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Biofilms in food processing environments pose a potential risk of contamination by pathogenic and spoilage microorganisms, and Yersinia enterocolitica, known for its wide temperature adaptation, is the third most common zoonotic pathogen in the European Union. This study investigates the inhibitory effects of protocatechuic acid (PCA) at sub-inhibitory concentrations on various stages of Y. enterocolitica biofilm formation. Core genes are identified through transcriptome data and weighted gene co-expression network analysis (WGCNA), and the potential binding sites are predicted using molecular docking. Additionally, polyvinyl alcohol (PVA) films containing PCA are prepared to assess their antibacterial and mechanical properties. PCA is found to inhibit biofilm formation by up to 50.5% without affecting bacterial growth, reducing flagella-mediated motility, hydrophobicity, cohesion, and extracellular polysaccharides (EPS) content, and inhibiting the production of acyl-homoserine lactones (AHLs). Transcriptome data reveal that PCA inhibits biofilm formation by suppressing the quorum sensing system and reducing the expression of genes such as CsrD, yenR, FlhD, and FlhC. The PVA-PCA antimicrobial film exhibits robust mechanical properties, effectively reducing total colonies in pork and slowing down the deterioration rate. This study enhances our understanding of PCA's inhibitory effects on Y. enterocolitica and suggests its potential application as a quorum-sensing inhibitor in antimicrobial films for the food industry. [ABSTRACT FROM AUTHOR]

Bacterial biofilms have been implicated in several chronic infections. After initial attachment, a critical first step in biofilm formation is a cell inducing a surface-sensing response. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, two second messengers, cyclic diguanylate monophosphate (c-di-GMP) and cyclic adenosine monophosphate (cAMP), are produced by different surface-sensing mechanisms. However, given the disparate cellular behaviors regulated by these second messengers, how newly attached cells coordinate these pathways remains unclear. Some of the uncertainty relates to studies using different strains, experimental systems, and usually focusing on a single second messenger. In this study, we developed a tricolor reporter system to simultaneously gauge c-di-GMP and cAMP levels in single cells. Using PAO1, we show that c-di-GMP and cAMP are selectively activated in two commonly used experimental systems to study surface sensing. By further examining the conditions that differentiate a c-di-GMP or cAMP response, we demonstrate that an agarose-air interface activates cAMP signaling through type IV pili and the Pil-Chp system. However, a liquid-agarose interface favors the activation of c-di-GMP signaling. This response is dependent on flagellar motility and correlated with higher swimming speed. Collectively, this work indicates that c-di-GMP and cAMP signaling responses are dependent on the surface context. [ABSTRACT FROM AUTHOR]

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

Bacillus velezensis is a promising candidate for biocontrol applications. A common second messenger molecule, bis-(3,5)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has the ability to regulate a range of physiological functions that impact the effectiveness of biocontrol. However, the status of the c-di-GMP signaling pathway in biocontrol strain LQ-3 remains unknown. Strain LQ-3, which was isolated from wheat rhizosphere soil, has shown effective control of wheat sharp eyespot and has been identified as B. velezensis through whole-genome sequencing analyses. In this study, we investigated the intracellular c-di-GMP signaling pathway of LQ-3 and further performed a comparative genomic analysis of LQ-3 and 29 other B. velezensis strains. The results revealed the presence of four proteins containing the GGDEF domain, which is the conserved domain for c-di-GMP synthesis enzymes. Additionally, two proteins were identified with the EAL domain, which represents the conserved domain for c-di-GMP degradation enzymes. Furthermore, one protein was found to possess a PilZ domain, indicative of the conserved domain for c-di-GMP receptors in LQ-3. These proteins are called DgcK, DgcP, YybT, YdaK, PdeH, YkuI, and DgrA, respectively; they are distributed in a similar manner across the strains and belong to the signal transduction family. We selected five genes from the aforementioned seven genes for further study, excluding YybT and DgrA. They all play a role in regulating the motility, biofilm formation, and colonization of LQ-3. This study reveals the c-di-GMP signaling pathway associated with biocontrol features in B. velezensis LQ-3, providing guidance for the prevention and control of wheat sharp eyespot by LQ-3. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus. [ABSTRACT FROM AUTHOR]

This paper presents the work performed to transition a lab-scale synthesis (1 g) to a large-scale (400 g) synthesis of the 3-5-diamino-1H-Pyrazole Disperazol, a new pharmaceutical for treatment of antibiotic-resistant Pseudomonas aeruginosa biofilm infections. The potentially hazardous diazotisation step in the lab-scale synthesis was transformed to a safe and easy-to-handle flow chemistry step. Additionally, the paper presents an OSHA-recommended safety assessment of active compound E, as performed by Fauske and Associates, LLC, Burr Ridge, IL, USA. [ABSTRACT FROM AUTHOR]

Aims Klebsiella pneumoniae , an important opportunistic pathogen of nosocomial inflection, is known for its ability to form biofilm. The purpose of the current study is to assess how co- or mono-cultured probiotics affect K. pneumoniae 's ability to produce biofilms and investigate the potential mechanisms by using a polyester nonwoven chemostat and a Caco-2 cell line. Methods and Results Compared with pure cultures of Lactobacillus rhamnosus and Lactobacillus sake , the formation of K. pneumoniae biofilm was remarkably inhibited by the mixture of L. rhamnosus, L. sake , and Bacillus subtilis at a ratio of 5:5:1 by means of qPCR and FISH assays. In addition, Lactobacillus in combination with B. subtilis could considerably reduce the adherence of K. pneumoniae to Caco-2 cells by using inhibition, competition, and displacement assays. According to the RT-PCR assay, the adsorption of K. pneumoniae to Caco-2 cells was effectively inhibited by the co-cultured probiotics, leading to significant reduction in the expression of proinflammatory cytokines induced by K. pneumoniae. Furthermore, the HPLC and RT-PCR analyses showed that the co-cultured probiotics were able to successfully prevent the expression of the biofilm-related genes of K. pneumoniae by secreting plenty of organic acids as well as the second signal molecule (c-di-GMP), resulting in inhibition on biofilm formation. Conclusion Co-culture of L. sake, L. rhamnosus , and B. subtilis at a ratio of 5:5:1 could exert an antagonistic effect on the colonization of pathogenic K. pneumoniae by down-regulating the expression of biofilm-related genes. At the same time, the co-cultured probiotics could effectively inhibit the adhesion of K. pneumoniae to Caco-2 cells and block the expression of proinflammatory cytokines induced by K. pneumoniae. [ABSTRACT FROM AUTHOR]

