creation of novel sensor-effector systems for enriching the optogenetic toolbox., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species., (open access.)

Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyclic di-GMP (c-di-GMP) is a second messenger of intracellular communication in bacterial species, which widely modulates diverse cellular processes. However, little is known about the c-di-GMP network in filamentous multicellular cyanobacteria. In this study, we preliminarily investigated the c-di-GMP turnover proteins in Arthrospira based on published protein data. Bioinformatics results indicate the presence of at least 149 potential turnover proteins in five Arthrospira subspecies. Some proteins are highly conserved in all tested Arthrospira , whereas others are specifically found only in certain subspecies. To further validate the protein catalytic activity, we constructed a riboswitch-based c-di-GMP expression assay system in Escherichia coli and confirmed that a GGDEF domain protein, Adc11, exhibits potential diguanylate cyclase activity. Moreover, we also evaluated a protein with a conserved HD-GYP domain, Ahd1, the expression of which significantly improved the swimming ability of E. coli . Enzyme-linked immunosorbent assay also showed that overexpression of Ahd1 reduced the intracellular concentration of c-di-GMP, which is presumed to exhibit phosphodiesterase activity. Notably, meta-analyses of transcriptomes suggest that Adc11 and Ahd1 are invariable. Overall, this work confirms the possible existence of a functional c-di-GMP network in Arthrospira , which will provide support for the revelation of the biological function of the c-di-GMP system in Arthrospira .

Background: Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4., Results: Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells., Conclusions: The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Phototrophic bacteria face diurnal variations of environmental conditions such as light and osmolarity that affect their carbon metabolism and ability to generate organic compounds. The model cyanobacterium, Synechocystis sp. PCC 6803 forms a biofilm when it encounters extreme conditions like high salt stress, but the molecular mechanisms involved in perception of environmental changes that lead to biofilm formation are unknown. Here, we studied two two-component regulatory systems (TCSs) that contain diguanylate cyclases (DGCs), which produce the second messenger c-di-GMP, as potential components of the biofilm-inducing signaling pathway in Synechocystis. Analysis of single mutants provided evidence for involvement of the response regulators, Rre2 and Rre8 in biofilm formation. A bacterial two-hybrid assay showed that Rre2 and Rre8 each formed a TCS with a specific histidine kinase, Hik12 and Hik14, respectively. The in vitro assay showed that Rre2 had DGC activity regardless of its de/phosphorylation status, whereas Rre8 required phosphorylation for DGC activity. Hik14-Rre8 likely functioned as an inducible sensing system in response to environmental change. Biofilm assays with Synechocystis mutants suggested that pairs of hik12-rre2 and hik14-rre8 responded to high salinity-induced biofilm formation. Inactivation of hik12-rre2 and hik14-rre8 did not affect the performance of the light reactions of photosynthesis. These data suggest that Hik12-Rre2 and Hik14-Rre8 participate in biofilm formation in Synechocystis by regulating c-di-GMP production via the DGC activity of Rre2 and Rre8., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

c-di-GMP primarily controls motile to sessile transitions in bacteria. Diguanylate cyclases (DGCs) catalyze the synthesis of c-di-GMP from two GTP molecules. Typically, bacteria encode multiple DGCs that are activated by specific environmental signals. Their catalytic activity is modulated by c-di-GMP binding to autoinhibitory sites (I-sites). YfiN is a conserved inner membrane DGC that lacks these sites. Instead, YfiN activity is directly repressed by periplasmic YfiR, which is inactivated by redox stress. In Escherichia coli, an additional envelope stress causes YfiN to relocate to the mid-cell to inhibit cell division by interacting with the division machinery. Here, we report a third activity for YfiN in E. coli, where cell growth is inhibited without YfiN relocating to the division site. This action of YfiN is only observed when the bacteria are cultured on gluconeogenic carbon sources, and is dependent on absence of the autoinhibitory sites. Restoration of I-site function relieves the growth-arrest phenotype, and disabling this function in a heterologous DGC causes acquisition of this phenotype. Arrested cells are tolerant to a wide range of antibiotics. We show that the likely cause of growth arrest is depletion of cellular GTP from run-away synthesis of c-di-GMP, explaining the dependence of growth arrest on gluconeogenic carbon sources that exhaust more GTP during production of glucose. This is the first report of c-di-GMP-mediated growth arrest by altering metabolic flow. IMPORTANCE The c-di-GMP signaling network in bacteria not only controls a variety of cellular processes such as motility, biofilms, cell development, and virulence, but does so by a dizzying array of mechanisms. The DGC YfiN singularly represents the versatility of this network in that it not only inhibits motility and promotes biofilms, but also arrests growth in Escherichia coli by relocating to the mid-cell and blocking cell division. The work described here reveals that YfiN arrests growth by yet another mechanism in E. coli, changing metabolic flow. This function of YfiN, or of DGCs without autoinhibitory I-sites, may contribute to antibiotic tolerant persisters in relevant niches such as the gut where gluconeogenic sugars are found.

Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to various stresses. C-di-AMP is synthesized by diadenylate cyclases that contain the DAC (DisA_N) domain, which was originally characterized as the N-terminal domain in the DNA integrity scanning protein DisA. In other experimentally studied diadenylate cyclases, DAC domain is typically located at the protein C termini and its enzymatic activity is controlled by one or more N-terminal domains. As in other bacterial signal transduction proteins, these N-terminal modules appear to sense environmental or intracellular signals through ligand binding and/or protein-protein interactions. Studies of bacterial and archaeal diadenylate cyclases also revealed numerous sequences with uncharacterized N-terminal regions. This work provides a comprehensive review of the N-terminal domains of bacterial and archaeal diadenylate cyclases, including the description of five previously undefined domains and three PK_C-related domains of the DacZ_N superfamily. These data are used to classify diadenylate cyclases into 22 families, based on their conserved domain architectures and the phylogeny of their DAC domains. Although the nature of the regulatory signals remains obscure, the association of certain dac genes with anti-phage defense CBASS systems and other phage-resistance genes suggests that c-di-AMP might also be involved in the signaling of phage infection.

Transitions between motile and biofilm lifestyles are highly regulated and fundamental to microbial pathogenesis. H-NOX (heme-nitric oxide/oxygen-binding domain) is a key regulator of bacterial communal behaviors, such as biofilm formation. A predicted bifunctional cyclic di-GMP metabolizing enzyme, composed of diguanylate cyclase and phosphodiesterase (PDE) domains ( avi _3097), is annotated downstream of an hnoX gene in Agrobacterium vitis S4. Here, we demonstrate that av H-NOX is a nitric oxide (NO)-binding hemoprotein that binds to and regulates the activity of avi _3097 ( av HaCE; H-NOX-associated cyclic di-GMP processing enzyme). Kinetic analysis of av HaCE indicates a ∼four-fold increase in PDE activity in the presence of NO-bound av H-NOX. Biofilm analysis with crystal violet staining reveals that low concentrations of NO reduce biofilm growth in the wild-type A. vitis S4 strain, but the mutant Δ hnoX strain has no NO phenotype, suggesting that H-NOX is responsible for the NO biofilm phenotype in A. vitis . Together, these data indicate that av H-NOX enhances cyclic di-GMP degradation to reduce biofilm formation in response to NO in A. vitis .

A previous proteomic study uncovered a relationship between nutritional stress and fluctuations in levels of diadenylate cyclases (DACs) and other proteins that regulate DAC activity, degrade, or interact with c-di-AMP, suggesting a possible role of this second messenger in B. subtilis stress-associated mutagenesis (SAM). Here, we investigated a possible role of c-di-AMP in SAM and growth-associated mutagenesis (GAM). Our results showed that in growing cells of B. subtilis YB955 ( hisC952 , metB25 and leuC427 ), the DACs CdaA and DisA, which play crucial roles in cell wall homeostasis and chromosomal fidelity, respectively, counteracted spontaneous and Mitomycin-C-induced mutagenesis. However, experiments in which hydrogen peroxide was used to induce mutations showed that single deficiencies in DACs caused opposite effects compared to each other. In contrast, in the stationary-phase, DACs promoted mutations in conditions of nutritional stress. These results tracked with intracellular levels of c-di-AMP, which are significantly lower in cdaA - and disA -deficient strains. The restoration of DAC-deficient strains with single functional copies of the cdaA and/or disA returned SAM and GAM levels to those observed in the parental strain. Taken together, these results reveal a role for c-di-AMP in promoting genetic diversity in growth-limiting conditions in B. subtilis . Finally, we postulate that this novel function of c-di-AMP can be exerted through proteins that possess binding domains for this second messenger and play roles in DNA repair, ion transport, transcriptional regulation, as well as oxidative stress protection.

Cyclic diguanosine monophosphate (c-di-GMP) is widely used by bacteria to control biological functions in response to diverse signals or cues. A previous study showed that potential c-di-GMP metabolic enzymes play a role in the regulation of biofilm formation and motility in Acinetobacter baumannii . However, it was unclear whether and how A. baumannii cells use c-di-GMP signaling to modulate biological functions. Here, we report that c-di-GMP is an important intracellular signal in the modulation of biofilm formation, motility, and virulence in A. baumannii . The intracellular level of c-di-GMP is principally controlled by the diguanylate cyclases (DGCs) A1S_1695, A1S_2506, and A1S_3296 and the phosphodiesterase (PDE) A1S_1254. Intriguingly, we revealed that A1S_2419 (an elongation factor P [EF-P]), is a novel c-di-GMP effector in A. baumannii . Response to a c-di-GMP signal boosted A1S_2419 activity to rescue ribosomes from stalling during synthesis of proteins containing consecutive prolines and thus regulate A. baumannii physiology and pathogenesis. Our study presents a unique and widely conserved effector that controls bacterial physiology and virulence by sensing the second messenger c-di-GMP.

