Cyclic Diguanosine Monophosphate Research Articles

Extracellular c-di-GMP Plays a Role in Biofilm Formation and Dispersion of Campylobacter jejuni.

Cyclic diguanosine monophosphate (c-diGMP) is a ubiquitous second messenger involved in the regulation of many signalling systems in bacteria, including motility and biofilm formation. Recently, it has been reported that c-di-GMP was detected in C. jejuni DRH212; however, the presence and the role of c-di-GMP in other C. jejuni strains are unknown. Here, we investigated extracellular c-di-GMP as an environmental signal that potentially triggers biofilm formation in C. jejuni NCTC 11168 using a crystal violet-based assay, motility-based plate assay, RT-PCR and confocal laser scanning microscopy (CLSM). We found that, in presence of extracellular c-di-GMP, the biofilm formation was significantly reduced (>50%) and biofilm dispersion enhanced (up to 60%) with no effect on growth. In addition, the presence of extracellular c-di-GMP promoted chemotactic motility, inhibited the adherence of C. jejuni NCTC 11168-O to Caco-2 cells and upregulated the expression of Cj1198 (luxS, encoding quarum sensing pathway component, autoinducer-2), as well as chemotaxis genes Cj0284c (cheA) and Cj0448c (tlp6). Unexpectedly, the expression of Cj0643 (cbrR), containing a GGDEF-like domain and recently identified as a potential diguanylate cyclase gene, required for the synthesis of c-di-GMP, was not affected. Our findings suggest that extracellular c-di-GMP could be involved in C. jejuni gene regulation, sensing and biofilm dispersion.

Elongation factor P modulates Acinetobacter baumannii physiology and virulence as a cyclic dimeric guanosine monophosphate effector

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.

Single-molecule investigations of single-chain cellulose biosynthesis

Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.

Research progress of c-di-GMP in the regulation of Escherichia coli biofilm

Escherichia coli biofilm is a complex membrane aggregation produced by the adhesion and secretion of extracellular polymeric substances by E. coli cells aggregated on specific media. Pathogenic E. coli will evade the immune system and the impact of various harmful factors in the environment after the formation of biofilm, causing sustained and even fatal damage to the host. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger ubiquitous in bacteria and plays a crucial role in regulating biofilm formation. This paper reviewed the recent studies about the role of c-di-GMP in the movement, adhesion, and EPS production mechanism of E. coli during biofilm formation, aiming to provide a basis for inhibiting E. coli biofilm from the perspective of c-di-GMP.

Microparticle Delivery of a STING Agonist Enables Indirect Activation of NK Cells by Antigen-Presenting Cells.

Natural killer (NK) cells are an important member of the innate immune system and can participate in direct tumor cell killing in response to immunotherapies. One class of immunotherapy is stimulator of interferon gene (STING) agonists, which result in a robust type I interferon (IFN-I) response. Most mechanistic studies involving STING have focused on macrophages and T cells. Nevertheless, NK cells are also activated by IFN-I, but the effect of STING activation on NK cells remains to be adequately investigated. We show that both direct treatment with soluble STING agonist cyclic di-guanosine monophosphate-adenosine monophosphate (cGAMP) and indirect treatment with cGAMP encapsulated in microparticles (MPs) result in NK cell activation in vitro, although the former requires 100× more cGAMP than the latter. Additionally, direct activation with cGAMP leads to NK cell death. Indirect activation with cGAMP MPs does not result in NK cell death but rather cell activation and cell killing in vitro. In vivo, treatment with soluble cGAMP and cGAMP MPs both cause short-term activation, whereas only cGAMP MP treatment produces long-term changes in NK cell activation markers. Thus, this work indicates that treatment with an encapsulated STING agonist activates NK cells more efficiently than that with soluble cGAMP. In both the in vitro and in vivo systems, the MP delivery system results in more robust effects at a greatly reduced dosage. These results have potential applications in aiding the improvement of cancer immunotherapies.

Cyclic-di-GMP stimulates keratinocyte innate immune responses and attenuates methicillin-resistant Staphylococcus aureus colonization in a murine skin wound infection model

BackgroundStaphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model.ResultsStimulation of HaCaT cells with 125 μM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human β-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 μM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice.ConclusionsThese results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.

The Vibrio cholerae master regulator for the activation of biofilm biogenesis genes, VpsR, senses both cyclic di-GMP and phosphate.