Solid surfaces submerged in liquid in natural environments alter bacterial swimming behavior and serve as platforms for bacteria to form biofilms. In the initial stage of biofilm formation, bacteria detect surfaces and increase the intracellular level of the second messenger c-di-GMP, leading to a reduction in swimming speed. The impact of this speed reduction on bacterial surface swimming remains unclear. In this study, we utilized advanced microscopy techniques to examine the effect of swimming speed on bacterial surface swimming behavior. We found that a decrease in swimming speed reduces the cell-surface distance and prolongs the surface trapping time. Both these effects would enhance bacterial surface sensing and increase the likelihood of cells adhering to the surface, thereby promoting biofilm formation. We also examined the surface-escaping behavior of wild-type Escherichia coli and Pseudomonas aeruginosa, noting distinct surface-escaping mechanisms between the two bacterial species. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

Biofilms are complex bacterial communities characterized by a high persister prevalence, which contributes to chronic and relapsing infections. Historically, persister formation in biofilms has been linked to constraints imposed by their dense structures. However, we observed an elevated persister frequency accompanying the stage of cell adhesion, marking the onset of biofilm development. Subsequent mechanistic studies uncovered a comparable type of toxin-antitoxin (TA) module (TA-like system) triggered by cell adhesion, which is responsible for this elevation. In this module, the toxin HipH acts as a genotoxic deoxyribonuclease, inducing DNA double strand breaks and genome instability. While the second messenger c-di-GMP functions as the antitoxin, exerting control over HipH expression and activity. The dynamic interplay between c-di-GMP and HipH levels emerges as a crucial determinant governing genome stability and persister generation within biofilms. These findings unveil a unique TA system, where small molecules act as the antitoxin, outlining a biofilm-specific molecular mechanism influencing genome stability and antibiotic persistence, with potential implications for treating biofilm infections.

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

Type IV pili (TFP) play a crucial role in the sensing of the external environment for several bacteria. This surface sensing is essential for the lifestyle transitions of several bacteria and involvement in pathogenesis. However, the precise mechanisms underlying TFP's integration of environmental cues, particularly in regulating the TFP-Chp system and its effects on Xanthomonas physiology, social behavior, and virulence, remain poorly understood. In this study, we focused on investigating Clp, a global transcriptional regulator similar to CRP-like proteins, in Xanthomonas oryzae pv. oryzae, a plant pathogen. Our findings reveal that Clp integrates environmental cues detected through diffusible signaling factor (DSF) quorum sensing into the TFP-Chp regulatory system. It accomplishes this by directly binding to TFP-Chp promoters in conjunction with intracellular levels of cyclic-di-GMP, a ubiquitous bacterial second messenger, thereby controlling TFP expression. Moreover, Clp-mediated regulation is involved in regulating several cellular processes, including the production of virulence-associated functions. Collectively, these processes contribute to host colonization and disease initiation. Our study elucidates the intricate regulatory network encompassing Clp, environmental cues, and the TFP-Chp system, providing insights into the molecular mechanisms that drive bacterial virulence in Xanthomonas spp. These findings offer valuable knowledge regarding Xanthomonas pathogenicity and present new avenues for innovative strategies aimed at combating plant diseases caused by these bacteria. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Nucleotide second messengers are highly versatile signaling molecules that regulate a variety of key biological processes in bacteria. The best‐studied examples are cyclic AMP (cAMP) and bis‐(3′–5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP), which both act as global regulators. Global regulatory frameworks of c‐di‐GMP and cAMP in bacteria show several parallels but also significant variances. In this review, we illustrate the global regulatory models of the two nucleotide second messengers, compare the different regulatory frameworks between c‐di‐GMP and cAMP, and discuss the mechanisms and physiological significance of cross‐regulation between c‐di‐GMP and cAMP. c‐di‐GMP responds to numerous signals dependent on a great number of metabolic enzymes, and it regulates various signal transduction pathways through its huge number of effectors with varying activities. In contrast, due to the limited quantity, the cAMP metabolic enzymes and its major effector are regulated at different levels by diverse signals. cAMP performs its global regulatory function primarily by controlling the transcription of a large number of genes via cAMP receptor protein (CRP) in most bacteria. This review can help us understand how bacteria use the two typical nucleotide second messengers to effectively coordinate and integrate various physiological processes, providing theoretical guidelines for future research.

β-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent β-lactam resistance in Enterobacter cloacae. However, NagZ’s role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ’s function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

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

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. [ABSTRACT FROM AUTHOR]

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production. [ABSTRACT FROM AUTHOR]

ABSTRACT The communication between plants and their microbiota is highly dynamic and involves a complex network of signal molecules. Among them, the auxin indole-3-acetic acid (IAA) is a critical phytohormone that not only regulates plant growth and development, but is emerging as an important inter- and intra-kingdom signal that modulates many bacterial processes that are important during interaction with their plant hosts. However, the corresponding signaling cascades remain largely unknown. Here, we advance our understanding of the largely unknown mechanisms by which IAA carries out its regulatory functions in plant-associated bacteria. We showed that IAA caused important changes in the global transcriptome of the rhizobacterium Serratia plymuthica and multidisciplinary approaches revealed that IAA sensing interferes with the signaling mediated by other pivotal plant-derived signals such as amino acids and 4-hydroxybenzoic acid. Exposure to IAA caused large alterations in the transcript levels of genes involved in amino acid metabolism, resulting in significant metabolic alterations. IAA treatment also increased resistance to toxic aromatic compounds through the induction of the AaeXAB pump, which also confers resistance to IAA. Furthermore, IAA promoted motility and severely inhibited biofilm formation; phenotypes that were associated with decreased c-di-GMP levels and capsule production. IAA increased capsule gene expression and enhanced bacterial sensitivity to a capsule-dependent phage. Additionally, IAA induced the expression of several genes involved in antibiotic resistance and led to changes in the susceptibility and responses to antibiotics with different mechanisms of action. Collectively, our study illustrates the complexity of IAA-mediated signaling in plant-associated bacteria.IMPORTANCESignal sensing plays an important role in bacterial adaptation to ecological niches and hosts. This communication appears to be particularly important in plant-associated bacteria since they possess a large number of signal transduction systems that respond to a wide diversity of chemical, physical, and biological stimuli. IAA is emerging as a key inter- and intra-kingdom signal molecule that regulates a variety of bacterial processes. However, despite the extensive knowledge of the IAA-mediated regulatory mechanisms in plants, IAA signaling in bacteria remains largely unknown. Here, we provide insight into the diversity of mechanisms by which IAA regulates primary and secondary metabolism, biofilm formation, motility, antibiotic susceptibility, and phage sensitivity in a biocontrol rhizobacterium. This work has important implications for our understanding of bacterial ecology in plant environments and for the biotechnological and clinical applications of IAA, as well as related molecules.