Understanding the relationship between protein sequence, structure and function is one of the fundamental challenges in biochemistry. A direct correlation, however, is often not trivial since protein dynamics also play an important functional role-especially in signal transduction processes. In a subfamily of bacterial light sensors, phytochrome-activated diguanylate cyclases (PadCs), a characteristic coiled-coil linker element connects photoreceptor and output module, playing an essential role in signal integration. Combining phylogenetic analyses with biochemical characterisations, we were able to show that length and composition of this linker determine sensor-effector function and as such are under considerable evolutionary pressure. The linker length, together with the upstream PHY-specific domain, influences the dynamic range of effector activation and can even cause light-induced enzyme inhibition. We demonstrate phylogenetic clustering according to linker length, and the development of new linker lengths as well as new protein function within linker families. The biochemical characterisation of PadC homologs revealed that the functional coupling of PHY dimer interface and linker element defines signal integration and regulation of output functionality. A small subfamily of PadCs, characterised by a linker length breaking the coiled-coil pattern, shows a markedly different behaviour from other homologs. The effect of the central helical spine on PadC function highlights its essential role in signal integration as well as direct regulation of diguanylate cyclase activity. Appreciation of sensor-effector linkers as integrator elements and their coevolution with sensory modules is a further step towards the use of functionally diverse homologs as building blocks for rationally designed optogenetic tools., (© 2022. The Author(s).)

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates biofilm formation and pathogenicity. To study the global regulatory effect of individual components of the c-di-GMP metabolic system, we deleted all 12 diguanylate cyclase (dgc) and phosphodiesterase (pde)-encoding genes in E. amylovora Ea1189 (Ea1189Δ12). Ea1189Δ12 was impaired in surface attachment due to a transcriptional dysregulation of the type IV pilus and the flagellar filament. A transcriptomic analysis of surface-exposed WT Ea1189 and Ea1189Δ12 cells indicated that genes involved in metabolism, appendage generation and global transcriptional/post-transcriptional regulation were differentially regulated in Ea1189Δ12. Biofilm formation was regulated by all 5 Dgcs, whereas type III secretion and disease development were differentially regulated by specific Dgcs. A comparative transcriptomic analysis of Ea1189Δ8 (lacks all five enzymatically active dgc and 3 pde genes) against Ea1189Δ8 expressing specific dgcs, revealed the presence of a dual modality of spatial and global regulatory frameworks in the c-di-GMP signaling network., Competing Interests: The authors have declared that no competing interests exist.

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC, were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Psychiatric manifestations in patients with tetrahydrobiopterin (BH4) defects are common, and may occur even with treatment of the underlying disorder. The neurobiological background of these conditions has been linked to abnormalities of neurotransmitters, such as dopamine, serotonin, norepinephrine, and gamma-aminobutyric acid. Here, we review the psychiatric profile of all patients with BH4 defects followed in the pediatric and adult metabolic clinics at our center. Three patients with autosomal recessive (AR) guanosine triphosphate cyclohydrolase (GTPCH) deficiency and three patients with 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency were reviewed.All patients had behavioral disturbances and two had significant psychiatric comorbidities. These included attention deficit/hyperactivity disorder, anxiety, depression, aggression, or oppositional defiant disorder. One patient with PTPS deficiency had a severe psychiatric presentation, requiring inpatient admission and temporary placement into foster care for intensive behavioral therapy. Another with AR GTPCH deficiency was diagnosed with aggressive behavioral dysregulation requiring intensive psychiatric treatment. Management of the psychiatric manifestations of BH4 defects can be challenging, due to lack of information and studies of interactions between psychiatric medications on the deficient neurotransmitters and their receptors in these conditions. Further studies are needed to establish safety and efficacy of these treatments., Competing Interests: None declared., (Thieme. All rights reserved.)

