Diguanylate Cyclase Research Articles

Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli.

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

Genomic insights into the c-di-GMP signaling and biofilm development in the saprophytic spirochete Leptospira biflexa.

C-di-GMP is a bacterial second messenger with central role in biofilm formation. Spirochete bacteria from Leptospira genus present a wide diversity, with species of medical importance and environmental species, named as saprophytic. Leptospira form biofilms in the rat's reservoir kidneys and in the environment. Here, we performed genomic analyses to identify enzymatic and effector c-di-GMP proteins in the saprophytic biofilm-forming species Leptospira biflexa serovar Patoc. We identified 40 proteins through local alignments. Amongst them, 16 proteins are potentially functional diguanylate cyclases, phosphodiesterases, or hybrid proteins. We also identified nine effectors, including PilZ proteins. Enrichment analyses suggested that c-di-GMP interacts with cAMP signaling system, CsrA system, and flagella assembly regulation during biofilm development of L. biflexa. Finally, we identified eight proteins in the pathogen Leptospira interrogans serovar Copenhageni that share high similarity with L. biflexa c-di-GMP-related proteins. This work revealed proteins related to c-di-GMP turnover and cellular response in Leptospira and their potential roles during biofilm development.

Two cyanobacterial response regulators with diguanylate cyclase activity, Rre2 and Rre8, participate in biofilm formation.

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.

Blue light promotes zero-valent sulfur production in a deep-sea bacterium.

Increasing evidence has shown that light exists in a diverse range of deep-sea environments. We unexpectedly found that blue light is necessary to produce excess zero-valent sulfur (ZVS) in Erythrobacter flavus 21-3, a bacterium that has been recently isolated from a deep-sea cold seep. E.flavus 21-3 is able to convert thiosulfate to ZVS using a novel thiosulfate oxidation pathway comprising a thiosulfate dehydrogenase (TsdA) and a thiosulfohydrolase (SoxB). Using proteomic, bacterial two-hybrid and heterologous expression assays, we found that the light-oxygen-voltage histidine kinase LOV-1477 responds to blue light and activates the diguanylate cyclase DGC-2902 to produce c-di-GMP. Subsequently, the PilZ domain-containing protein mPilZ-1753 binds to c-di-GMP and activates TsdA through direct interaction. Finally, Raman spectroscopy and gene knockout results verified that TsdA and two SoxB homologs cooperate to regulate ZVS production. As ZVS is an energy source for E.flavus 21-3, we propose that deep-sea blue light provides E.flavus 21-3 with a selective advantage in the cold seep, suggesting a previously unappreciated relationship between light-sensing pathways and sulfur metabolism in a deep-sea microorganism.

Analysis of cellulose synthesis in a high-producing acetic acid bacterium Komagataeibacter hansenii

Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high cellulose-producing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC.Key points• BcsAB1 induces formation of microcrystalline cellulose fibers.• Modifications by BcsAB2 and BcsAB3 alter diameter of cellulose fibers.• Complex regulatory network of DGCs on cellulose pellicle formation and motility.

Bacterial cellulose: A highly versatile nanomaterial.

Bacterial cellulose: A highly versatile nanomaterial.

H-NOX Regulates Biofilm Formation in Agrobacterium Vitis in Response to NO.

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 avH-NOX is a nitric oxide (NO)-binding hemoprotein that binds to and regulates the activity of avi_3097 (avHaCE; H-NOX-associated cyclic di-GMP processing enzyme). Kinetic analysis of avHaCE indicates a ∼four-fold increase in PDE activity in the presence of NO-bound avH-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 ΔhnoX strain has no NO phenotype, suggesting that H-NOX is responsible for the NO biofilm phenotype in A. vitis. Together, these data indicate that avH-NOX enhances cyclic di-GMP degradation to reduce biofilm formation in response to NO in A. vitis.

KidA, a novel interactor of the cyanobacterial circadian oscillator that tunes its period.