Abstract Background Oligoribonuclease (orn) of P. aeruginosa is a highly conserved exonuclease, which can regulate the global gene expression levels of bacteria through regulation of both the nanoRNA and c-di-GMP. NanoRNA can regulate the expression of the bacterial global genome as a transcription initiator, and c-di-GMP is the most widely second messenger in bacterial cells. Objective This study seeks to elucidate on the regulation by orn on pathogenicity of P. aeruginosa. Methods P. aeruginosa with orn deletion was constructed by suicide plasmid homologous recombination method. The possible regulatory process of orn was analyzed by TMT quantitative labeling proteomics. Then experiments were conducted to verify the changes of Δorn on bacterial motility, virulence and biofilm formation. Bacterial pathogenicity was further detected in cell and animal skin trauma models. ELISA detection c-di-GMP concentration and colony aggregation and biofilm formation were observed by scanning electron microscope. Results orn deletion changed the global metabolism of P. aeruginosa and reduced intracellular energy metabolism. It leads to the disorder of the quorum sensing system, the reduction of bacterial motility and virulence factors pyocyanin and rhamnolipids. But, orn deletion enhanced pathogenicity in vitro and in vivo, a high level of c-di-GMP and biofilm development of P. aeruginosa. Conclusion orn regulates the ability of P. aeruginosa to adapt to the external environment.

Vibrio spp. are opportunistic human and animal pathogens found ubiquitously in marine environments. Globally, there is a predicted rise in the prevalence of Vibrio spp. due to increasing ocean temperatures, which carries significant implications for public health and the seafood industry. Consequently, there is an urgent need for enhanced strategies to control Vibrio spp. and prevent contamination, particularly in aquaculture and seafood processing facilities. Presently, these industries employ various disinfectants, including benzalkonium chloride (BAC), as part of their management strategies. While higher concentrations of BAC may be effective against these pathogens, inadequate rinsing post-disinfection could result in residual concentrations of BAC in the surrounding environment. This study aimed to investigate the adaptation and survival of Vibrio spp. exposed to varying concentrations of BAC residues. Results revealed that Vibrio bacteria, when exposed, exhibited a phenotypic adaptation characterized by an increase in biofilm biomass. Importantly, this effect was found to be strainspecific rather than species-specific. Exposure to BAC residues induced physiological changes in Vibrio biofilms, leading to an increase in the number of injured and alive cells within the biofilm. The exact nature of the "injured" bacteria remains unclear, but it is postulated that BAC might heighten the risk of viable but non-culturable (VBNC) bacteria development. These VBNC bacteria pose a significant threat, especially since they cannot be detected using the standard culture-based methods commonly employed for microbiological risk assessment in aquaculture and seafood industries. The undetected presence of VBNC bacteria could result in recurrent contamination events and subsequent disease outbreaks. This study provides evidence regarding the role of c-di-GMP signaling pathways in Vibrio adaptation mechanisms and suggests that c-di-GMP mediated repression is a potential avenue for further research. The findings underscore that the misuse and overuse of BAC may increase the risk of biofilm development and bacterial survival within the seafood processing chain. [ABSTRACT FROM AUTHOR]

Background: Oligoribonuclease (orn) of P. aeruginosa is a highly conserved exonuclease, which can regulate the global gene expression levels of bacteria through regulation of both the nanoRNA and c-di-GMP. NanoRNA can regulate the expression of the bacterial global genome as a transcription initiator, and c-di-GMP is the most widely second messenger in bacterial cells. Objective: This study seeks to elucidate on the regulation by orn on pathogenicity of P. aeruginosa. Methods: P. aeruginosa with orn deletion was constructed by suicide plasmid homologous recombination method. The possible regulatory process of orn was analyzed by TMT quantitative labeling proteomics. Then experiments were conducted to verify the changes of Δorn on bacterial motility, virulence and biofilm formation. Bacterial pathogenicity was further detected in cell and animal skin trauma models. ELISA detection c-di-GMP concentration and colony aggregation and biofilm formation were observed by scanning electron microscope. Results: orn deletion changed the global metabolism of P. aeruginosa and reduced intracellular energy metabolism. It leads to the disorder of the quorum sensing system, the reduction of bacterial motility and virulence factors pyocyanin and rhamnolipids. But, orn deletion enhanced pathogenicity in vitro and in vivo, a high level of c-di-GMP and biofilm development of P. aeruginosa. Conclusion: orn regulates the ability of P. aeruginosa to adapt to the external environment. [ABSTRACT FROM AUTHOR]