As a key bacterial second messenger, cyclic di-GMP (c-di-GMP) regulates various physiological processes, such as motility, biofilm formation, and virulence. Cellular c-di-GMP levels are regulated by the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). Beyond that, the enzymatic activities of c-di-GMP metabolizing proteins are controlled by a variety of extracellular signals and intracellular physiological conditions. Here, we report that pdcA ( BTH_II2363 ), pdcB ( BTH_II2364 ), and pdcC ( BTH_II2365 ) are cotranscribed in the same operon and are involved in a regulatory cascade controlling the cellular level of c-di-GMP in Burkholderia thailandensis. The GGDEF domain-containing protein PdcA was found to be a DGC that modulates biofilm formation, motility, and virulence in B. thailandensis. Moreover, the DGC activity of PdcA was inhibited by phosphorylated PdcC, a single-domain response regulator composed of only the phosphoryl-accepting REC domain. The phosphatase PdcB affects the function of PdcA by dephosphorylating PdcC. The observation that homologous operons of pdcABC are widespread among betaproteobacteria and gammaproteobacteria suggests a general mechanism by which the intracellular concentration of c-di-GMP is modulated to coordinate bacterial behavior and virulence. IMPORTANCE The transition from planktonic cells to biofilm cells is a successful strategy adopted by bacteria to survive in diverse environments, while the second messenger c-di-GMP plays an important role in this process. Cellular c-di-GMP levels are mainly controlled by modulating the activity of c-di-GMP-metabolizing proteins via the sensory domains adjacent to their enzymatic domains. However, in most cases how c-di-GMP-metabolizing enzymes are modulated by their sensory domains remains unclear. Here, we reveal a new c-di-GMP signaling cascade that regulates motility, biofilm formation, and virulence in B. thailandensis. While pdcA , pdcB , and pdcC constitute an operon, the phosphorylated PdcC binds the PAS sensory domain of PdcA to inhibit its DGC activity, with PdcB dephosphorylating PdcC to derepress the activity of PdcA. We also show this c-di-GMP regulatory model is widespread in the phylum Proteobacteria . Our study expands the current knowledge of how bacteria regulate intracellular c-di-GMP levels.

The biosynthetic gene cluster of atlantinone B ( 10 ) was discovered in Penicillium chrysogenum MT-40. A multifunctional cytochrome P450 (AtlD) encoded by the cluster is responsible for the formation of the unique lactone-bridged ring and the 16β-hydroxyl of atlantinone B, and a new terpene cyclase (AtlC) can unprecedentedly accept the demethylated substrate epoxyfarnesyl-DMOA ( 4a ) to generate three bicyclic meroterpenoids ( 5a - 5c ). This study paves the way for combinatorial synthesis of structurally diverse meroterpenoids for drug discovery.

Cyclic diguanylate (c-di-GMP) signal transduction systems provide bacteria with the ability to sense changing cell status or environmental conditions and then execute suitable physiological and social behaviors in response. In this review, we provide a comprehensive census of the stimuli and receptors that are linked to the modulation of intracellular c-di-GMP. Emerging evidence indicates that c-di-GMP networks sense light, surfaces, energy, redox potential, respiratory electron acceptors, temperature, and structurally diverse biotic and abiotic chemicals. Bioinformatic analysis of sensory domains in diguanylate cyclases and c-di-GMP-specific phosphodiesterases as well as the receptor complexes associated with them reveals that these functions are linked to a diverse repertoire of protein domain families. We describe the principles of stimulus perception learned from studying these modular sensory devices, illustrate how they are assembled in varied combinations with output domains, and summarize a system for classifying these sensor proteins based on their complexity. Biological information processing via c-di-GMP signal transduction not only is fundamental to bacterial survival in dynamic environments but also is being used to engineer gene expression circuitry and synthetic proteins with à la carte biochemical functionalities.

Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found that extracellular polysaccharide (EPS) production, regulation of motility, and induction of stress response pathways contribute to biofilm tolerance during such insults. However, little is known regarding the mechanism(s) whereby biofilm maintenance is regulated when exposed to such environmental challenges. Here, we provide evidence that the diguanylate cyclase YfiN is important for the regulation of biofilm maintenance when exposed to peroxide. We find that compared to the wild type (WT), static biofilms of the Δ yfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H 2 O 2 ); this defect can be rescued through genetic complementation. Additionally, we found that the Δ yfiN mutant biofilms produce less c-di-GMP than WT and that H 2 O 2 treatment enhanced motility of surface-associated bacteria and increased cell death for the Δ yfiN mutant grown as a biofilm compared to WT biofilms. These data provide evidence that YfiN is required for biofilm maintenance by P. aeruginosa, via c-di-GMP signaling, to limit motility and protect viability in response to peroxide stress. These findings add to the growing recognition that biofilm maintenance by P. aeruginosa is an actively regulated process that is controlled, at least in part, by the wide array of c-di-GMP metabolizing enzymes found in this microbe. IMPORTANCE We build on previous findings that suggest that Pseudomonas aeruginosa utilizes c-di-GMP metabolizing enzymes to actively maintain a mature biofilm. Here, we explore how the diguanylate cyclase YfiN contributes to the regulation of biofilm maintenance during peroxide exposure. We find that mature P. aeruginosa biofilms require YfiN to synthesize c-di-GMP, regulate motility, and ensure viability during peroxide stress. These findings provide further evidence that the modulation of c-di-GMP in response to environmental signals is an important mechanism by which biofilms are maintained.