Composed of only three proteins, KaiA, KaiB, and KaiC, the cyanobacterial circadian clock is the simplest known biochemical oscillator that can be reconstituted in vitro. While the molecular mechanism of the three proteins has been studied in depth, how they are connected to the rest of the cellular physiology remains unclear. In order to identify previously unknown players in the broader network of the circadian system, we performed a coimmunoprecipitation (co-IP)/mass spec experiment on S. elongatus PCC 7942 cell lysate. Using KaiB in its nighttime form (fold-switched KaiB) as bait, we identified a novel clock interactor protein we named Kai-Interacting Diguanylate cyclase A (KidA), which is predicted to have four N-terminal PAS domains and C-terminal GGDEF/EAL domains. We found that KidA shortens the period of the in vivo rhythms in a dose-dependent manner and alters the entrainment to light-dark cycles. We found that an N-terminal triple-PAS domain fragment is sufficient for the period-shortening phenotype. Consistently, adding purified triple-PAS fragment to the in vitro Kai oscillator reconstituted the dose-dependent shortening of the period. Our in vitro co-IP showed that the N-terminal PAS domains of KidA can directly and specifically bind to the fold-switched form of KaiB. To understand the potential mechanism, we used a computational modeling approach where a fold-switched KaiB binder was added to the Kai oscillator model. The results showed that KidA can tune the period by shifting the equilibrium of KaiB fold-switching. The discovery of KidA suggests a potential role of PAS domain in prokaryotic timekeeping, which has been well documented only in eukaryotic clock systems. Future studies could unveil more potential of KidA, such as integration of time-of-day information and other signals via multiple PAS domains and regulation of cellular processes via cyclic-di-GMP.

The Oxidative Stress-Induced Hypothetical Protein PG_0686 in Porphyromonas gingivalis W83 Is a Novel Diguanylate Cyclase.

The survival/adaptation of Porphyromonas gingivalis to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Several functional classes of genes, depending on the severity and duration of the exposure, were induced in P. gingivalis under H2O2-induced oxidative stress. The PG_0686 gene was highly upregulated under prolonged oxidative stress. PG_0686, annotated as a hypothetical protein of unknown function, is a 60 kDa protein that carries several domains including hemerythrin, PAS10, and domain of unknown function (DUF)-1858. Although PG_0686 showed some relatedness to several diguanylate cyclases (DGCs), it is missing the classical conserved, active site sequence motif (GGD[/E]EF), commonly observed in other bacteria. PG_0686-related proteins are observed in other anaerobic bacterial species. The isogenic mutant P. gingivalis FLL361 (ΔPG_0686::ermF) showed increased sensitivity to H2O2, and decreased gingipain activity compared to the parental strain. Transcriptome analysis of P. gingivalis FLL361 showed the dysregulation of several gene clusters/operons, known oxidative stress resistance genes, and transcriptional regulators, including PG_2212, CdhR and PG_1181 that were upregulated under normal anaerobic conditions. The intracellular level of c-di-GMP in P. gingivalis FLL361 was significantly decreased compared to the parental strain. The purified recombinant PG_0686 (rPG_0686) protein catalyzed the formation of c-di-GMP from GTP. Collectively, our data suggest a global regulatory property for PG_0686 that may be part of an unconventional second messenger signaling system in P. gingivalis. Moreover, it may coordinately regulate a pathway(s) vital for protection against environmental stress, and is significant in the pathogenicity of P. gingivalis and other anaerobes. IMPORTANCE Porphyromonas gingivalis is an important etiological agent in periodontitis and other systemic diseases. There is still a gap in our understanding of the mechanisms that P. gingivalis uses to survive the inflammatory microenvironment of the periodontal pocket. The hypothetical PG_0686 gene was highly upregulated under prolonged oxidative stress. Although the tertiary structure of PG_0686 showed little relatedness to previously characterized diguanylate cyclases (DGCs), and does not contain the conserved GGD(/E)EF catalytic domain motif sequence, an ability to catalyze the formation of c-di-GMP from GTP is demonstrated. The second messenger pathway for c-di-GMP was previously predicted to be absent in P. gingivalis. PG_0686 paralogs are identified in other anaerobic bacteria. Thus, PG_0686 may represent a novel class of DGCs, which is yet to be characterized. In conclusion, we have shown, for the first time, evidence for the presence of c-di-GMP signaling with environmental stress protective function in P. gingivalis.

Challenges in using transcriptome data to study the c-di-GMP signaling network in Pseudomonas aeruginosa clinical isolates.

In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts nonoverlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding DGCs. This approach might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, while we demonstrate that the selection of subgroups of clinical isolates with high versus low expression of sigma factor encoding genes served the identification of their downstream regulons, we were unable to confirm the predicted DGC regulons, because the high c-di-GMP associated phenotypes were rapidly lost in the clinical isolates,. Further studies are needed to determine the specific mechanisms underlying the loss of cyclase activity upon prolonged cultivation of clinical P. aeruginosa isolates.

Local signaling enhances output specificity of bacterial c-di-GMP signaling networks.