Biofilm dispersal contributes to bacterial spread and disease transmission. However, its exact mechanism, especially that in the pathogen Mycobacterium tuberculosis, is unclear. In this study, the cellulase activity of the M. tuberculosis Rv0062 protein was characterized, and its effect on mycobacterial biofilm dispersal was analyzed by observation of the structure and components of Rv0062-treated biofilm in vitro. Meanwhile, the metabolite factors that induced cellulase-related biofilm dispersal were also explored with metabolome analysis and further validations. The results showed that Rv0062 protein had a cellulase activity with a similar optimum pH (6.0) and lower optimum temperature (30 °C) compared to the cellulases from other bacteria. It promoted mycobacterial biofilm dispersal by hydrolyzing cellulose, the main component of extracellular polymeric substrates of mycobacterial biofilm. A metabolome analysis revealed that 107 metabolites were significantly altered at different stages of M. smegmatis biofilm development. Among them, a decrease in gamma-aminobutyric acid (GABA) promoted cellulase-related biofilm dispersal, and this effect was realized with the down-regulation of the bacterial signal molecule c-di-GMP. All these findings suggested that cellulase promotes mycobacterial biofilm dispersal and that this process is closely associated with biofilm metabolite alterations. [ABSTRACT FROM AUTHOR]

In bacteria, the second messenger cyclic di-GMP (c-di-GMP) is synthesized and degraded by multiple diguanylate cyclases (DGCs) and phosphodiesterases. A high level of c-di-GMP induces biofilm formation and represses motility. WspR, a hybrid response regulator DGC, produces c-di-GMP when it is phosphorylated. FlhF, a signal recognition particle-type GTPase, is initially localized to the cell poles and is indispensable for polar flagellar localization in Pseudomonas aeruginosa. In this study, we report that deletion of flhF affected biofilm formation and the c-di-GMP level in P. aeruginosa. Phenotypic analysis of a flhF knockout mutant revealed increased biofilm formation, wrinkled colonies on Congo red agar, and an elevated c-di-GMP level compared to the wild-type strain, PAO1. Yeast and bacterial two-hybrid systems showed that FlhF binds to the response regulator HsbR, and HsbR binds to WspR. Deletion of hsbR or wspR in the ΔflhF background abolished the phenotype of ΔflhF. In addition, confocal microscopy demonstrated that WspR-GFP was distributed throughout the cytoplasm and formed a visible cluster at one cell pole in PAO1 and ΔhsbR, but it was mainly distributed as visible clusters at the lateral side of the periplasm and with visible clusters at both cell poles in ΔflhF. These findings suggest that FlhF influences the subcellular cluster and localization of WspR and negatively modulates WspR DGC activity in a manner dependent on HsbR. Together, our findings demonstrate a novel mechanism for FlhF modulating the lifestyle transition between motility and biofilm via HsbR to regulate the DGC activity of WspR. [ABSTRACT FROM AUTHOR]

Acyl-homoserine lactones (AHLs) are quorum-sensing signaling molecules in Gram-negative bacteria and positively regulate biofilm formation in Salmonella under specific conditions. In this study, biofilm formation in Salmonella enterica was evaluated at 28 and 37 °C, under aerobic and anaerobic conditions. Additionally, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on biofilm formation and the expression of genes related to the synthesis of structural components, regulation, and quorum sensing was assessed under anaerobiosis at 28 and 37 °C. Biofilm formation was found not to be influenced by the atmospheric conditions at 28 °C. However, it was reduced at 37 °C under anaerobiosis. C12-HSL enhanced biofilm formation at 37 °C under anaerobiosis and increased the expression of the adrA and luxS genes, suggesting an increase in c-di-GMP, a second messenger that controls essential physiological functions in bacteria. These results provide new insights into the regulation of biofilm formation in Salmonella under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa can cause a wide array of chronic and acute infections associated with its ability to rapidly switch between planktonic, biofilm, and dispersed lifestyles, each with a specific arsenal for bacterial survival and virulence. At the cellular level, many of the physiological transitions are orchestrated by the intracellular second messenger c-di-GMP and its receptor-effector FleQ. A bacterial enhancer binding protein, FleQ acts as a master regulator of both flagellar motility and adherence factor secretion and uses remarkably different transcription activation mechanisms depending on its dinucleotide loading state, adenosine triphosphatase (ATPase) activity, interactions with polymerase sigma (s) factors, and complexation with a second ATPase, FleN. How the FleQ-FleN tandem can exert diverse effects through recognition of a conserved FleQ binding consensus has remained enigmatic. Here, we provide cryogenic electron microscopy (cryo-EM) structures of both c-di-GMP-bound and c-di-GMP-free FleQ-FleN complexes which deepen our understanding of the proteins' (di)nucleotide-dependent conformational switching and fine-tuned roles in gene expression regulation. [ABSTRACT FROM AUTHOR]

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

Abstract Cyclic bis-(3', 5')-dimeric guanosine monophosphate (c-di-GMP) is ubiquitous in many bacterial species, where it functions as a nucleotide-based secondary messenger and is a vital regulator of numerous biological processes. Due to its ubiquity, most bacterial species possess a wide range of downstream receptors that has a binding affinity to c-di-GMP and elicit output responses. In eukaryotes, several enzymes and riboswitches operate as receptors that interact with c-di-GMP and transduce cellular or environmental signals. This review examines the functional variety of receptors in prokaryotic and eukaryotic systems that exhibit distinct biological responses after interacting with c-di-GMP. Evolutionary relationships and similarities in distance among the c-di-GMP receptors in various bacterial species were evaluated to understand their specificities. Furthermore, residues of receptors involved in c-di-GMP binding are summarized. This review facilitates the understanding of how distinct receptors from different origins bind c-di-GMP equally well, yet fulfill diverse biological roles at the interspecies, intraspecies, and interkingdom levels. Furthermore, it also highlights c-di-GMP receptors as potential therapeutic targets, particularly those found in pathogenic microorganisms. Video Abstract

Bacillus velezensis is a promising candidate for biocontrol applications. A common second messenger molecule, bis-(3,5)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has the ability to regulate a range of physiological functions that impact the effectiveness of biocontrol. However, the status of the c-di-GMP signaling pathway in biocontrol strain LQ-3 remains unknown. Strain LQ-3, which was isolated from wheat rhizosphere soil, has shown effective control of wheat sharp eyespot and has been identified as B. velezensis through whole-genome sequencing analyses. In this study, we investigated the intracellular c-di-GMP signaling pathway of LQ-3 and further performed a comparative genomic analysis of LQ-3 and 29 other B. velezensis strains. The results revealed the presence of four proteins containing the GGDEF domain, which is the conserved domain for c-di-GMP synthesis enzymes. Additionally, two proteins were identified with the EAL domain, which represents the conserved domain for c-di-GMP degradation enzymes. Furthermore, one protein was found to possess a PilZ domain, indicative of the conserved domain for c-di-GMP receptors in LQ-3. These proteins are called DgcK, DgcP, YybT, YdaK, PdeH, YkuI, and DgrA, respectively; they are distributed in a similar manner across the strains and belong to the signal transduction family. We selected five genes from the aforementioned seven genes for further study, excluding YybT and DgrA. They all play a role in regulating the motility, biofilm formation, and colonization of LQ-3. This study reveals the c-di-GMP signaling pathway associated with biocontrol features in B. velezensis LQ-3, providing guidance for the prevention and control of wheat sharp eyespot by LQ-3.

