Cyclic Diguanylate Research Articles

Functional analysis of cyclic diguanylate-modulating proteins in Vibrio fischeri.

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid (Euprymna scolopes) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri. The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V. fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions.

Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Cyclic Diguanylate in the Wild: Roles During Plant and Animal Colonization.

Cyclic diguanylate (c-di-GMP) is a near-ubiquitous signaling molecule that regulates the motility-to-sessility transition in many bacterial species. Among the phenotypes influenced by c-di-GMP are biofilm formation, motility, cell cycle, and virulence. The hallmark phenotypes regulated by c-di-GMP-biofilm formation and motility-are key determinants of host-bacterial interactions. A large body of research has identified the roles of c-di-GMP in regulating phenotypes in culture. While numerous studies have investigated roles for c-di-GMP during the establishment and maintenance of pathogenic host-bacterial associations, considerably less attention has been devoted to defining the roles of c-di-GMP during beneficial and commensal associations. This review describes the known roles of c-di-GMP in regulating phenotypes that contribute to host colonization, with a focus on knowledge gaps and future prospects for examining c-di-GMP during beneficial colonization.

The LuxO-OpaR quorum-sensing cascade differentially controls Vibriophage VP882 lysis-lysogeny decision making in liquid and on surfaces.

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to synchronize collective behaviors. QS relies on the production, release, and group-wide detection of extracellular signaling molecules called autoinducers. Vibrios use two QS systems: the LuxO-OpaR circuit and the VqmA-VqmR circuit. Both QS circuits control group behaviors including biofilm formation and surface motility. The Vibrio parahaemolyticus temperate phage φVP882 encodes a VqmA homolog (called VqmAφ). When VqmAφ is produced by φVP882 lysogens, it binds to the host-produced autoinducer called DPO and launches the φVP882 lytic cascade. This activity times induction of lysis with high host cell density and presumably promotes maximal phage transmission to new cells. Here, we explore whether, in addition to induction from lysogeny, QS controls the initial establishment of lysogeny by φVP882 in naïve host cells. Using mutagenesis, phage infection assays, and phenotypic analyses, we show that φVP882 connects its initial lysis-lysogeny decision to both host cell density and whether the host resides in liquid or on a surface. Host cells in the low-cell-density QS state primarily undergo lysogenic conversion. The QS regulator LuxO~P promotes φVP882 lysogenic conversion of low-cell-density planktonic host cells. By contrast, the ScrABC surface-sensing system regulates lysogenic conversion of low-cell-density surface-associated host cells. ScrABC controls the abundance of the second messenger molecule cyclic diguanylate, which in turn, modulates motility. The scrABC operon is only expressed when its QS repressor, OpaR, is absent. Thus, at low cell density, QS-dependent derepression of scrABC drives lysogenic conversion in surface-associated host cells. These results demonstrate that φVP882 integrates cues from multiple sensory pathways into its lifestyle decision making upon infection of a new host cell.

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.

Looking for Resistance to Soft Rot Disease of Potatoes Facing Environmental Hypoxia.