Many bacteria use cyclic dimeric guanosine monophosphate (c-di-GMP) to control changes in lifestyle. The molecule, synthesized by proteins having diguanylate cyclase activity, is often a signal to transition from motile to sedentary behaviour. In Vibrio cholerae, c-di-GMP can exert its effects via the transcription factors VpsT and VpsR. Together, these proteins activate genes needed for V. cholerae to form biofilms. In this work, we have mapped the genome-wide distribution of VpsT in a search for further regulatory roles. We show that VpsT binds 23 loci and recognises a degenerate DNA palindrome having the consensus 5'-W-5R-4[CG]-3Y-2W-1W+1R+2[GC]+3Y+4W+5-3'. Most genes targeted by VpsT encode functions related to motility, biofilm formation, or c-di-GMP metabolism. Most notably, VpsT activates expression of the vpvABC operon that encodes a diguanylate cyclase. This creates a positive feedback loop needed to maintain intracellular levels of c-di-GMP. Mutation of the key VpsT binding site, upstream of vpvABC, severs the loop and c-di-GMP levels fall accordingly. Hence, as well as relaying the c-di-GMP signal, VpsT impacts c-di-GMP homeostasis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Pseudomonas putida KT2440, cfcR encodes an orphan multidomain response regulator with diguanylate cyclase activity, which is responsible for the synthesis of c-di-GMP, a second messenger key in the transition from planktonic to sessile bacterial lifestyles. When overexpressed, cfcR enhances biofilm formation and causes other phenotype alterations. The cfcA gene, encoding a membrane-anchored multisensory CHASE3/GAF hybrid histidine kinase (HK), is required to develop this pleiotropic phenotype. Here we show autophosphorylation of CfcA through HisKA/HATPase_c domains and then transfer of the phosphoryl group to an internal receiver (REC) domain. CfcA REC domains are nonessential for phosphotransfer from CfcA~P to the REC domain of CfcR. CfcA~P also phosphorylates the REC domain of CfcD, a second HK encoded in the same gene cluster as CfcA, which negatively regulates the CfcA/CfcR pathway. To evaluate the impact of CfcA domains on CfcR activity, a battery of mutants with in-frame domain deletions was generated, whose CfcA protein locations were also examined. CfcA membrane anchorage contributes to protein stability and CfcR activation. Salt enhances c-di-GMP levels through CfcR, a response which is hampered by alteration of a presumed ligand-binding motif in the CHASE3 sensor domain. Thus, in P. putida, c-di-GMP is salt-regulated through the CfcA/CfcR/CfcD system., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A major target of c-di-GMP signaling is the production of biofilm-associated extracellular polymeric substances (EPS), which in Escherichia coli K-12 include amyloid curli fibers, phosphoethanolamine-modified cellulose, and poly- N -acetylglucosamine. However, the characterized c-di-GMP-binding effector systems are largely outnumbered by the 12 diguanylate cyclases (DGCs) and 13 phosphodiesterases (PDEs), which synthetize and degrade c-di-GMP, respectively. E. coli possesses a single protein with a potentially c-di-GMP-binding MshEN domain, NfrB, which-together with the outer membrane protein NfrA-is known to serve as a receptor system for phage N4. Here, we show that NfrB not only binds c-di-GMP with high affinity but, as a novel c-di-GMP-controlled glycosyltransferase, synthesizes a secreted EPS, which can impede motility and is required as an initial receptor for phage N4 infection. In addition, a systematic screening of the 12 DGCs of E. coli K-12 revealed that specifically DgcJ is required for the infection with phage N4 and interacts directly with NfrB. This is in line with local signaling models, where specific DGCs and/or PDEs form protein complexes with particular c-di-GMP effector/target systems. Our findings thus provide further evidence that intracellular signaling pathways, which all use the same diffusible second messenger, can act in parallel in a highly specific manner. IMPORTANCE Key findings in model organisms led to the concept of "local" signaling, challenging the dogma of a gradually increasing global intracellular c-di-GMP concentration driving the motile-sessile transition in bacteria. In our current model, bacteria dynamically combine both global and local signaling modes, in which specific DGCs and/or PDEs team up with effector/target systems in multiprotein complexes. The present study highlights a novel example of how specificity in c-di-GMP signaling can be achieved by showing NfrB as a novel c-di-GMP binding effector in E. coli, which is controlled in a local manner specifically by DgcJ. We further show that NfrB (which was initially found as a part of a receptor system for phage N4) is involved in the production of a novel exopolysaccharide. Finally, our data shine new light on host interaction of phage N4, which uses this exopolysaccharide as an initial receptor for adsorption.