Vibrio spp. are opportunistic human and animal pathogens found ubiquitously in marine environments. Globally, there is a predicted rise in the prevalence of Vibrio spp. due to increasing ocean temperatures, which carries significant implications for public health and the seafood industry. Consequently, there is an urgent need for enhanced strategies to control Vibrio spp. and prevent contamination, particularly in aquaculture and seafood processing facilities. Presently, these industries employ various disinfectants, including benzalkonium chloride (BAC), as part of their management strategies. While higher concentrations of BAC may be effective against these pathogens, inadequate rinsing post-disinfection could result in residual concentrations of BAC in the surrounding environment. This study aimed to investigate the adaptation and survival of Vibrio spp. exposed to varying concentrations of BAC residues. Results revealed that Vibrio bacteria, when exposed, exhibited a phenotypic adaptation characterized by an increase in biofilm biomass. Importantly, this effect was found to be strain-specific rather than species-specific. Exposure to BAC residues induced physiological changes in Vibrio biofilms, leading to an increase in the number of injured and alive cells within the biofilm. The exact nature of the “injured” bacteria remains unclear, but it is postulated that BAC might heighten the risk of viable but non-culturable (VBNC) bacteria development. These VBNC bacteria pose a significant threat, especially since they cannot be detected using the standard culture-based methods commonly employed for microbiological risk assessment in aquaculture and seafood industries. The undetected presence of VBNC bacteria could result in recurrent contamination events and subsequent disease outbreaks. This study provides evidence regarding the role of c-di-GMP signaling pathways in Vibrio adaptation mechanisms and suggests that c-di-GMP mediated repression is a potential avenue for further research. The findings underscore that the misuse and overuse of BAC may increase the risk of biofilm development and bacterial survival within the seafood processing chain.

ABSTRACTSpecies of the phylum Actinobacteria have long time been thought mostly non-motile and non-flagellated, in spite of their extraordinary diversity in morphology, physiology, and ecology. It remains unclear how very few actinobacterial species acquired their flagellar genes and the role of flagellar motility in the evolution of different lineages within this large phylum. Here, we performed a comprehensive phylogenomic analysis of flagellar components in all actinobacterial species, including the deepest branches of this phylum identified recently. Our results provide robust evidence that all actinobacterial flagella shared a common origin and likely evolved from their last common flagellated ancestor by vertical inheritance. Later lineages underwent massive flagellar losses with the ecological switch from aquatic to terrestrial land or host-associated environments. In addition, the actinobacterial flagella also showed degenerative evolution in terms of their composition, leading to the simplest rod known so far. Furthermore, these evolutionary changes of flagella were accompanied by noteworthy variations in the chemosensory system and c-di-GMP mediated signaling network. Overall, we present here a detailed picture of flagellar evolution in Actinobacteria, which provides new insights into the evolution of major actinobacterial lineages and previously unobserved links between flagella and other biological features. The simplest flagellar rod as a product of degenerative evolution could possibly serve as a model for future nanomachine reconstruction in synthetic biology.IMPORTANCEFlagellar motility plays an important role in the environmental adaptation of bacteria and is found in more than 50% of known bacterial species. However, this important characteristic is sparsely distributed within members of the phylum Actinobacteria, which constitutes one of the largest bacterial groups. It is unclear why this important fitness organelle is absent in most actinobacterial species and the origin of flagellar genes in other species. Here, we present detailed analyses of the evolution of flagellar genes in Actinobacteria, in conjunction with the ecological distribution and cell biological features of major actinobacterial lineages, and the co-evolution of signal transduction systems. The results presented in addition to clarifying the puzzle of sporadic distribution of flagellar motility in Actinobacteria, also provide important insights into the evolution of major lineages within this phylum.

ABSTRACT Dickeya dadantii is a Gram-negative bacterium that causes soft rot diseases in a variety of plants. This pathogen secretes pectate lyase (Pel) enzymes to degrade the plant cell wall, a process controlled by the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP). VfmH is a Fis-family response regulator of the Vfm quorum sensing system, which has been previously reported to positively regulate pel transcription. Our results demonstrated that VfmH upregulated the production of Pel at low c-di-GMP levels, which was suppressed by c-di-GMP via deleting gene egcpB, encoding a phosphodiesterase that degrades c-di-GMP. Multiple sequence alignments between FleQ, a known c-di-GMP binding protein, and VfmH revealed several potential c-di-GMP binding motifs. We further validated these motifs via single amino acid substitutions. Indeed, derivatives of VfmH, including VfmHR195A, VfmHR279A, and VfmHR344A, abolished the c-di-GMP binding ability and the corresponding regulation in Pel. We found that VfmH exhibited ATPase activity, and this could be hindered by c-di-GMP binding. Interestingly, our results showed that VfmH interacted with CRP, suggesting that the cyclic AMP (cAMP) signaling pathway is also involved in the VfmH-mediated Pel regulation. Taken together, our study suggested that D. dadantii amalgamates both c-di-GMP and cAMP signaling networks along with the Vfm quorum sensing pathway to control Pel. IMPORTANCE Bacteria respond to environmental changes and adapt to host systems. The response regulator VfmH of the Vfm quorum sensing system regulates a crucial virulence factor, pectate lyase (Pel), in Dickeya dadantii. At high c-di-GMP concentrations, VfmH binds c-di-GMP, resulting in the loss of its activation property in the Pel and virulence regulation in D. dadantii. VfmH binds to c-di-GMP via three conserved arginine residues, and mutations of these residues eliminate the c-di-GMP-related phenotypes of VfmH in Pel synthesis. Our data also show that VfmH interacts with CRP to regulate pelD transcription, thus integrating cyclic AMP and c-di-GMP signaling pathways to control virulence in D. dadantii. We propose that VfmH is an important intermediate factor incorporating quorum sensing and nucleotide signaling pathways for the collective regulation of D. dadantii pathogenesis.

