Control of biofilm formation by an Agrobacterium tumefaciens pterin-binding periplasmic protein conserved among diverse Proteobacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages.

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution.

Monitoring changes of signaling molecules and metabolites with high temporal resolution is key to understanding dynamic biological systems. Here, we use directed evolution to develop a genetically encoded ratiometric biosensor for c-di-GMP, a ubiquitous bacterial second messenger regulating important biological processes like motility, surface attachment, virulence and persistence. The resulting biosensor, cdGreen2, faithfully tracks c-di-GMP in single cells and with high temporal resolution over extended imaging times, making it possible to resolve regulatory networks driving bimodal developmental programs in different bacterial model organisms. We further adopt cdGreen2 as a simple tool for in vitro studies, facilitating high-throughput screens for compounds interfering with c-di-GMP signaling and biofilm formation. The sensitivity and versatility of cdGreen2 could help reveal c-di-GMP dynamics in a broad range of microorganisms with high temporal resolution. Its design principles could also serve as a blueprint for the development of similar, orthogonal biosensors for other signaling molecules, metabolites and antibiotics.

A Label-Free Approach for Relative Spatial Quantitation of c-di-GMP in Microbial Biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.

Regulatory roles of the second messenger c-di-GMP in beneficial plant-bacteria interactions.

The rhizosphere system of plants hosts a diverse consortium of bacteria that confer beneficial effects on plant, such as plant growth-promoting rhizobacteria (PGPR), biocontrol agents with disease-suppression activities, and symbiotic nitrogen fixing bacteria with the formation of root nodule. Efficient colonization in planta is of fundamental importance for promoting of these beneficial activities. However, the process of root colonization is complex, consisting of multiple stages, including chemotaxis, adhesion, aggregation, and biofilm formation. The secondary messenger, c-di-GMP (cyclic bis-(3'-5') dimeric guanosine monophosphate), plays a key regulatory role in a variety of physiological processes. This paper reviews recent progress on the actions of c-di-GMP in plant beneficial bacteria, with a specific focus on its role in chemotaxis, biofilm formation, and nodulation.

ccdC Regulates Biofilm Dispersal in Bacillus velezensis FZB42.

Bacillus velezensis FZB42 is a plant growth-promoting rhizobacterium (PGPR) and a model microorganism for biofilm studies. Biofilms are required for the colonization and promotion of plant growth in the rhizosphere. However, little is known about how the final stage of the biofilm life cycle is regulated, when cells regain their motility and escape the mature biofilm to spread and colonize new niches. In this study, the non-annotated gene ccdC was found to be involved in the process of biofilm dispersion. We found that the ccdC-deficient strain maintained a wrinkled state at the late stage of biofilm formation in the liquid-gas interface culture, and the bottom solution showed a clear state, indicating that no bacterial cells actively escaped, which was further evidenced by the formation of a cellular ring (biofilm pellicle) located on top of the preformed biofilm. It can be concluded that dispersal, a biofilm property that relies on motility proficiency, is also positively affected by the unannotated gene ccdC. Furthermore, we found that the level of cyclic diguanylate (c-di-GMP) in the ccdC-deficient strain was significantly greater than that in the wild-type strain, suggesting that B. velezensis exhibits a similar mechanism by regulating the level of c-di-GMP, the master regulator of biofilm formation, dispersal, and cell motility, which controls the fitness of biofilms in Pseudomonas aeruginosain. In this study, we investigated the mechanism regulating biofilm dispersion in PGPR.

Cultivation of phosphate-accumulating biofilm: Study of the effects of acyl-homoserine lactones (AHLs) and cyclic dimeric guanosine monophosphate (c-di-GMP) on the formation of biofilm and the enhancement of phosphate metabolism capacity.