The marine bacterium Vibrio fischeri colonizes its host, the Hawaiian bobtail squid, in a manner requiring both bacterial biofilm formation and motility. The decision to switch between sessile and motile states is often triggered by environmental signals and regulated by the widespread signaling molecule c-di-GMP. Calcium is an environmental signal previously shown to affect both biofilm formation and motility by V. fischeri. In this study, we investigated the link between calcium and c-di-GMP, determining that calcium increases intracellular c-di-GMP dependent on a specific diguanylate cyclase, ca lcium- s ensing protein A (CasA). CasA is activated by calcium, dependent on residues in an N-terminal sensory domain, and synthesizes c-di-GMP through an enzymatic C-terminal domain. CasA is responsible for calcium-dependent inhibition of motility and activation of cellulose-dependent biofilm formation. Calcium regulates cellulose biofilms at the level of transcription, which also requires the transcription factor VpsR. Finally, the Vibrio cholerae CasA homolog, CdgK, is unable to complement CasA and may be inhibited by calcium. Collectively, these results identify CasA as a calcium-responsive regulator, linking an external signal to internal decisions governing behavior, and shed light on divergence between Vibrio spp. IMPORTANCE Biofilm formation and motility are often critical behaviors for bacteria to colonize a host organism. Vibrio fischeri is the exclusive colonizer of its host's symbiotic organ and requires both biofilm formation and motility to initiate successful colonization, providing a relatively simple model to explore complex behaviors. In this study, we determined how the environmental signal calcium alters bacterial behavior through production of the signaling molecule c-di-GMP. Calcium activates the diguanylate cyclase CasA to synthesize c-di-GMP, resulting in inhibition of motility and activation of cellulose production. These activities depend on residues in CasA's N-terminal sensory domain and C-terminal enzymatic domain. These findings thus identify calcium as a signal recognized by a specific diguanylate cyclase to control key bacterial phenotypes. Of note, CasA activity is seemingly inverse to that of the homologous V. cholerae protein, CdgK, providing insight into evolutionary divergence between closely related species.

Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O 2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria., Competing Interests: Declaration of interests R.S. is a scientific cofounder and advisor of BiomX, Pantheon Bioscience, and Ecophage., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli, and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-GMP (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. c-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri. In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae, in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation and reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes. IMPORTANCE The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance among Shigella species is on the rise. Here, we explored how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We found that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella 's ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

The striking multiplicity, signal input diversity, and output specificity of c-di-GMP signaling proteins in many bacteria has brought second messenger signaling back onto the agenda of contemporary microbiology. How can several signaling pathways act in parallel in a specific manner if all of them use the same diffusible second messenger present at a certain global cellular concentration? Recent research has now shown that bacteria achieve this by flexibly combining modes of local and global c-di-GMP signaling in complex signaling networks. Three criteria have to be met to define local c-di-GMP signaling: specific knockout phenotypes, direct interactions between proteins involved, and actual cellular c-di-GMP levels remaining below the K d of effectors. Adaptive changes in signaling network architecture can further enhance signaling flexibility., Competing Interests: Declaration of interests There are no interests to declare., (Copyright © 2021. Published by Elsevier Ltd.)

The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium -soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis. IMPORTANCE The ubiquitous second messenger c-di-GMP is well-known for its role in biofilm formation and host adaptation of pathogens, whereas it is less investigated in mutualistic symbioses. Here, we reveal a cocktail of eight functional diguanylate cyclases (DGCs) catalyzing the biosynthesis of c-di-GMP in a broad-host-range Sinorhizobium that can establish nitrogen-fixing nodules on soybean and many other legumes. These functional DGCs are generally transcribed at low levels in soybean nodules compared to free-living conditions. The engineered nodule-specific upregulation of DGC can elevate the c-di-GMP level and cause symbiotic defects, while the upregulation of a phosphodiesterase that quenches c-di-GMP has no detectable symbiotic defects. Moreover, eight functional DGCs located on two different replicons are all directly repressed in nodules by a global silencer, MucR1, that is essential for Sinorhizobium -soybean symbiosis. These findings represent a novel mechanism of a strategic regulation of the c-di-GMP biosynthesis arsenal in prokaryote-eukaryote interactions.