The rapid emergence of bacterial resistance to antibiotics in current use is occurring worldwide and poses a significant threat to global healthcare systems. Recent research to identify new effective anti-bacterial agents has focused on regulatory pathways as targets for interference. Regulatory mechanisms employing intracellular Bis-(3',5') cyclic di-guanylate (c-di-GMP) as a secondary messenger represent a distinct category of subjects. This molecule, c-di-GMP, is present in nearly all bacterial species and plays a pivotal role in governing various biological processes, encompassing antibiotic resistance, biofilm formation, and virulence. Alteration of the cellular concentrations of the nucleotide through modulation of associated signaling pathways has the potential to reduce biofilm formation or increase susceptibility of the biofilm bacteria to antibiotics. Here, we have developed a screen for compounds that alter c-di-GMP levels in Pseudomonas aeruginosa in co-culture with bronchial epithelial cells. Through the assay of 200 natural compounds, we were able to identify several substances showing promising effects on P. aeruginosa in a host biofilm infection model. Importantly, we detected compounds that inhibit c-di-GMP levels and showed significant influence on biofilm formation and virulence in P. aeruginosa in vitro and in vivo. Consequently, we offer proof-of-concept information regarding swift and practical drug screening assays, suitable for medium- to high-throughput applications, which target the c-di-GMP signaling pathways in this significant Gram-negative pathogen. [ABSTRACT FROM AUTHOR]

Aeromonas veronii. a ubiquitous of zoonotic disease pathogen, depends on adhesion as the crucial way to colonize the gastrointestinal tract of humans and animals, which further causes severe gastrointestinal diseases and parenteral infections. However, the adherence mechanism of A. veronii has not been fully characterized. Therefore, we investigate the effect of autoinducer-2 (AI-2) on adherence of A. veronii through facilitating the expression of mannose-sensitive hemagglutinin (MSHA) type IV pili genes mediated by cyclic diguanosine monophosphate (c-di-GMP). The deficiency of AI-2 significantly lowered the adherence of A. veronii to erythrocytes and intestinal mucus, and the complement of AI-2 could increase its adherence ability. The deficiency of AI-2 only limited the formation of pili, instead of outer-membrane proteins and lipopolysaccharide, through reducing the expression levels of MSHA type IV pili genes due to the decline of c-di-GMP. The addition of guanosine triphosphate (GTP) could increase the content of c-di-GMP and the expression of MSHA type IV pili genes, and further promote adherence of A. veronii. Therefore, this study reveals, for the first time, adherence mediated by c-di-GMP with MshE as the c-di-GMP receptor is positively regulated by AI-2 in A. veronii, which increases the understanding of colonization strategy of pathogen and may facilitate control of A. veronii infection to host. IMPORTANCE Aeromonas veronii can adhere to host cells through different adherence factors including outer-membrane proteins (OMPs), lipopolysaccharide (LPS), and pili, but its adherence mechanisms are still unclear. Here, we evaluated the effect of autoinducer-2 (AI-2) on adherence of A. veronii and its regulation mechanism. After determination of the promotion effect of AI-2 on adherence, we investigated which adherence factor was regulated by AI-2, and the results show that AI-2 only limits the formation of pili. Among the four distinct pili systems, only the mannose-sensitive hemagglutinin (MSHA) type IV pili genes were significantly downregulated after deficiency of AI-2. MshE, an ATPase belonged to MSHA type IV pilin, was confirmed as c-di-GMP receptor, that can bind with c-di-GMP which is positively regulated by AI-2, and the increase of c-di-GMP can promote the expression of MSHA type IV pili genes and adherence of A. veronii. Therefore, this study confirms that c-di-GMP positively regulated by AI-2 binds with MshE, then increases the expression of MSHA pili genes, finally promoting adherence of A. veronii, suggesting a multilevel positive regulatory adhesion mechanism that is responsible for A. veronii adherence. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner–Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self‐flocculated, the bacterial cells are more productive, since they can self‐immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self‐flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c‐di‐GMP) on self‐flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c‐di‐GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c‐di‐GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c‐di‐GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self‐flocculating morphology to boost production but also understanding mechanism underlying c‐di‐GMP biosynthesis and degradation in the bacterium. [ABSTRACT FROM AUTHOR]

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

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 AVA4033p4033. 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. [ABSTRACT FROM AUTHOR]

Cyclic bis-(3', 5')-dimeric guanosine monophosphate (c-di-GMP) is ubiquitous in many bacterial species, where it functions as a nucleotide-based secondary messenger and is a vital regulator of numerous biological processes. Due to its ubiquity, most bacterial species possess a wide range of downstream receptors that has a binding affinity to c-di-GMP and elicit output responses. In eukaryotes, several enzymes and riboswitches operate as receptors that interact with c-di-GMP and transduce cellular or environmental signals. This review examines the functional variety of receptors in prokaryotic and eukaryotic systems that exhibit distinct biological responses after interacting with c-di-GMP. Evolutionary relationships and similarities in distance among the c-di-GMP receptors in various bacterial species were evaluated to understand their specificities. Furthermore, residues of receptors involved in c-di-GMP binding are summarized. This review facilitates the understanding of how distinct receptors from different origins bind c-di-GMP equally well, yet fulfill diverse biological roles at the interspecies, intraspecies, and interkingdom levels. Furthermore, it also highlights c-di-GMP receptors as potential therapeutic targets, particularly those found in pathogenic microorganisms. C5iF5BkbNAVqYPEX5S1oMB Video Abstract [ABSTRACT FROM AUTHOR]