For many years the surprising multiplicity, signal input diversity, and output specificity of c-di-GMP signaling proteins has intrigued researchers studying bacterial second messengers. How can several signaling pathways act in parallel to produce specific outputs despite relying on the same diffusible second messenger maintained at a certain global cellular concentration? Such high specificity and flexibility arise from combining modes of local and global c-di-GMP signaling in complex signaling networks. Local c-di-GMP signaling can be experimentally shown by three criteria being met: (i) highly specific knockout phenotypes for particular c-di-GMP-related enzymes, (ii) actual cellular c-di-GMP levels that remain unchanged by such mutations and/or below the Kd's of the relevant c-di-GMP-binding effectors, and (iii) direct interactions between the signaling proteins involved. Here, we discuss the rationale behind these criteria and present well-studied examples of local c-di-GMP signaling in Escherichia coli and Pseudomonas. Relatively simple systems just colocalize a local source and/or a local sink for c-di-GMP, i.e. a diguanylate cyclase (DGC) and/or a specific phosphodiesterase (PDE), respectively, with a c-di-GMP-binding effector/target system. More complex systems also make use of regulatory protein interactions, e.g. when a "trigger PDE" responds to locally provided c-di-GMP, and thereby serves as a c-di-GMP-sensing effector that directly controls a target's activity, or when a c-di-GMP-binding effector recruits and directly activates its own "private" DGC. Finally, we provide an outlook into how cells can combine local and global signaling modes of c-di-GMP and possibly integrate those into other signaling nucleotides networks.

Polarity of c-di-GMP synthesis and degradation.

The bacterial cell pole has long been recognized as a defined compartment for enzymatic activities that are important or even vital for the cell. Polarity of diguanylate cyclases and phosphodiesterases, enzymes that synthesize and degrade the second messenger c-di-GMP, has now been demonstrated for several bacterial systems. Here we review these polar regulatory systems and show how the asymmetry of c-di-GMP production and turnover in concert with different modes of activation and deactivation creates heterogeneity in cellular c-di-GMP levels. We highlight how this heterogeneity generates a diverse set of phenotypic identities or states and how this may benefit the cell population, and we discuss reasons why the polarity of c-di-GMP signaling is probably widespread among bacteria.

Exploring Innovative Approaches to Isolate a One-Component c-di-GMP Transducer: A Pilot Study.

Environmental nutrients control bacterial biofilm homeostasis, by regulating the intracellular levels of c-di-GMP. One component transducers can sense different classes of small molecules through a periplasmic domain; the nutrient recognition triggers the subsequent regulation of the downstream cytosolic diguanylate cyclase (GGDEF) or phosphodiesterase (EAL) domains, via transmembrane helix(ces), to finally change c-di-GMP levels.Protein studies on such transducers have been mainly carried out on isolated domains due to the presence of the transmembrane portion. Nevertheless, the cleavage of GGDEF and EAL-containing proteins could be detrimental since both tertiary and quaternary structures could be allosterically controlled; to by-pass this limitation, studies on the corresponding full-length proteins are highly desired.We have in silico selected a GGDEF-EAL transducer from Dyella thiooxydans (ann. A0A160N0B7), whose periplasmic binding domain was predicted to bind to arginine, a nutrient often associated with chronic infections and biofilm. This protein has been used as an in vitro tool for the identification of the best approach for its isolation, including (i) protein engineering to produce a water-soluble version via QTY (Glutamine, Threonine, and Tyrosine) code or (ii) nanodiscs assembly. The results on this "prototype" may represent the proof-of-concept for future isolation of other transmembrane proteins sharing the same architecture, including more complex nutrient-based transducers controlling c-di-GMP levels.

In Vitro Measurement of Gas-Dependent and Redox-Sensitive Diguanylate Cyclase Activity.

Bacteria sense and respond to gaseous ligand changes in the environment to regulate a multitude of behaviors, including the production of the secondary messengers cyclic di-GMP. Gas sensing can be difficult to measure due to the high concentration of the oxygen in the atmosphere, particularly in redox-sensitive systems. Here, we describe a method for anaerobic quantification of cyclic di-GMP production which can be used to measure the impact of molecular oxygen, nitric oxide, and carbon monoxide on the catalysis of a diguanylate cyclase-containing protein and the possible pitfalls in the experimental procedure.