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

Cyanobacteriochromes (CBCRs) are spectrally diverse photosensors from cyanobacteria distantly related to phytochromes that exploit photoisomerization of linear tetrapyrrole (bilin) chromophores to regulate associated signaling output domains. Unlike phytochromes, a single CBCR domain is sufficient for photoperception. CBCR domains that regulate the production or degradation of cyclic nucleotide second messengers are becoming increasingly well characterized. Cyclic di-guanosine monophosphate (c-di-GMP) is a widespread small-molecule regulator of bacterial motility, developmental transitions, and biofilm formation whose biosynthesis is regulated by CBCRs coupled to GGDEF (diguanylate cyclase) output domains. In this study, we compare the properties of diverse CBCR-GGDEF proteins with those of synthetic CBCR-GGDEF chimeras. Our investigation shows that natural diversity generates promising candidates for robust, broad spectrum optogenetic applications in live cells. Since light quality is constantly changing during plant development as upper leaves begin to shade lower leaves-affecting elongation growth, initiation of flowering, and responses to pathogens, these studies presage application of CBCR-GGDEF sensors to regulate orthogonal, c-di-GMP-regulated circuits in agronomically important plants for robust mitigation of such deleterious responses under natural growing conditions in the field., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mammary gland-derived Escherichia coli is an important pathogen causing dairy cow mastitis. Mammary gland mucosal immunity against infectious E. coli mainly depends on recognition of pathogen-associated molecular patterns by innate receptors. Stimulator of interferon (IFN) gene (STING) has recently been the dominant mediator in reacting to bacterial intrusion and preventing inflammatory disorders. In this study, we first proved that the diguanylate cyclase YeaJ relieves mouse mammary gland pathological damage by changing E. coli phenotypic and host STING-dependent innate immunity responses. YeaJ decreases mammary gland circular vacuoles, bleeding, and degeneration in mice. In addition, YeaJ participates in STING-IRF3 signaling to regulate inflammation in vivo . In vitro , YeaJ decreases damage to macrophages (RAW264.7) but not to mouse mammary epithelial cells (EpH4-Ev). Consistent with the results in mouse mammary glands, YeaJ significantly activates the STING/TBK1/IRF3 pathway in RAW264.7 macrophages as well. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo . This study highlights a novel role for YeaJ in E. coli infection, which provides a better understanding of host-bacterium interactions and potential prophylactic strategies for infections. IMPORTANCE E. coli is the etiological agent of environmental mastitis in dairy cows, which causes massive financial losses worldwide. However, the pathophysiological role of YeaJ in the interaction between E. coli and host remains unclear. We found that YeaJ significantly influences various biological characteristics and suppresses severe inflammatory response as well as greater damage. YeaJ alleviates damage to macrophages (RAW264.7) and mouse mammary gland. Moreover, these effects of YeaJ are achieved at least partial by mediating the STING-IRF3 signaling pathway. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo . This study is the basis for further research to better understand host-bacterium interactions and provides potential prophylactic strategies for infections.

Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in Pseudomonas aeruginosa . Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling., Competing Interests: GC, YZ, WH, JP, ML, YZ, TW, LZ, LZ, HL None, JZ none, (© 2021, Chen et al.)

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) from Paenibacillus 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)-O 2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in 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., Competing Interests: The authors declare no competing interest.

Aloe polysaccharides (APs) are indigestible bioactive polysaccharides, while can be fermented by colonic microbiota. Although plant polysaccharides can alleviate subacute ulcerative colitis (SUC), the mechanisms APs regulated SUC via colonic microbiota have not been fully explored. Hence, to elucidate the complex interactions between the novel APs, colonic microbiota, SCFAs, and inflammation, the SUC mouse model and in-depth analysis were performed, including multiple bioinformatics analysis and structural equation modeling (SEM). After APs intervention, SCFAs and SCFAs-producing genus, including Akkermansia and Blautia, were increased in colon, and the colonic inflammation and barrier dysfunction were alleviated significantly in SUC mice. Spearman analysis found positive correlations between microbiota and SCFAs. PICRUSt2 and KEGG analysis revealed 6-pyruvoyltetra hydropterin synthase in folate biosynthesis metabolism pathway was activated, while phosphotransferase system was inhibited. SEM results further proved APs was beneficial to gut micro-ecological balance in mice via SCFAs metabolism and anti-inflammatory functions. Together, APs could be exploited to alleviate SUC as dietary therapeutics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Streptococcus mitis is a commensal bacterial species of the oral cavity, with the potential for opportunistic pathogenesis. For successful colonization, S. mitis must be able to adhere to surfaces of the oral cavity and survive and adapt to frequently changing environmental conditions. Cyclic-di-AMP (c-di-AMP) is a nucleotide second messenger, involved in the regulation of stress responses and biofilm formation in several bacterial species. Cyclic-di-AMP is produced by diadenylate cyclases and degraded by phosphodiesterases. We have previously shown that in S. mitis, one diadenylate cyclase (CdaA) and at least two phosphodiesterases (Pde1 and Pde2) regulate the intracellular concentration of c-di-AMP. In this study, we utilized S. mitis deletion mutants of cdaA, pde1, and pde2 to analyze the role of c-di-AMP signaling in various stress responses, biofilm formation, and adhesion to eukaryotic cells. Here, we demonstrate that the Δpde1 mutant displayed a tendency toward increased susceptibility to acetic acid at pH 4.0. Deletion of cdaA increases auto-aggregation of S. mitis but reduces biofilm formation on an abiotic surface. These phenotypes are more pronounced under acidic extracellular conditions. Inactivation of pde1 or pde2 reduced the tolerance to ciprofloxacin, and UV radiation and the Δpde1 mutant was more susceptible to Triton X-100, indicating a role for c-di-AMP signaling in responses to DNA damage and cell membrane perturbation. Finally, the Δpde2 mutant displayed a tendency toward a reduced ability to adhere to oral keratinocytes. Taken together, our results indicate an important role for c-di-AMP signaling in cellular processes important for colonization of the mouth., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Protein dynamics play a major role for the catalytic function of enzymes, the interaction of protein complexes or signal integration in regulatory proteins. In the context of multi-domain proteins involved in light-regulation of enzymatic effectors, the central role of conformational dynamics is well established. Light activation of sensory modules is followed by long-range signal transduction to different effectors; rather than domino-style structural rearrangements, a complex interplay of functional elements is required to maintain functionality. One family of such sensor-effector systems are red-light-regulated phytochromes that control diguanylate cyclases involved in cyclic-dimeric-GMP formation. Based on structural and functional studies of one prototypic family member, the central role of the coiled-coil sensor-effector linker was established. Interestingly, subfamilies with different linker lengths feature strongly varying biochemical characteristics. The dynamic interplay of the domains involved, however, is presently not understood. Here we show that the PHY domain dimer interface plays an essential role in signal integration, and that a functional coupling with the coiled-coil linker element is crucial. Chimaeras of two biochemically different family members highlight the phytochrome-spanning helical spine as an essential structural element involved in light-dependent upregulation of enzymatic turnover. However, isolated structural elements can frequently not be assigned to individual characteristics, which further emphasises the importance of global conformational dynamics. Our results provide insights into the intricate processes at play during light signal integration and transduction in these photosensory systems and thus provide additional guidelines for a more directed design of novel sensor-effector combinations with potential applications as optogenetic tools., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Dietary lipid composition has been shown to impact brain morphology, brain development, and neurologic function. However, how diet uniquely regulates brain lipid homeostasis compared with lipid homeostasis in peripheral tissues remains largely uncharacterized. To evaluate the lipid response to dietary changes in the brain, we assessed actively translating mRNAs in astrocytes and neurons across multiple diets. From this data, ethanolamine phosphate phospholyase (Etnppl) was identified as an astrocyte-specific fasting-induced gene. Etnppl catabolizes phosphoethanolamine (PEtN), a prominent headgroup precursor in phosphatidylethanolamine (PE) also found in other classes of neurologically relevant lipid species. Altered Etnppl expression has also previously been associated with humans with mood disorders. We evaluated the relevance of Etnppl in maintaining brain lipid homeostasis by characterizing Etnppl across development and in coregulation with PEtN-relevant genes, as well as determining the impact to the brain lipidome after Etnppl loss. We found that Etnppl expression dramatically increased during a critical window of early brain development in mice and was also induced by glucocorticoids. Using a constitutive knockout of Etnppl (Etnppl KO ), we did not observe robust changes in expression of PEtN-related genes. However, loss of Etnppl altered the phospholipid profile in the brain, resulting in increased total abundance of PE and in polyunsaturated fatty acids within PE and phosphatidylcholine species in the brain. Together, these data suggest that brain phospholipids are regulated by the phospholyase action of the enzyme Etnppl, which is induced by dietary fasting in astrocytes., Competing Interests: Conflict of interest The authors have no competing financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no competing interest.