This study investigated the mechanisms of microbial growth and metabolism during biofilm cultivation in the biofilm sequencing batch reactor (BSBR) process for phosphate (P) enrichment. The results showed that the sludge discharge was key to biofilm growth, as it terminated the competition for carbon (C) source between the nascent biofilm and the activated sludge. For the tested reactor, after the sludge discharge on 18 d, P metabolism and C source utilization improved significantly, and the biofilm grew rapidly. The P concentration of the recovery liquid reached up to 157.08 mg/L, which was sufficient for further P recovery via mineralization. Meta-omics methods were used to analyze metabolic pathways and functional genes in microbial growth during biofilm cultivation. It appeared that the sludge discharge activated the key genes of P metabolism and inhibited the key genes of C metabolism, which strengthened the polyphosphate-accumulating metabolism (PAM) as a result. The sludge discharge not only changed the types of polyphosphate-accumulating organisms (PAOs) but also promoted the growth of dominant PAOs. Before the sludge discharge, the necessary metabolic abilities that were spread among different microorganisms gradually concentrated into a small number of PAOs, and after the sludge discharge, they further concentrated into Candidatus_Contendobacter (P3) and Candidatus_Accumulibacter (P17). The messenger molecule C-di-GMP, produced mostly by P3 and P17, facilitated P enrichment by regulating cellular P and C metabolism. The glycogen-accumulating organism (GAO) Candidatus_Competibacter secreted N-Acyl homoserine lactones (AHLs), which stimulated the secretion of protein in extracellular polymeric substances (EPS), thus promoting the adhesion of microorganisms to biofilm and improving P metabolism via EPS-based P adsorption. Under the combined action of the dominant GAOs and PAOs, AHLs and C-di-GMP mediated QS to promote biofilm development and P enrichment. The research provides theoretical support for the cultivation of biofilm and its wider application.

Bacterial c-di-GMP signaling gene affects mussel larval metamorphosis through outer membrane vesicles and lipopolysaccharides.

Biofilms serve as crucial cues for settlement and metamorphosis in marine invertebrates. Within bacterial systems, c-di-GMP functions as a pivotal signaling molecule regulating both biofilm formation and dispersion. However, the molecular mechanism of how c-di-GMP modulates biofilm-induced larval metamorphosis remains elusive. Our study reveals that the deletion of a c-di-GMP related gene in Pseudoalteromonas marina led to an increase in the level of bacterial c-di-GMP by knockout technique, and the mutant strain had an enhanced ability to produce more outer membrane vesicles (OMVs) and lipopolysaccharides (LPS). The mutant biofilms had higher induction activity for larval metamorphosis in mussels Mytilus coruscus, and OMVs play a major role in the induction activity. We further explored the function of LPS in OMVs. Extracted LPS induced high larval metamorphosis rate, and LPS content were subject to c-di-GMP and LPS-biosynthesis gene. Thus, we postulate that the impact of c-di-GMP on biofilm-induced metamorphosis is mediated through OMVs and LPS.

Decoding the influence of low temperature on biofilm development: The hidden roles of c-di-GMP.

Biofilms are widely used and play important roles in biological processes. Low temperature of wastewater inhibits the development of biofilms derived from wastewater activated sludge. However, the specific mechanism of temperature on biofilm development is still unclear. This study explored the mechanism of temperature on biofilm development and found a feasible method to enhance biofilm development at low temperature. The amount of biofilm development decreased by approximately 66 % and 55 % at 4 °C and 15 °C, respectively, as compared to 28 °C. The cyclic dimeric guanosine monophosphate (c-di-GMP) concentration also decreased at low temperature and was positively correlated with extracellular polymeric substance (EPS) content, formation, and adhesion strength. Microbial community results showed that low temperature inhibited the normal survival of most microorganisms, but promoted the growth of some psychrophile bacteria like Sporosarcina, Caldilineaceae, Gemmataceae, Anaerolineaceae and Acidobacteriota. Further analysis of functional genes demonstrated that the abundance of functional genes related to the synthesis of c-di-GMP (K18968, K18967 and K13590) decreased at low temperature. Subsequently, the addition of exogenous spermidine increased the level of intracellular c-di-GMP and alleviated the inhibition effect of low temperature on biofilm development. Therefore, the possible mechanism of low temperature on biofilm development could be the inhibition of the microorganism activity and reduction of the communication level between cells, which is the closely related to the EPS content, formation, and adhesion strength. The enhancement of c-di-GMP level through the exogenous addition of spermidine provides an alternative strategy to enhance biofilm development at low temperatures. The results of this study enhance the understanding of the influence of temperature on biofilm development and provide possible strategies for enhancing biofilm development at low temperatures.

Local control: a hub-based model for the c-di-GMP network.

The genome of Pseudomonas fluorescens encodes >50 proteins predicted to play a role in bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)-mediated biofilm formation. We built a network representation of protein-protein interactions and extracted key information via multidimensional scaling (i.e., principal component analysis) of node centrality measures, which measure features of proteins in a network. Proteins of different domain types (diguanylate cyclase, dual domain, phosphodiesterase, PilZ) exhibit unique network behavior and can be accurately classified by their network centrality values (i.e., roles in the network). The predictive power of protein-protein interactions in biofilm formation indicates the possibility of localized pools of c-di-GMP. A regression model showed a statistically significant impact of protein-protein interactions on the extent of biofilm formation in various environments. These results highlight the importance of a localized c-di-GMP signaling, extend our understanding of signaling by this second messenger beyond the current "Bow-tie Model," support a newly proposed "Hub Model," and suggest future avenues of investigation.