Bacteria are single-celled organisms that carry a comparatively small set of genetic information, typically consisting of a few thousand genes that can be selectively activated or repressed in an energy-efficient manner and transcribed to encode various biological functions in accordance with environmental changes. Research over the last few decades has uncovered various ingenious molecular mechanisms that allow bacterial pathogens to sense and respond to different environmental cues or signals to activate or suppress the expression of specific genes in order to suppress host defenses and establish infections. In the setting of infection, pathogenic bacteria have evolved various intelligent mechanisms to reprogram their virulence to adapt to environmental changes and maintain a dominant advantage over host and microbial competitors in new niches. This review summarizes the bacterial virulence programming mechanisms that enable pathogens to switch from acute to chronic infection, from local to systemic infection, and from infection to colonization. It also discusses the implications of these findings for the development of new strategies to combat bacterial infections. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for acute and chronic, hard to treat infections. Persistence of P. aeruginosa is due to its ability to develop into biofilms, which are sessile bacterial communities adhered to substratum and encapsulated in layers of self-produced exopolysaccharides. These biofilms provide enhanced protection from the host immune system and resilience towards antibiotics, which poses a challenge for treatment. Various strategies have been expended for combating biofilms, which involve inhibiting biofilm formation or promoting their dispersal. The current remediation approaches offer some hope for clinical usage, however, treatment and eradication of preformed biofilms is still a challenge. Thus, identifying novel targets and understanding the detailed mechanism of biofilm regulation becomes imperative. Structure-based drug discovery (SBDD) provides a powerful tool that exploits the knowledge of atomic resolution details of the targets to search for high affinity ligands. This review describes the available structural information on the putative target protein structures that can be utilized for high throughput in silico drug discovery against P. aeruginosa biofilms. Integrating available structural information on the target proteins in readily accessible format will accelerate the process of drug discovery. [ABSTRACT FROM AUTHOR]

Biofilm cells have a different physiology than planktonic cells, which has been the focus of most research. Biofilms are complex biostructures that form on any surface that comes into contact with water on a regular basis. They are dynamic, structurally complex systems having characteristics of multicellular animals and multiple ecosystems. The three themes covered in this review are biofilm ecology, biofilm reactor technology and design, and biofilm modeling. Membrane-supported biofilm reactors, moving bed biofilm reactors, granular sludge, and integrated fixed-film activated sludge processes are all examples of biofilm reactors used for water treatment. Biofilm control and/or beneficial application in membrane processes are improving. Biofilm models have become critical tools for biofilm foundational research as well as biofilm reactor architecture and design. At the same time, the differences between biofilm modeling and biofilm reactor modeling methods are acknowledged. [ABSTRACT FROM AUTHOR]

ABSTRACT Some marine bacteria can swim at speeds exceeding 50× their body length per second as they actively seek out fleeting microscale nutrient patches. Yet, even in vast marine environments, most micro-organisms live in surface-associated biofilm communities. Such astonishing speeds can pose a serious challenge to initiating biofilm formation, and our understanding of the mechanisms that regulate deceleration is lacking. Calcium, a major constituent of marine ecosystems, promotes biofilm formation in the marine bacterium and opportunistic human pathogen Vibrio vulnificus by increasing cellular levels of the key secondary signaling molecule bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we show that elevated c-di-GMP levels inhibit V. vulnificus swimming motility and identify a PilZ domain protein, PlzD, that mediates this effect. PlzD is localized at the flagellar pole in slow-moving, initially adherent, and mature biofilm cells. This positioning was partly dependent on the flagellar stator protein PomA and was regulated by c-di-GMP. Mutating the conserved RXXXR c-di-GMP binding motif of the PilZ domain abrogated PlzD activity. Single-cell tracking of bacterial swimming trajectories revealed that PlzD altered the foraging behavior of cells by slowing swimming speed and by decreasing the number of directional changes, ultimately limiting exploration of the surrounding 3D space. The cumulative effects were increased biofilm formation, aggregation, oyster colonization, and attenuated virulence in mice, phenotypes underpinning the evolution of V. vulnificus as a resilient environmental organism and potent human pathogen. IMPORTANCE Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates biofilm formation, a sessile lifestyle that is a nearly universal surface colonization paradigm in bacteria. In general, motility and biofilm formation are inversely regulated by the intracellular second messenger bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we identify a protein, PlzD, bearing a conserved c-di-GMP binding PilZ domain that localizes to the flagellar pole in a c-di-GMP-dependent manner and alters the foraging behavior, biofilm, and virulence characteristics of the opportunistic human pathogen, Vibrio vulnificus. Our data suggest that PlzD interacts with components of the flagellar stator to decrease bacterial swimming speed and changes in swimming direction, and these activities are enhanced when cellular c-di-GMP levels are elevated. These results reveal a physical link between a second messenger (c-di-GMP) and an effector (PlzD) that promotes transition from a motile to a sessile state in V. vulnificus.

ABSTRACT Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is a filamentous cyanobacterium, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen fixation. A widely used type strain [wild type (WT) in this article] of this species has not been reported to show any motile activity. However, we isolated a spontaneous mutant strain that shows active motility (gliding activity) to give rise to complicated colony patterns, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations in the genome of the mutant strain. We confirmed that inactivation of the candidate gene dgc2 (LBDG_02920) in the WT background was sufficient to give rise to motility and morphologically complex colony patterns. This gene encodes a protein containing the GGDEF motif which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the dgc2 mutant significantly facilitated biofilm formation, suggesting a role for the dgc2 gene in suppressing both gliding motility and biofilm formation. Thus, Leptolyngbya is expected to be an excellent genetic model for studying dynamic colony pattern formation and to provide novel insights into the role of DGC family genes in biofilm formation. IMPORTANCE Self-propelled bacteria often exhibit complex collective behaviors, such as formation of dense-moving clusters, which are exemplified by wandering comet-like and rotating disk-like colonies; however, the molecular details of how these structures are formed are scant. We found that a strain of the filamentous cyanobacterium Leptolyngbya deficient in the GGDEF protein gene dgc2 elicits motility and complex and dynamic colony pattern formation, including comet-like and disk-like clusters. Although c-di-GMP has been reported to activate biofilm formation in some bacterial species, disruption of dgc2 unexpectedly enhanced it, suggesting a novel role for this GGDEF protein for inhibiting both colony pattern formation and biofilm formation.