R elapsing f ever (RF), caused by spirochetes of the genus Borrelia , is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. C yclic di - AMP (c-di-AMP), synthesized by d i a denylate c yclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium Borrelia turicatae , a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole-genome sequencing was performed on cdaA mutants, and single-nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium Borrelia burgdorferi Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes., (Copyright © 2021 Jackson-Litteken et al.)

In the soil gammaproteobacterium Lysobacter enzymogenes, a natural fungal predator, the response regulator PilR controls type IV pili (T4P)-mediated twitching motility as well as synthesis of the heat-stable antifungal factor (HSAF). Earlier we showed that PilR acts via the second messenger, c-di-GMP; however, the mechanism remained unknown. Here, we describe how PilR, c-di-GMP signalling, and HSAF synthesis are connected. We screened genes for putative diguanylate cyclases (c-di-GMP synthases) and found that PilR binds to the promoter region of lchD and down-regulates its transcription. The DNA-binding affinity of PilR, and therefore its repressor function, are enhanced by phosphorylation by its cognate histidine kinase, PilS. The lchD gene product is a diguanylate cyclase, and the decrease in LchD levels shifts the ratio of c-di-GMP-bound and c-di-GMP-free transcription factor Clp, a key activator of the HSAF biosynthesis operon expression. Furthermore, Clp directly interacts with LchD and enhances its diguanylate cyclase activity. Therefore, the PilS-PilR two-component system activates T4P-motility while simultaneously decreasing c-di-GMP levels and promoting HSAF production via the highly specific LchD-c-di-GMP-Clp pathway. Coordinated increase in motility and secretion of the "long-distance" antifungal weapon HSAF is expected to ensure safer grazing of L. enzymogenes on soil or plant surfaces, unimpeded by fungal competitors, or to facilitate bacterial preying on killed fungal cells. This study uncovered the mechanism of coregulated pili-based motility and production of an antifungal antibiotic in L. enzymogenes, showcased the expanded range of functions of the PilS-PilR system, and highlighted exquisite specificity in c-di-GMP-mediated circuits., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5'-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of diguanylate cyclases. Full-length crystal structures reveal that BeF 3 - pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many diguanylate cyclases with N-terminal coiled-coil linkers.