Plants are exposed to various stressors, including pathogens, requiring specific environmental conditions to provoke/induce plant disease. This phenomenon is called the "disease triangle" and is directly connected with a particular plant-pathogen interaction. Only a virulent pathogen interacting with a susceptible plant cultivar will lead to disease under specific environmental conditions. This may seem difficult to accomplish, but soft rot Pectobacteriaceae (SRPs) is a group virulent of pathogenic bacteria with a broad host range. Additionally, waterlogging (and, resulting from it, hypoxia), which is becoming a frequent problem in farming, is a favoring condition for this group of pathogens. Waterlogging by itself is an important source of abiotic stress for plants due to lowered gas exchange. Therefore, plants have evolved an ethylene-based system for hypoxia sensing. Plant response is coordinated by hormonal changes which induce metabolic and physiological adjustment to the environmental conditions. Wetland species such as rice (Oryza sativa L.), and bittersweet nightshade (Solanum dulcamara L.) have developed adaptations enabling them to withstand prolonged periods of decreased oxygen availability. On the other hand, potato (Solanum tuberosum L.), although able to sense and response to hypoxia, is sensitive to this environmental stress. This situation is exploited by SRPs which in response to hypoxia induce the production of virulence factors with the use of cyclic diguanylate (c-di-GMP). Potato tubers in turn reduce their defenses to preserve energy to prevent the negative effects of reactive oxygen species and acidification, making them prone to soft rot disease. To reduce the losses caused by the soft rot disease we need sensitive and reliable methods for the detection of the pathogens, to isolate infected plant material. However, due to the high prevalence of SRPs in the environment, we also need to create new potato varieties more resistant to the disease. To reach that goal, we can look to wild potatoes and other Solanum species for mechanisms of resistance to waterlogging. Potato resistance can also be aided by beneficial microorganisms which can induce the plant's natural defenses to bacterial infections but also waterlogging. However, most of the known plant-beneficial microorganisms suffer from hypoxia and can be outcompeted by plant pathogens. Therefore, it is important to look for microorganisms that can withstand hypoxia or alleviate its effects on the plant, e.g., by improving soil structure. Therefore, this review aims to present crucial elements of potato response to hypoxia and SRP infection and future outlooks for the prevention of soft rot disease considering the influence of environmental conditions.

The second messenger (cyclic diguanylate and autoinducer-2) promotes N-acylated-homoserine lactones-based quorum sensing for enhanced recovery of stored aerobic granular sludge

Efficient quorum sensing (QS) response is the premise for recovering the activities of stored aerobic granular sludge (AGS). This study aims to explore the crosstalk between the secondary messenger and the N-acylated-homoserine lactones (AHLs) to yield protein-rich granules efficiently from stored AGS by enhancing its QS efficiency selectively. 80 nmol/L cyclic diguanylate (c-di-GMP) with 20 nmol/L AHLs could increase the activity of isocitrate lyase activity (ICD) by 89 % and isocitrate dehydrogenase activity (ICDHc) by 113.5 %, to accelerate the tricarboxylic acid (TCA) cycle for yielding excess proteins by 166.4 %. In contrast, 80 nmol/L autoinducer-2 (AI-2) with 20 nmol/L AHLs could increase the activities of ICD and ICDHc by 485 % and 54.5 %, respectively, accelerating the glyoxylate (GCA) cycle to activate fat acid synthesis for stimulating polysaccharides (PS) secretion by 137.9 %. The strategy with c-di-GMP successfully recovers the refrigerated-stored and dried-stored AGS into proteins-rich AGS, with enriched functional strains for the PN secretion.

Aerobic granular sludge for complex heavy metal-containing wastewater treatment: characterization, performance, and mechanisms analysis.

Complex heavy metal (HM)-containing wastewater discharges pose substantial risks to global water ecosystems and human health. Aerobic granular sludge (AGS) has attracted increased attention as an efficient and low-cost adsorbent in HM-containing wastewater treatment. Therefore, this study systematically evaluates the effect of Cu(II), Ni(II), and Cr(III) addition on the characteristics, performance and mechanism of AGS in complex HM-containing wastewater treatment process by means of fourier transform infrared spectroscopy, inductively coupled plasma spectrocopcy, confocal laser scanning microscopy, extracellular polymeric substances (EPS) fractions detection and scanning electron microscope-energy dispersive X-ray. The results showed that AGS efficiently eliminated Cu(II), Ni(II), and Cr(III) by the orchestrated mechanisms of ion exchange, three-layer EPS adsorption [soluble microbial products EPS (SMP-EPS), loosely bound EPS (LB-EPS), tightly bound EPS (TB-EPS)], and inner-sphere adsorption; notably, almost 100% of Ni(II) was removed. Three-layer EPS adsorption was the dominant mechanism through which the HM were removed, followed by ion exchange and inner-sphere adsorption. SMP-EPS and TB-EPS were identified as the key EPS fractions for adsorbing Cr(III) and Cu(II), respectively, while Ni(II) was adsorbed evenly on SMP-EPS, TB-EPS, and LB-EPS. Moreover, the rates at which the complex HM penetrated into the granule interior and their affinity for EPS followed the order Cu(II) > Ni(II) > Cr(III). Ultimately, addition of complex HM stimulated microorganisms to excrete massive phosphodiesterases (PDEs), leading to a pronounced decrease in cyclic diguanylate (c-di-GMP) levels, which subsequently suppressed EPS secretion due to the direct linkage between c-di-GMP and EPS. This study unveils the adaptability and removal mechanism of AGS in the treatment of complex HM-containing wastewater, which is expected to provide novel insights for addressing the challenges posed by intricate real wastewater scenarios.