Vibrio cholerae biofilm formation/maintenance is controlled by myriad factors; chief among these are the regulator VpsR and cyclic di-guanosine monophosphate (c-di-GMP). VpsR has strong sequence similarity to enhancer binding proteins (EBPs) that activate RNA polymerase containing sigma factor σ54. However, we have previously shown that transcription from promoters within the biofilm biogenesis/maintenance pathways uses VpsR, c-di-GMP and RNA polymerase containing the primary sigma factor (σ70). Previous work suggested that phosphorylation of VpsR at a highly conserved aspartate, which is phosphorylated in other EBPs, might also contribute to activation. Using the biofilm biogenesis promoter PvpsL, we show that in the presence of c-di-GMP, either wild type or the phospho-mimic VpsR D59E activates PvpsL transcription, while the phospho-defective D59A variant does not. Furthermore, when c-di-GMP levels are low, acetyl phosphate (Ac∼P) is required for significant VpsR activity in vivo and in vitro. Although these findings argue that VpsR phosphorylation is needed for activation, we show that VpsR is not phosphorylated or acetylated by Ac∼P and either sodium phosphate or potassium phosphate, which are not phosphate donors, fully substitutes for Ac∼P. We conclude that VpsR is an unusual regulator that senses phosphate directly, rather than through phosphorylation, to aid in the decision to form/maintain biofilm.

Quantification of cyanobacterial cyclic di-guanosine monophosphate (c-di-GMP) by liquid chromatography electrospray ionization tandem mass spectrometry

Cyclic di-guanosine monophosphate (c-di-GMP) is a second messenger found ubiquitously in bacteria. This signaling molecule regulates a variety of physiological activities such as phototaxis and flocculation in cyanobacteria and is critical for their environmental adaptation. Although genes encoding the enzymes for synthesis and/or degradation of c-di-GMP are found in the genomes of both multicellular and unicellular cyanobacteria, little is known about the biological functions of these enzymes in cyanobacterial cells. Here we have established a robust and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method for c-di-GMP quantification using a cost-effective solvent, methanol. Quantification methods were validated by measuring c-di-GMP in the cyanobacterium Synechococcus elongatus PCC 7942 through spiking and recovery assays after which the method was applied to examine short-term changes in cellular levels of c-di-GMP in response to a transition from light to dark or from dark to light in S. elongatus. Results showed that a transient increase in c-di-GMP upon transitioning from light to dark was occurring which resembled responses involving cyclic adenosine monophosphate and other second messengers in cyanobacteria. These findings demonstrated that our method enabled relatively specific and sensitive quantification of c-di-GMP in cyanobacteria at lower cost.

Signaling events that occur when cells of Escherichia coli encounter a glass surface

Bacterial cells interact with solid surfaces and change their lifestyle from single free-swimming cells to sessile communal structures (biofilms). Cyclic di-guanosine monophosphate (c-di-GMP) is central to this process, yet we lack tools for direct dynamic visualization of c-di-GMP in single cells. Here, we developed a fluorescent protein-based c-di-GMP-sensing system for Escherichia coli that allowed us to visualize initial signaling events and assess the role played by the flagellar motor. The sensor was pH sensitive, and the events that appeared on a seconds' timescale were alkaline spikes in the intracellular pH. These spikes were not apparent when signals from different cells were averaged. Instead, a signal appeared on a minutes' timescale that proved to be due to an increase in intracellular c-di-GMP. This increase, but not the alkaline spikes, depended upon a functional flagellar motor. The kinetics and the amplitude of both the pH and c-di-GMP responses displayed cell-to-cell variability indicative of the distinct ways the cells approached and interacted with the surface. The energetic status of a cell can modulate these events. In particular, the alkaline spikes displayed an oscillatory behavior and the c-di-GMP increase was modest in the presence of glucose.

BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP.

The widespread bacterial second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups.

PA0575 (RmcA) interacts with other c-di-GMP metabolizing proteins in Pseudomonas aeruginosa PAO1.

As a central signaling molecule, c-di-GMP (bis-(3,5)-cyclic diguanosine monophosphate) is becoming the focus for research in bacteria physiology. Pseudomonas aeruginosa PAO1 genome contains highly complicated c-di-GMP metabolizing genes and a number of these proteins have been identified and investigated. Especially, a sophisticated network of these proteins is emerging. In current study, mainly through Bacteria-2-Hybrid assay, we found PA0575 (RmcA), a GGDEF-EAL dual protein, to interact with two other dual proteins of PA4601 (MorA) and PA4959 (FimX). These observations imply the intricacy of c-di-GMP metabolizing protein interactions. Our work thus provides one piece of data to increase the understandings to c-di-GMP signaling.

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius.

Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3′,5′-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3′,5′-cAMP, cyclic guanosine monophosphate (3′,5′-cGMP), 5′-phosphoadenylyl-3′,5′-adenosine (5′-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2′,3′-cyclic isomers of all four RNA building blocks (2′,3′-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3′,5′-cCMP), cyclic uridine monophosphate (3′,5′-cUMP) and cyclic diadenosine monophosphate (3′,5′-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3′,5′-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

Regulation of Biofilm Exopolysaccharide Production by Cyclic Di-Guanosine Monophosphate.

Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly-N-acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c-di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP.

Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence.

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

The Insights and Perspectives of Nitric Oxide-mediated Biofilm Eradication.

Biofilms are among the most important causes of nosocomial and recurrent infections as biofilms confer antibiotic resistance to pathogenic bacteria and protect them from the host's immune system. Thus, it is imperative to investigate effective therapeutic agents to counteract biofilms. As an important signaling molecule, Nitric Oxide (NO) plays a crucial role in various biological and pathological processes. NO could disperse biofilm and restore the drug sensitivity by reducing intracellular cyclic-diguanosine monophosphate (c-di-GMP) levels. This review highlights recent advances on antibacterial and antibiofilm effects of NO when NO was co-administered with other antimicrobial agents. A significant improvement in drug permeability and biofilm cell targeting and reduced cytotoxicity could be attained with this strategy. In this review, we briefly lay out challenges and propose future directions in this appealing avenue of research on NO-based therapy for biofilm eradication.

Comparative molecular dynamics simulation analysis of G20 and C92 mutations in c-di-GMP I riboswitch and the wild type with docked c-di-GMP ligand.

Riboswitch, a bacterial regulatory RNA consists of an aptamer (specific ligand binding unit) and an expression platform (gene expression modulation unit), which act as a potential drug target as it regulates critical genes. Therefore, it is of interest to glean information on the binding of c-di-GMP ligand to mutated conserved G20 and C92 residues of cyclic diguanosine monophosphate I (c-di-GMP I) riboswitch using molecular dynamics simulation. The result shows that the binding energy of wild/native type riboswitch-ligand complex (3IRW) is lower than the mutant complexes suggesting that the binding affinity for c-di-GMP ligand decreases in case of mutant riboswitches. The hydrogen bonding interactions analysis also showed a high number of hydrogen bonds formation in the wild type riboswitch-ligand complex as compared to the mutant complexes illustrating stronger interaction of ligand to wild type riboswitch than the mutants. The simulation result shows that the mutations affected riboswitch-ligand interactions. The residues G14, G21, C46, A47, and U92 were identified as the key residues which contributed effectively to the binding of c-di-GMP I riboswitch with the natural ligand.

Mechanism of nitrite transporter NirC in motility, biofilm formation, and adhesion of avian pathogenic Escherichia coli.

The Escherichia coli (E. coli) nirC gene encodes a nitrite transporter, which involved in transporting toxic nitrite (NO2-) from the environment into the bacteria. Although the deletion of nirC gene could cause changes in motility, adhesion in the previous study, and the virulence involved in the specified mechanism for pathogenic E. coli remains to be known. In the present work, we aimed to evaluate the role of NirC in a serotype O2:K1:H7 avian pathogenic Escherichia coli (APEC) strain. For this purpose, we generated a NirC-deficient mutant of APEC XM strain and examined its biological characteristics. The nirC gene deletion mutant enhanced ability of motility, decreased in biofilm formation, and it markedly reduced ability to adhere mouse brain microvascular endothelial cell b.End3 cells. For understanding its mechanism, sequentially we detected and found the stress regulator rpoS and its downstream genes csrA were up-regulated in NirC-deficient mutant while diguanylate cyclase gene dgcT was down-regulated. By high-performance liquid chromatography (HPLC) experiment, we demonstrated the concentration of intracellular 3',5'-cyclic diguanosine monophosphate (c-di-GMP) significantly decrease in nirC gene deletion mutant. Taken data together, we may make a conclusion with a possible signal pathway clue, due to NirC mutation, environmental NO2- accumulation leads to nitrite stress and inactivates c-di-GMP synthesis by stimulating the stress regulator RpoS, resulting in changes of biological characteristics.

Natural diversity provides a broad spectrum of cyanobacteriochrome-based diguanylate cyclases

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.

Exploration of Ligand-receptor Binding and Mechanisms for Alginate Reduction and Phenotype Reversion by Mucoid Pseudomonas aeruginosa.

Bacteria in general can develop a wide range of phenotypes under different conditions and external stresses. The phenotypes that reside in biofilms, overproduce exopolymers, and show increased motility often exhibit drug tolerance and drug persistence. In this work, we describe a class of small molecules that delay and inhibit the overproduction of alginate by a non-swarming mucoid Pseudomonas aeruginosa. Among these molecules, selected benzophenone-derived alkyl disaccharides cause the mucoid bacteria to swarm on hydrated soft agar gel and revert the mucoid to a nonmucoid phenotype. The sessile (biofilm) and motile (swarming) phenotypes are controlled by opposing signaling pathways with high and low intracellular levels of bis-(3',5')-cyclic diguanosine monophosphate (cdG), respectively. As our molecules control several of these phenotypes, we explored a protein receptor, pilin of the pili appendages, that is consistent with controlling these bioactivities and signaling pathways. To test this binding hypothesis, we developed a bacterial motility-enabled binding assay that uses the interfacial properties of hydrated gels and bacterial motility to conduct label-free ligand-receptor binding studies. The structure-activity correlation and receptor identification reveal a plausible mechanism for reverting mucoid to nonmucoid phenotypes by binding pili appendages with ligands capable of sequestering and neutralizing reactive oxygen species.

Cyclic di-GMP sensing histidine kinase PdtaS controls mycobacterial adaptation to carbon sources.

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.