Bacterial synthase-dependent exopolysaccharide secretion: a focus on cellulose.

Bacterial biofilms are a prevalent multicellular life form in which individual members can undergo significant functional differentiation and are typically embedded in a complex extracellular matrix of proteinaceous fimbriae, extracellular DNA, and exopolysaccharides (EPS). Bacteria have evolved at least four major mechanisms for EPS biosynthesis, of which the synthase-dependent systems for bacterial cellulose secretion (Bcs) represent not only key biofilm determinants in a wide array of environmental and host-associated microbes, but also an important model system for the studies of processive glycan polymerization, cyclic diguanylate (c-di-GMP)-dependent synthase regulation, and biotechnological polymer applications. The secreted cellulosic chains can be decorated with additional chemical groups or can pack with various degrees of crystallinity depending on dedicated enzymatic complexes and/or cytoskeletal scaffolds. Here, I review recent progress in our understanding of synthase-dependent EPS biogenesis with a focus on common and idiosyncratic molecular mechanisms across diverse cellulose secretion systems.

Environmental purines decrease Pseudomonas aeruginosa biofilm formation by disrupting c-di-GMP metabolism.

Cyclic di-guanosine monophosphate (c-di-GMP) is a bacterial second messenger that governs the lifestyle switch between planktonic and biofilm states. While substantial investigation has focused on the proteins that produce and degrade c-di-GMP, less attention has been paid to the potential for metabolic control of c-di-GMP signaling. Here, we show that micromolar levels of specific environmental purines unexpectedly decrease c-di-GMP and biofilm formation in Pseudomonas aeruginosa. Using a fluorescent genetic reporter, we show that adenosine and inosine decrease c-di-GMP even when competing purines are present. We confirm genetically that purine salvage is required for c-di-GMP decrease. Furthermore, we find that (p)ppGpp prevents xanthosine and guanosine from producing an opposing c-di-GMP increase, reinforcing a salvage hierarchy that favors c-di-GMP decrease even at the expense of growth. We propose that purines can act as a cue for bacteria to shift their lifestyle away from the recalcitrant biofilm state via upstream metabolic control of c-di-GMP signaling.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

Biofilm formation of Hafnia paralvei induced by c-di-GMP through facilitating bcsB gene expression promotes spoilage of Yellow River carp (Cyprinus carpio).

Hafnia paralvei, a Gram-negative foodborne pathogen, is found ubiquitously in various aquatic animals and seafoods, which can form biofilm as a dominant virulence factor that contributes to its pathogenesis. However, the biofilm formation mechanism of H. paralvei and its effect on food spoilage has not been fully characterized. Here we show that biofilm formation, is regulated by c-di-GMP which mediated by bcsB, can increase the spoilage ability of H. paralvei. We found that GTP was added exogenously to enhance the synthesis of c-di-GMP, which further promoted biofilm formation. The gene dgcC, one of 11 genes encoding GGDEF domain-containing proteins in H. paralvei, was significantly upregulated with GTP as substrate. The upregulation of dgcC contributes to a significant increase of c-di-GMP and the formation of biofilm. In addition, the overexpression of dgcC induced upregulation of bcsB, a reported effector protein encoding gene, which was further demonstrated that overexpression of bcsB can encourage the synthesis of bacterial cellulose and biofilm formation. The effect of biofilm formation induced by c-di-GMP on spoilage of Yellow River carp (Cyprinus carpio) was evaluated by sensory evaluation, the total viable count, and the total volatile basic nitrogen, which showed that biofilm formation can significantly increase the spoilage ability of H. paralvei on C. carpio. Our findings provide the regulation of c-di-GMP on expression of bcsB, that can contribute to biofilm formation and spoilage ability of H. paralvei, which is favor to understanding the pathogenesis of Hafnia paralvei and its role in food spoilage.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

A c-di-GMP binding effector STM0435 modulates flagellar motility and pathogenicity in Salmonella.

Flagella play a crucial role in the invasion process of Salmonella and function as a significant antigen that triggers host pyroptosis. Regulation of flagellar biogenesis is essential for both pathogenicity and immune escape of Salmonella. We identified the conserved and unknown function protein STM0435 as a new flagellar regulator. The ∆stm0435 strain exhibited higher pathogenicity in both cellular and animal infection experiments than the wild-type Salmonella. Proteomic and transcriptomic analyses demonstrated dramatic increases in almost all flagellar genes in the ∆stm0435 strain compared to wild-type Salmonella. In a surface plasmon resonance assay, purified STM0435 protein-bound c-di-GMP had an affinity of ~8.383 µM. The crystal structures of apo-STM0435 and STM0435&c-di-GMP complex were determined. Structural analysis revealed that R33, R137, and D138 of STM0435 were essential for c-di-GMP binding. A Salmonella with STM1987 (GGDEF protein) or STM4264 (EAL protein) overexpression exhibits completely different motility behaviours, indicating that the binding of c-di-GMP to STM0435 promotes its inhibitory effect on Salmonella flagellar biogenesis.

A c-di-GMP signaling module controls responses to iron in Pseudomonas aeruginosa.

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a bacterial second messenger that modulates various processes including biofilm formation, motility, and host-microbe symbiosis. Numerous studies have conducted comprehensive analysis of c-di-GMP. However, the mechanisms by which certain environmental signals such as iron control intracellular c-di-GMP levels are unclear. Here, we show that iron regulates c-di-GMP levels in Pseudomonas aeruginosa by modulating the interaction between an iron-sensing protein, IsmP, and a diguanylate cyclase, ImcA. Binding of iron to the CHASE4 domain of IsmP inhibits the IsmP-ImcA interaction, which leads to increased c-di-GMP synthesis by ImcA, thus promoting biofilm formation and reducing bacterial motility. Structural characterization of the apo-CHASE4 domain and its binding to iron allows us to pinpoint residues defining its specificity. In addition, the cryo-electron microscopy structure of ImcA in complex with a c-di-GMP analog (GMPCPP) suggests a unique conformation in which the compound binds to the catalytic pockets and to the membrane-proximal side located at the cytoplasm. Thus, our results indicate that a CHASE4 domain directly senses iron and modulates the crosstalk between c-di-GMP metabolic enzymes.

Essential role of ROS - 8-Nitro-cGMP signaling in long-term memory of motor learning and cerebellar synaptic plasticity.

Although reactive oxygen species (ROS) are known to have harmful effects in organisms, recent studies have demonstrated expression of ROS synthases at various parts of the organisms and the controlled ROS generation, suggesting possible involvement of ROS signaling in physiological events of individuals. However, physiological roles of ROS in the CNS, including functional roles in higher brain functions or neuronal activity-dependent ROS production, remain to be elucidated. Here, we demonstrated involvement of ROS - 8-NO2-cGMP signaling in motor learning and synaptic plasticity in the cerebellum. In the presence of inhibitors of ROS signal or ROS synthases, cerebellar motor learning was impaired, and the stimulus inducing long-term depression (LTD), cellular basis for the motor learning, failed to induce LTD but induced long-term potentiation (LTP)-like change at cerebellar synapses. Furthermore, ROS was produced by LTD-inducing stimulus in enzyme-dependent manner, and excess administration of the antioxidant vitamin E impaired cerebellar motor learning, suggesting beneficial roles of endogenous ROS in the learning. As a downstream signal, involvement of 8-NO2-cGMP in motor learning and cerebellar LTD were also revealed. These findings indicate that ROS - 8-NO2-cGMP signal is activated by neuronal activity and is essential for cerebellum-dependent motor learning and synaptic plasticity, demonstrating involvement of the signal in physiological function of brain systems.

Initial cyclic-di-GMP upregulation triggers sporadic cellular expansion leading to improved cellular survival.

Bacterial second messenger c-di-GMP upregulation is associated with the transition from planktonic to sessile microbial lifestyle, inhibiting cellular motility, and virulence. However, in-depth elucidation of the cellular processes resulting from c-di-GMP upregulation has not been fully explored. Here, we report the role of upregulated cellular c-di-GMP in promoting planktonic cell growth of Escherichia coli K12 and Pseudomonas aeruginosa PAO1. We found a rapid expansion of cellular growth during initial cellular c-di-GMP upregulation, resulting in a larger planktonic bacterial population. The initial increase in c-di-GMP levels promotes bacterial swarming motility during the growth phase, which is subsequently inhibited by the continuous increase of c-di-GMP, and ultimately facilitates the formation of biofilms. We demonstrated that c-di-GMP upregulation triggers key bacterial genes linked to bacterial growth, swarming motility, and biofilm formation. These genes are mainly controlled by the master regulatory genes csgD and csrA. This study provides us a glimpse of the bacterial behavior of evading potential threats through adapting lifestyle changes via c-di-GMP regulation.