A number of multidrug‐resistant bacterial pathogens code for S1‐P1 nucleases with a poorly understood role. We have characterized a recombinant form of S1‐P1 nuclease from Stenotrophomonas maltophilia, an opportunistic pathogen. S. maltophilia nuclease 1 (SmNuc1) acts predominantly as an RNase and is active in a wide range of temperatures and pH. It retains a notable level of activity towards RNA and ssDNA at pH 5 and 9 and about 10% of activity towards RNA at 10 °C. SmNuc1 with very high catalytic rates outperforms S1 nuclease from Aspergillus oryzae and other similar nucleases on all types of substrates. SmNuc1 degrades second messenger c‐di‐GMP, which has potential implications for its role in the pathogenicity of S. maltophilia. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a significant food‐borne pathogen that is found in diverse aquatic habitats. Quorum sensing (QS), a signaling system for cell–cell communication, plays an important role in V. parahaemolyticus persistence. We characterized the function of three V. parahaemolyticus QS signal synthases, CqsAvp, LuxMvp, and LuxSvp, and show that they are essential to activate QS and regulate swarming. We found that CqsAvp, LuxMvp, and LuxSvp activate a QS bioluminescence reporter through OpaR. However, V. parahaemolyticus exhibits swarming defects in the absence of CqsAvp, LuxMvp, and LuxSvp, but not OpaR. The swarming defect of this synthase mutant (termed Δ3AI) was recovered by overexpressing either LuxOvpD47A, a mimic of dephosphorylated LuxOvp mutant, or the scrABC operon. CqsAvp, LuxMvp, and LuxSvp inhibit lateral flagellar (laf) gene expression by inhibiting the phosphorylation of LuxOvp and the expression of scrABC. Phosphorylated LuxOvp enhances laf gene expression in a mechanism that involves modulating c‐di‐GMP levels. However, enhancing swarming requires phosphorylated and dephosphorylated LuxOvp which is regulated by the QS signals that are synthesized by CqsAvp, LuxMvp, and LuxSvp. The data presented here suggest an important strategy of swarming regulation by the integration of QS and c‐di‐GMP signaling pathways in V. parahaemolyticus. [ABSTRACT FROM AUTHOR]

Expression of rdar (red, dry, and rough) colony morphology-based biofilm formation in Escherichia coli is highly variable. To investigate the molecular mechanisms of semi-constitutive rdar morphotype formation, we compared their cyclic di-GMP turnover protein content and variability to the highly regulated, temperature-dependent morphotype of the historical and modern ST10 isolates E. coli MG1655 and Fec10, respectively. Subsequently, we assessed the effects of cyclic di-GMP turnover protein variants of the EAL phosphodiesterases YcgG and YjcC and the horizontally transferred diguanylate cyclase DgcX on biofilm formation and motility. The two YcgG variants with truncations of the N-terminal CSS signaling domain were oppositely effective in targeting downregulation of rdar biofilm formation compared to the full-length reference protein. Expression of the C-terminal truncated variants YjcCFec67 and YjcCTob1 showed highly diminished apparent phosphodiesterase activity compared to the reference YjcCMG1655. For YjcCFec101, substitution of the C-terminus led to an apparently inactive enzyme. Overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but not to elevated expression of the major biofilm regulator csgD in the "classical" rdar-expressing commensal strain E. coli Fec10. Thus, the c-di-GMP regulating network is highly complex with protein variants displaying substantially different apparent enzymatic activities. [ABSTRACT FROM AUTHOR]

Aims Pseudomonas plecoglossicida (P. plecoglossicida) is the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea) and it causes severe economic loss to its industry. Biofilm formation, related to intracellular cyclic bis (3′–5′) diguanylic acid (c-di-GMP) levels, is essential for the lifestyle of P. plecoglossicida. This research aims to investigate the role of YfiR—a key regulator of the diguanylate cyclase YfiN to regulate c-di-GMP levels and reveal its regulatory function of bacterial virulence expression in P. plecoglossicida. Methods and results A genetic analysis was carried out to identify the yfiBNR operon for c-di-GMP regulation in P. plecoglossicida. Then, we constructed a yfiR mutant and observed increased c-di-GMP levels, enhanced biofilm formation, increased exopolysaccharides, and diminished swimming and swarming motility in this strain. Moreover, through establishing a yolk sac microinjection infection model in zebrafish larvae, an attenuated phenotype of yfiR mutant that manifested as restored survival and lower bacterial colonization was found. Conclusions YfiR is the key regulator of virulence in P. plecoglossicida , which contributes to c-di-GMP level, biofilm formation, exopolysaccharides production, swimming, swarming motility, and bacterial colonization in zebrafish model. [ABSTRACT FROM AUTHOR]

The widespread bacterial second messenger c-di-GMP is responsible for regulating many important physiological functions such as biofilm formation, motility, cell differentiation, and virulence. The synthesis and degradation of c-di-GMP in bacterial cells depend, respectively, on diguanylate cyclases and c-di-GMP-specific phosphodiesterases. Since c-di-GMP metabolic enzymes (CMEs) are often fused to sensory domains, their activities are likely controlled by environmental signals, thereby altering cellular c-di-GMP levels and regulating bacterial adaptive behaviors. Previous studies on c-di-GMP-mediated regulation mainly focused on downstream signaling pathways, including the identification of CMEs, cellular c-di-GMP receptors, and c-di-GMP-regulated processes. The mechanisms of CME regulation by upstream signaling modules received less attention, resulting in a limited understanding of the c-di-GMP regulatory networks. We review here the diversity of sensory domains related to bacterial CME regulation. We specifically discuss those domains that are capable of sensing gaseous or light signals and the mechanisms they use for regulating cellular c-di-GMP levels. It is hoped that this review would help refine the complete c-di-GMP regulatory networks and improve our understanding of bacterial behaviors in changing environments. In practical terms, this may eventually provide a way to control c-di-GMP-mediated bacterial biofilm formation and pathogenesis in general. [ABSTRACT FROM AUTHOR]