Exploring the Metabolic Response of Pseudomonas putida to L-arginine.

Beyond their role as protein-building units, amino acids are modulators of multiple behaviours in different microorganisms. In the root-colonizing beneficial bacterium Pseudomonas putida (recently proposed to be reclassified as alloputida) KT2440, current evidence suggests that arginine functions both as a metabolic indicator and as an environmental signal molecule, modulating processes such as chemotactic responses, siderophore-mediated iron uptake or the levels of the intracellular second messenger cyclic diguanylate (c-di-GMP). Using microcalorimetry and extracellular flux analysis, in this work we have studied the metabolic adaptation of P. putida KT2440 to the presence of L-arginine in the growth medium, and the influence of mutations related to arginine metabolism. Arginine causes rapid changes in the respiratory activity of P. putida, particularly magnified in a mutant lacking the transcriptional regulator ArgR. The metabolic activity of mutants affected in arginine transport and metabolism is also altered during biofilm formation in the presence of the amino acid. The results obtained here further support the role of arginine as a metabolic signal in P. putida and the relevance of ArgR in the adaptation to the amino acid. They also serve as proof of concept on the use of calorimetric and extracellular flux techniques to analyse metabolic responses in bacteria and the impact of different mutant backgrounds on such responses.

Bioinformatic and functional characterization of cyclic-di-GMP metabolic proteins in Vibrio alginolyticus unveils key diguanylate cyclases controlling multiple biofilm-associated phenotypes.

The biofilm lifestyle is critical for bacterial survival and proliferation in the fluctuating marine environment. Cyclic diguanylate (c-di-GMP) is a key second messenger during bacterial adaptation to various environmental signals, which has been identified as a master regulator of biofilm formation. However, little is known about whether and how c-di-GMP signaling regulates biofilm formation in Vibrio alginolyticus, a globally dominant marine pathogen. Here, a large set of 63 proteins were predicted to participate in c-di-GMP metabolism (biosynthesis or degradation) in a pathogenic V. alginolyticus strain HN08155. Guided by protein homology, conserved domains and gene context information, a representative subset of 22 c-di-GMP metabolic proteins were selected to determine which ones affect biofilm-associated phenotypes. By comparing phenotypic differences between the wild-type and mutants or overexpression strains, we found that 22 c-di-GMP metabolic proteins can separately regulate different phenotypic outputs in V. alginolyticus. The results indicated that overexpression of four c-di-GMP metabolic proteins, including VA0356, VA1591 (CdgM), VA4033 (DgcB) and VA0088, strongly enhanced rugose colony morphotypes and strengthened Congo Red (CR) binding capacity, both of which are indicators of biofilm matrix overproduction. Furthermore, rugose enhanced colonies were accompanied by increased transcript levels of extracellular polysaccharide (EPS) biosynthesis genes and decreased expression of flagellar synthesis genes compared to smooth colonies (WTpBAD control), as demonstrated by overexpression strains WTp4033 and ∆VA4033p4033. Overall, the high abundance of c-di-GMP metabolic proteins in V. alginolyticus suggests that c-di-GMP signaling and regulatory system could play a key role in its response and adaptation to the ever-changing marine environment. This work provides a robust foundation for the study of the molecular mechanisms of c-di-GMP in the biofilm formation of V. alginolyticus.

Bacterial c-di-GMP has a key role in establishing host-microbe symbiosis.