Interference of flagellar rotation up-regulates the expression of small RNA contributing to Bordetella pertussis infection

Bacterial small RNAs (sRNAs) posttranscriptionally regulate gene expressions involved in various biological processes, including pathogenicity. Our previous study identified sRNAs, the expression of which was up-regulated in Bordetella pertussis, the causative agent of whooping cough, upon tracheal colonization of the bacteria; however, their roles in bacterial infection remain unknown. Here, we found that one sRNA, Bpr4, contributes to B. pertussis infection by posttranscriptionally up-regulating filamentous hemagglutinin (FHA), a major adhesin of the bacteria. Bpr4 bound to the 5' untranslated region of fhaB mRNA encoding FHA and inhibited its degradation mediated by RNaseE. Our results demonstrated that Bpr4 up-regulation was triggered by the interference of flagellar rotation, which caused the disengagement of MotA, a flagellar stator. Subsequently, MotA activated a diguanylate cyclase to generate cyclic di-GMP, which plays a role in Bpr4 up-regulation through the RisK/RisA two-component system. Our findings indicate that a flagellum-triggered sensory system contributes to B. pertussis infection.

The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter

The ubiquitous Wsp (wrinkly spreader phenotype) chemosensory system and DSF (diffusible signal factor) quorum sensing are two important chemically associated signaling systems that mediate bacterial communications between the host and environment. Although these two systems individually control biofilm formation in pathogenic bacteria via the ubiquitous second messenger c-di-GMP, their crosstalk mechanisms remain elusive. Here we present a scenario from the plant-beneficial and antifungal bacterium Lysobacter enzymogenes OH11, where biofilm formation favors the colonization of this bacterium in fungal hyphae. We found that the Wsp system regulated biofilm formation via WspR-mediated c-di-GMP signaling, whereas DSF system did not depend on the enzymatic activity of RpfG to regulate biofilm formation. We further found that WspR, a diguanylate cyclase (DGC) responsible for c-di-GMP synthesis, could directly bind to one of the DSF signaling components, RpfG, an active phosphodiesterase (PDE) responsible for c-di-GMP degradation. Thus, the WspR-RpfG complex represents a previously undiscovered molecular linker connecting the Wsp and DSF systems. Mechanistically, RpfG could function as an adaptor protein to bind and inhibit the DGC activity of unphosphorylated WspR independent of its PDE activity. Phosphorylation of WspR impaired its binding affinity to RpfG and also blocked the ability of RpfG to act as an adaptor protein, which enabled the Wsp system to regulate biofilm formation in a c-di-GMP-dependent manner by dynamically integrating the DSF system. Our findings demonstrated a previously uncharacterized mechanism of crosstalk between Wsp and DSF systems in plant-beneficial and antifungal bacteria.

Patterns of abundance, chromosomal localization, and domain organization among c-di-GMP-metabolizing genes revealed by comparative genomics of five alphaproteobacterial orders

BackgroundBis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger that affects diverse processes in different bacteria, including the cell cycle, motility, and biofilm formation. Its cellular levels are controlled by the opposing activities of two types of enzymes, with synthesis by diguanylate cyclases containing a GGDEF domain and degradation by phosphodiesterases containing either an HD-GYP or an EAL domain. These enzymes are ubiquitous in bacteria with up to 50 encoded in some genomes, the specific functions of which are mostly unknown.ResultsWe used comparative analyses to identify genomic patterns among genes encoding proteins with GGDEF, EAL, and HD-GYP domains in five orders of the class Alphaproteobacteria. GGDEF-containing sequences and GGDEF-EAL hybrids were the most abundant and had the highest diversity of co-occurring auxiliary domains while EAL and HD-GYP containing sequences were less abundant and less diverse with respect to auxiliary domains. There were striking patterns in the chromosomal localizations of the genes found in two of the orders. The Rhodobacterales’ EAL-encoding genes and Rhizobiales’ GGDEF-EAL-encoding genes showed opposing patterns of distribution compared to the GGDEF-encoding genes. In the Rhodobacterales, the GGDEF-encoding genes showed a tri-modal distribution with peaks mid-way between the origin (ori) and terminus (ter) of replication and at ter while the EAL-encoding genes peaked near ori. The patterns were more complex in the Rhizobiales, but the GGDEF-encoding genes were biased for localization near ter.ConclusionsThe observed patterns in the chromosomal localizations of these genes suggest a coupling of synthesis and hydrolysis of c-di-GMP with the cell cycle. Moreover, the higher proportions and diversities of auxiliary domains associated with GGDEF domains and GGDEF-EAL hybrids compared to EAL or HD-GYP domains could indicate that more stimuli affect synthesis compared to hydrolysis of c-di-GMP.

Cyclic di-GMP regulates bacterial colonization and further biocontrol efficacy of Bacillus velezensis against apple ring rot disease via its potential receptor YdaK.

Bacillus species are among the most investigated beneficial bacteria and widely used in agricultural systems as biological control agents. Its biocontrol efficacy is controlled by diverse regulators. Cyclic diguanylate (c-di-GMP) is a nearly universal second messenger in bacteria and modulates various important physiological processes, including motility, biofilm formation, antifungal antibiotic production and host colonization. However, the impact of c-di-GMP on biocontrol efficacy of beneficial bacteria is unknown. Bacillus velezensis PG12 is an effective biocontrol strain against apple ring rot disease caused by Botryosphaeria dothidea. In this study, the contribution of c-di-GMP to biocontrol efficacy of B. velezensis PG12 was investigated. Deletion of single gene encoding diguanylate cyclase or phosphodiesterase did not affect its biocontrol efficacy against apple ring rot. However, artificial modulation of c-di-GMP level in the cells leads to a significant change of biocontrol efficacy, suggesting that c-di-GMP positively regulates biocontrol efficacy of B. velezensis PG12 against apple ring rot disease. More evidences indicate that c-di-GMP does not affect the antagonistic activity of B. velezensis PG12 against B. dothidea in vitro and in vivo, but positively regulates biofilm formation of B. velezensis PG12 and its colonization on apple fruits. Importantly, deletion of ydaK could rescue the inhibition of biofilm formation, bacterial colonization and biocontrol efficacy caused by low c-di-GMP level, indicating that YdaK is the potential c-di-GMP receptor to regulate biofilm formation, colonization and effective biological control. However, YdaK did not affect the antagonistic activity of B. velezensis PG12 against B. dothidea. Based on these findings, we propose that c-di-GMP regulates biofilm formation, subsequently the bacterial colonization on apple fruits and thus biocontrol efficacy of B. velezensis through its receptor YdaK. This is the first report showing that c-di-GMP plays a role in biocontrol efficacy of beneficial bacteria.

Bioelectronic cell-based device provides a strategy for the treatment of the experimental model of multiple sclerosis.

Wireless powered optogenetic cell-based implant provides a strategy to deliver subcutaneously therapeutic proteins. Immortalize Human Mesenchymal Stem Cells (hMSC-TERT) expressing the bacteriophytochrome diguanylate cyclase (DGCL) were validated for optogenetic controlled interferon-β delivery (Optoferon cells) in a bioelectronic cell-based implant. Optoferon cells transcriptomic profiling was used to elaborate an in-silico model of the recombinant interferon-β production. Wireless optoelectronic device integration was developed using additive manufacturing and injection molding. Implant cell-based optoelectronic interface manufacturing was established to integrate industrial flexible compact low-resistance screen-printed Near Field Communication (NFC) coil antenna. Optogenetic cell-based implant biocompatibility, and device performances were evaluated in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of multiple sclerosis.

Mutation of gdpS gene induces a viable but non-culturable state in Staphylococcus epidermidis and changes in the global transcriptional profile

BackgroundIn the genome of staphylococci, only the gdpS gene encodes the conserved GGDEF domain, which is the characteristic of diguanylate cyclases. In our previous study, we have demonstrated that the gdpS gene can modulate biofilm formation by positively regulating the expression of ica operon in Staphylococcus epidermidis. Moreover, this regulation seems to be independent of the c-di-GMP signaling pathway and the protein-coding function of this gene. Therefore, the biological function of the gdpS gene remains to be further investigated.ResultsIn the present study, it was observed that mutation of the gdpS gene induced S. epidermidis to enter into a presumed viable but nonculturable state (VBNC) after cryopreservation with glycerol. Similarly, when moved from liquid to solid culture medium, the gdpS mutant strain also exhibited a VBNC state. Compared with the wild-type strain, the gdpS mutant strain autolyzed more quickly during storage at 4℃, indicating its increased susceptibility to low temperature. Transcriptional profiling analysis showed that the gdpS mutation affected the transcription of 188 genes (92 genes were upregulated and 96 genes were downregulated). Specifically, genes responsible for glycerol metabolism were most markedly upregulated and most of the altered genes in the mutant strain are those involved in nitrogen metabolism. In addition, the most significantly downregulated genes included the betB gene, whose product catalyzes the synthesis of glycine betaine and confers tolerance to cold.ConclusionThe preliminary results suggest that the gdpS gene may participate in VBNC formation of S. epidermidis in face of adverse environmental factors, which is probably achieved by regulating expression of energy metabolism genes. Besides, the gdpS gene is critical for S. epidermidis to survive low temperature, and the underlying mechanism may be partly explained by its influence on expression of betB gene.