Most microbes evolve faster than their hosts and should therefore drive evolution of host-microbe interactions. However, relatively little is known about the characteristics that define the adaptive path of microbes to host association. Here we identified microbial traits that mediate adaptation to hosts by experimentally evolving the free-living bacterium Pseudomonas lurida with the nematode Caenorhabditis elegans as its host. After ten passages, we repeatedly observed the evolution of beneficial host-specialist bacteria, with improved persistence in the nematode being associated with increased biofilm formation. Whole-genome sequencing revealed mutations that uniformly upregulate the bacterial second messenger, cyclic diguanylate (c-di-GMP). We subsequently generated mutants with upregulated c-di-GMP in different Pseudomonas strains and species, which consistently increased host association. Comparison of pseudomonad genomes from various environments revealed that c-di-GMP underlies adaptation to a variety of hosts, from plants to humans. This study indicates that c-di-GMP is fundamental for establishing host association.

Cyclic Diguanylate G-Quadruplex Inducer-Nitric Oxide Donor Conjugate as a Bifunctional Antibiofilm Agent and Antibacterial Synergist against Pseudomonas aeruginosa with a Hyperbiofilm Phenotype.

Antibiotic resistance caused by biofilm formation is a clinical challenge. Nitric oxide (NO) can effectively disperse a mature biofilm and can also synergistically influence the level of cyclic diguanylate (c-di-GMP), a universal secondary messenger that plays an important role in biofilm formation in bacteria. Based on our previous finding that c-di-GMP G-quadruplex inducers are effective biofilm formation inhibitors, we designed and synthesized a c-di-GMP G-quadruplex inducer-NO donor conjugate (A11@NO) as a bifunctional antibiofilm agent after obtaining the c-di-GMP G-quadruplex inducer (A11), which has an amino group capable of binding to a nitroso group (NO donor). The conjugate A11@NO showed better biofilm inhibition efficiency than A11, and it can also eradicate mature biofilm. Additionally, it exhibited good antimicrobial synergism against Pseudomonas aeruginosa and helped elevate the bactericidal efficiency of tobramycin against biofilm-formed bacteria. In combination with tobramycin, A11@NO also improved the survival rate of Caenorhabditis elegans in a hyperbiofilm environment.

Systematic Analysis of Two Tandem GGDEF/EAL Domain Genes Regulating Antifungal Activities in Pseudomonas glycinae MS82

Cyclic diguanylate (c-di-GMP) affects bacterial physiological and biochemical functions like biofilm, motility, virulence, and bacterial secretion systems. GGDEF/EAL-domain proteins, participating in c-di-GMP synthesis and degradation, are widely present in Pseudomonas, with various structures and functions. Pseudomonas glycinae MS82 is a rhizosphere bacterium that protects mushroom against the pathogenic fungi. Although 14 genes encoding GGDEF/EAL-domain proteins have been identified in the genome of MS82, c-di-GMP regulation is poorly understood as a facilitator or repressor of physiological phenotypes. Here, PafQ and PafR, encoding the proteins with the tandem GGDEF/EAL domain, were functionally analyzed and found to regulate antifungal activity. Individual deletion mutants of PafQ and PafR were constructed in P. glycinae MS82 through biparental conjugation and homologous recombination. Subsequently, antifungal activity, biofilm formation, motility, and expression of the genes related to antifungal substance synthesis were examined and contrasted with those of wild-type P. glycinae MS82. Most phenotypes of physiological activities were significantly reduced after knocking out PafQ or PafR. In other members of the genus Pseudomonas, homologous genes of PafQ and PafR possess different functions in c-di-GMP regulation. In P. glycinae, the positive regulation of PafQ and PafR on fungistatic substance synthesis, biofilm formation, and motility is crucial in the biocontrol of mushroom diseases.

In Situ Investigation of Pseudomonas aeruginosa Biofilm Development: Interplay between Flow, Growth Medium, and Mechanical Properties of Substrate.

To better understand the impact of biomaterial mechanical properties and growth medium on bacterial adhesion and biofilm formation under flow, we investigated the biofilm formation ability of Pseudomonas aeruginosa in different media on polydimethylsiloxane (PDMS) of different stiffness in real time using a microfluidic platform. P. aeruginosa colonization was recorded with optical microscopy and automated image analysis. The bacterial intracellular level of cyclic diguanylate (c-di-GMP), which regulates biofilm formation, was monitored using the transcription of the putative adhesin gene (cdrA) as a proxy. Contrary to the previous supposition, we revealed that PDMS material stiffness within the tested range has negligible impact on biofilm development and biofilm structures, whereas culture media not only influence the kinetics of biofilm development but also affect the biofilm morphology and structure dramatically. Interestingly, magnesium rather than previously reported calcium was identified here to play a decisive role in the formation of dense P. aeruginosa aggregates and high levels of c-di-GMP. These results demonstrate that although short-term adhesion assays bring valuable insight into bacterial and material interactions, long-term evaluations are essential to better predict overall biofilm outcome. The microfluidic system developed here presents a valuable application potential for studying biofilm development in situ. .

Impact of c-di-GMP on the Extracellular Proteome of Rhizobium etli.

Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second messenger cyclic diguanylate (cdG). We have performed quantitative proteomics to determine the extracellular protein contents of a Rhizobium etli strain expressing high cdG intracellular levels. cdG promoted the exportation of proteins that likely participate in adhesion and biofilm formation: the rhizobial adhesion protein RapA and two previously undescribed likely adhesins, along with flagellins. Unexpectedly, cdG also promoted the selective exportation of cytoplasmic proteins. Nearly 50% of these cytoplasmic proteins have been previously described as moonlighting or candidate moonlighting proteins in other organisms, often found extracellularly. Western blot assays confirmed cdG-promoted export of two of these cytoplasmic proteins, the translation elongation factor (EF-Tu) and glyceraldehyde 3-phosphate dehydrogenase (Gap). Transmission Electron Microscopy immunolabeling located the Gap protein in the cytoplasm but was also associated with cell membranes and extracellularly, indicative of an active process of exportation that would be enhanced by cdG. We also obtained evidence that cdG increases the number of extracellular Gap proteoforms, suggesting a link between cdG, the post-translational modification and the export of cytoplasmic proteins.

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.

Dual GGDEF/EAL-Domain Protein RmcA Controls the Type III Secretion System of Pseudomonas aeruginosa by Interaction with CbrB.

Cyclic diguanylate (c-di-GMP) is a major bacterial secondary signaling molecule that controls a multitude of cellular processes. More than 40 genes encoding diguanylate cyclases and phosphodiesterases have been identified in Pseudomonas aeruginosa, and many of them have been intensively investigated. However, the mechanism through which they achieve signaling specificity remains unclear. Here, we revealed that the absence of the dual GGDEF/EAL-domain protein RmcA significantly affected biofilm formation of P. aeruginosa PAO1 and led to upregulated expression of the type III secretion system (T3SS) genes; overexpression of RmcA strongly reduced the expression of T3SS. Further investigation showed that the regulatory function of RmcA was independent of the Gac/Rsm pathway. To identify the interaction partners of RmcA involved in this process, bacterial two-hybrid library screening was performed. We found that RmcA directly interacts with a two-component response regulator CbrB, which is involved in the regulation of biofilm formation and T3SS expression by RmcA. These findings reveal that the dual-domain GGDEF/EAL protein RmcA could achieve specificity of action through physical interaction with CbrB, which extends understanding the complex regulatory network of the c-di-GMP signaling.

H-NS Represses Biofilm Formation and c-di-GMP Synthesis in Vibrio parahaemolyticus

ObjectiveThis study aimed to investigate the regulation of histone-like nucleoid structuring protein (H-NS) on biofilm formation and cyclic diguanylate (c-di-GMP) synthesis in Vibrio parahaemolyticus RIMD2210633. MethodsRegulatory mechanisms were analyzed by the combined utilization of crystal violet staining, quantification of c-di-GMP, quantitative real-time polymerase chain reaction, LacZ fusion, and electrophoretic-mobility shift assay. ResultsThe deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V. parahaemolyticus RIMD2210633. H-NS can bind the upstream promoter–proximal DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979 to repress their transcription. These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism. ConclusionOne of the mechanisms by which H-NS represses the biofilm formation by V. parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

High Levels of Cyclic Diguanylate Interfere with Beneficial Bacterial Colonization.

ABSTRACTDuring colonization of the Hawaiian bobtail squid (Euprymna scolopes), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low-c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high-c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a regulatory interaction where transcription of one polysaccharide locus is inhibited by the presence of the other polysaccharide. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates.