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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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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.

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., Importance: In the early phase of biofilm formation, bacteria identify surfaces and increase the intracellular level of the second messenger c-di-GMP, resulting in a decrease in swimming speed. Here, we utilized advanced microscopy techniques to investigate the impact of swimming speed on bacterial surface swimming, focusing on Escherichia coli and Pseudomonas aeruginosa . We found that an increase in swimming speed led to an increase in the radius of curvature and a decrease in surface detention time. These effects were explained through hydrodynamic modeling as a result of an increase in the cell-surface distance with increasing swimming speed. We also observed distinct surface-escaping mechanisms between the two bacterial species. Our study suggests that a decrease in swimming speed could enhance the likelihood of cells adhering to the surface, promoting biofilm formation. This sheds light on the role of reduced swimming speed in the transition from motile to sedentary bacterial lifestyles., Competing Interests: The authors declare no conflict of interest.

Cyclic di-guanosine monophosphate (c-di-GMP) is a bacterial second messenger that governs the lifestyle switch between planktonic and biofilm states. While substantial investigation has focused on the proteins that produce and degrade c-di-GMP, less attention has been paid to the potential for metabolic control of c-di-GMP signaling. Here, we show that micromolar levels of specific environmental purines unexpectedly decrease c-di-GMP and biofilm formation in Pseudomonas aeruginosa. Using a fluorescent genetic reporter, we show that adenosine and inosine decrease c-di-GMP even when competing purines are present. We confirm genetically that purine salvage is required for c-di-GMP decrease. Furthermore, we find that (p)ppGpp prevents xanthosine and guanosine from producing an opposing c-di-GMP increase, reinforcing a salvage hierarchy that favors c-di-GMP decrease even at the expense of growth. We propose that purines can act as a cue for bacteria to shift their lifestyle away from the recalcitrant biofilm state via upstream metabolic control of c-di-GMP signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen causing chronic infections that are associated with the sessile/biofilm mode of growth rather than the free-living/planktonic mode of growth. The transcriptional regulator FleQ contributes to both modes of growth by functioning both as an activator and repressor and inversely regulating flagella genes associated with the planktonic mode of growth and genes contributing to the biofilm mode of growth. Here, we review findings that enhance our understanding of the molecular mechanism by which FleQ enables the transition between the two modes of growth. We also explore recent advances in the mechanism of action of FleQ to both activate and repress gene expression from a single promoter. Emphasis will be on the role of sigma factors, cyclic di-GMP, and the transcriptional regulator AmrZ in inversely regulating flagella and biofilm-associated genes and converting FleQ from a repressor to an activator., Competing Interests: The authors declare no conflict of interest.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZ R5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity., Competing Interests: The authors declare no conflict of interest.

Competing Interests: Declaration of interests No conflicts of interest are declared.

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 Δ flhF background abolished the phenotype of Δ flhF . 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 Δ hsbR , but it was mainly distributed as visible clusters at the lateral side of the periplasm and with visible clusters at both cell poles in Δ flhF . 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.IMPORTANCECyclic di-GMP (c-di-GMP) is a second messenger that controls flagellum biosynthesis, adhesion, virulence, motility, exopolysaccharide production, and biofilm formation in bacteria. Recent research has shown that distinct diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) produce highly specific outputs. Some DGCs and PDEs contribute to the total global c-di-GMP concentration, but others only affect local c-di-GMP in a microenvironment. However, the underlying mechanisms are unclear. Here, we report that FlhF affects the localization and DGC activity of WspR via HsbR and is implicated in local c-di-GMP signaling in Pseudomonas aeruginosa . This study establishes the link between the c-di-GMP signaling system and the flagellar localization and provides insight for understanding the complex regulatory network of c-di-GMP signaling., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

The ability of Pseudomonas aeruginosa to establish chronic infections is associated with an effective switch from a motile to a sessile lifestyle. This proficiency is controlled by intracellular levels of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Targeting the c-di-GMP network could be a strategy to interfere with P. aeruginosa pathogenicity. Therefore, the development of tools to profile c-di-GMP intracellular levels is crucial. Here, we describe a protocol for the in vivo measurement of c-di-GMP levels in P. aeruginosa., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Dispersion is an essential stage of the biofilm life cycle resulting in the release of bacteria from a biofilm into the surrounding environment. Dispersion contributes to bacterial survival by relieving overcrowding within a biofilm and allowing dissemination of cells into new habitats for colonization. Thus, dispersion can contribute to biofilm survival as well as disease progression and transmission. Cells dispersed from a biofilm rapidly lose their recalcitrant antimicrobial-tolerant biofilm phenotype and transition to a state that is susceptible to antibiotics. However, much of what is known about this biofilm developmental stage has been inferred from exogenously induced dispersion. Our findings provide the first evidence that native dispersion is coincident with reduced cyclic dimeric guanosine monophosphate levels, while also relying on at least some of the same factors that are central to the environmentally induced dispersion response, namely, BdlA, DipA, RbdA, and AmrZ. Additionally, we demonstrate for the first time that cis-DA signaling to induce dispersion is attributed to the two-component sensor/response regulator DspS, a homolog of the DSF sensor RpfC. Our findings also provide a path toward manipulating the native dispersion response as a novel and highly promising therapeutic intervention., Competing Interests: The authors declare no conflict of interest.

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 (σ) 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., Competing Interests: Competing interests statement: The authors declare no competing interest.

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter- gfp fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c -di-GMP m etabolism- r elated (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter- gfp fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter- gfp fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. IMPORTANCE The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter- gfp transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.

Dispersion is an active process exhibited by Pseudomonas aeruginosa during the late stages of biofilm development or in response to various cues, including nitric oxide and glutamate. Upon cue sensing, biofilm cells employ enzymes that actively degrade the extracellular matrix, thereby allowing individual cells to become liberated. While the mechanism by which P. aeruginosa senses and relays dispersion cues has been characterized, little is known about how dispersion cue sensing mechanisms result in matrix degradation. Considering that the alginate and motility regulator AmrZ has been reported to regulate genes that play a role in dispersion, including those affecting virulence, c-di-GMP levels, Pel and Psl abundance, and motility, we asked whether AmrZ contributes to the regulation of dispersion. amrZ was found to be significantly increased in transcript abundance under dispersion-inducing conditions, with the inactivation of amrZ impairing dispersion by P. aeruginosa biofilms in response to glutamate and nitric oxide. While the overexpression of genes encoding matrix-degrading enzymes pelA , pslG , and/or endA resulted in the dispersion of wild-type biofilms, similar conditions failed to disperse biofilms formed by dt amrZ . Likewise, the inactivation of amrZ abrogated the hyperdispersive phenotype of PAO1/pJN- bdlA _G31A biofilms, with dt amrZ -impaired dispersion being independent of the expression, production, and activation of BdlA. Instead, dispersion was found to require the AmrZ-target genes napB and PA1891. Our findings indicate that AmrZ is essential for the regulation of dispersion by P. aeruginosa biofilms, functions downstream of BdlA postdispersion cue sensing, and regulates the expression of genes contributing to biofilm matrix degradation as well as napB and PA1891. IMPORTANCE In P. aeruginosa, biofilm dispersion has been well-characterized with respect to dispersion cue perception, matrix degradation, and the consequences of dispersion. While the intracellular signaling molecule c-di-GMP has been linked to many of the phenotypic changes ascribed to dispersion, including the modulation of motility and matrix production, little is known about the regulatory mechanisms leading to matrix degradation and cells actively leaving the biofilm. In this study, we report for the first time an essential role of the transcriptional regulator AmrZ and two AmrZ-dependent genes, napB, and PA1891, in the dispersion response, thereby linking dispersion cue sensing via BdlA to the regulation of matrix degradation and to the ultimate liberation of bacterial cells from the biofilm.

Many bacterial species use the secondary messenger, c-di-GMP, to promote the production of biofilm matrix components. In Pseudomonas aeruginosa, c-di-GMP production is stimulated upon initial surface contact and generally remains high throughout biofilm growth. Transcription of several gene clusters, including the Sia signal transduction system, are induced in response to high cellular levels of c-di-GMP. The output of this system is SiaD, a diguanylate cyclase whose activity is induced in the presence of the detergent SDS. Previous studies demonstrated that Sia-mediated cellular aggregation is a key feature of P. aeruginosa growth in the presence of SDS. Here, we show that the Sia system is important for producing low levels of c-di-GMP when P. aeruginosa is growing planktonically. In addition, we show that Sia activity is important for maintaining cell-associated Psl in planktonic populations. We also demonstrate that Sia mutant strains have reduced cell-associated Psl and a surface attachment-deficient phenotype. The Sia system also appears to posttranslationally impact cell-associated Psl levels. Collectively, our findings suggest a novel role for the Sia system and c-di-GMP in planktonic populations by regulating levels of cell-associated Psl.

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.

Bacteria in biofilms are embedded in extracellular matrix and display low metabolic activity, partly due to insufficient diffusive exchange of metabolic substrate. The extracellular matrix and low metabolic activity both contribute to the high antibiotic tolerance-the hallmark of biofilm bacteria. The second messenger molecule, c-di-GMP, regulates biofilm development in Pseudomonas aeruginosa, where high internal levels lead to biofilm formation and low levels are associated with planktonic bacteria. Using a microcalorimetric approach, we show that c-di-GMP signaling is a major determinant of the metabolic activity of P. aeruginosa, both in planktonic culture and in two biofilm models. The high c-di-GMP content of biofilm bacteria forces them to rapidly spend a large amount of energy on the production of exopolysaccharides, resulting in a subsequent low metabolic state. This suggests that the low metabolic state of bacteria in mature biofilms, to some extent, is a consequence of a c-di-GMP-regulated survival strategy., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pseudomonas aeruginosa (P. aeruginosa) is a known bacterium that produces biofilms and causes severe infection. Furthermore, P. aeruginosa biofilms are extremely difficult to eradicate, leading to the development of chronic and antibiotic-resistant infections. Our previous study showed that a cathelicidin-related antimicrobial peptide (CRAMP) inhibits the formation of P. aeruginosa biofilms and markedly reduces the biomass of preformed biofilms, while the mechanism of eradicating bacterial biofilms remains elusive. Therefore, in this study, the potential mechanism by which CRAMP eradicates P. aeruginosa biofilms was investigated through an integrative analysis of transcriptomic, proteomic, and metabolomic data. The omics data revealed CRAMP functioned against P. aeruginosa biofilms by different pathways, including the Pseudomonas quinolone signal (PQS) system, cyclic dimeric guanosine monophosphate (c-di-GMP) signalling pathway, and synthesis pathways of exopolysaccharides and rhamnolipid. Moreover, a total of 2914 differential transcripts, 785 differential proteins, and 280 differential metabolites were identified. A series of phenotypic validation tests demonstrated that CRAMP reduced the c-di-GMP level with a decrease in exopolysaccharides, especially alginate, in P. aeruginosa PAO1 biofilm cells, improved bacterial flagellar motility, and increased the rhamnolipid content, contributing to the dispersion of biofilms. Our study provides new insight into the development of CRAMP as a potentially effective antibiofilm dispersant., (© 2022. The Author(s).)

Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second messenger, c-di-GMP, is a positive regulator of the production of various biofilm matrix components and is known to regulate alginate synthesis at the posttranslational level in P. aeruginosa. We provide evidence that c-di-GMP also regulates transcription of the alginate operon in P. aeruginosa. Previous work has shown that transcription of the alginate operon is regulated by nine different proteins, AmrZ, AlgP, IHFα, IHFβ, CysB, Vfr, AlgR, AlgB, and AlgQ, and we investigated if some of these proteins function as a c-di-GMP effector. We found that deletion of algP , algQ , IHFα , and IHFβ had only a marginal effect on the transcription of the alginate operon. Deletion of vfr and cysB led to decreased transcription of the alginate operon, and the dependence of the c-di-GMP level was less pronounced, indicating that Vfr and CysB could be partially required for c-di-GMP-mediated regulation of alginate operon transcription. Our experiments indicated that the AmrZ, AlgR, and AlgB proteins are absolutely required for transcription of the alginate operon. However, differential radial capillary action of ligand assay (DRaCALA) and site-directed mutagenesis indicated that c-di-GMP does not bind to any of the AmrZ, AlgR, and AlgB proteins. IMPORTANCE The proliferation of alginate-overproducing P. aeruginosa variants in the lungs of cystic fibrosis patients often leads to chronic infection. The alginate functions as a biofilm matrix that protects the bacteria against host immune defenses and antibiotic treatment. Knowledge about the regulation of alginate synthesis is important in order to identify drug targets for the development of medicine against chronic P. aeruginosa infections. We provide evidence that c-di-GMP positively regulates transcription of the alginate operon in P. aeruginosa. Moreover, we revisited the role of the known alginate regulators, AmrZ, AlgP, IHFα, IHFβ, CysB, Vfr, AlgR, AlgB, and AlgQ, and found that their effect on transcription of the alginate operon is highly varied. Deletion of algP , algQ , IHFα , or IHFβ only had a marginal effect on transcription of the alginate operon, whereas deletion of vfr or cysB led to decreased transcription and deletion of amrZ , algR , or algB abrogated transcription.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a Δ wspF mutant strain (high c-di-GMP levels), and a p lac - yhjH -containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo Δ wspF mutant compared to the in vivo p lac -yhjH -containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The Δ wspF mutant was less susceptible to host clearance than the p lac -yhjH -containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A Δ wspF mutant with a defective T3SS was more susceptible to host clearance than a Δ wspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Pseudomonas aeruginosa can cause various types of infections and is one of the most ubiquitous antibiotic-resistant pathogens found in healthcare settings. It is capable of adapting to adverse conditions by transforming its motile lifestyle to a sessile biofilm lifestyle, which induces a steady state of chronic infection. However, mechanisms triggering the lifestyle transition of P. aeruginosa strains with clinical significance are not very clear. In this study, we reported a recently isolated uropathogenic hyper-biofilm producer PA_HN002 and characterized its genome to explore genetic factors that may promote its transition into the biofilm lifestyle. We first showed that high intracellular c-di-GMP content in PA_HN002 gave rise to its attenuated motilities and extraordinary strong biofilm. Reducing the intracellular c-di-GMP content by overexpressing phosphodiesterases (PDEs) such as BifA or W909_14950 converted the biofilm and motility phenotypes. Whole genome sequencing and comprehensive analysis of all the c-di-GMP metabolizing enzymes led to the identification of multiple mutations within PDEs. Gene expression assays further indicated that the shifted expression profile of c-di-GMP metabolizing enzymes in PA_HN002 might mainly contribute to its elevated production of intracellular c-di-GMP and enhanced biofilm formation. Moreover, mobile genetic elements which might interfere the endogenous regulatory network of c-di-GMP metabolism in PA_HN002 were analyzed. This study showed a reprogrammed expression profile of c-di-GMP metabolizing enzymes which may promote the pathoadaption of clinical P. aeruginosa into biofilm producers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lin, Chen, Li, Cao, Li, Hu, Liao, Zhang and Xu.)

Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa.

C-di-GMP signaling can directly influence bacterial behavior by affecting the functionality of c-di-GMP-binding proteins. In addition, c-di-GMP can exert a global effect on gene transcription or translation, for example, via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c-di-GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c-di-GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c-di-GMP under growth conditions otherwise characterized by low c-di-GMP levels caused a switch to a non-motile, auto-aggregative P. aeruginosa phenotype. This phenotypic switch became apparent before any c-di-GMP-dependent role on transcription, translation, or protein abundance was observed. Our results suggest that rising global c-di-GMP pools first affects the motility phenotype of P. aeruginosa by altering protein functionality and only then global gene transcription., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Bacterial biofilm represents a multicellular community embedded within an extracellular matrix attached to a surface. This lifestyle confers to bacterial cells protection against hostile environments, such as antibiotic treatment and host immune response in case of infections. The Pseudomonas genus is characterised by species producing strong biofilms difficult to be eradicated and by an extraordinary metabolic versatility which may support energy and carbon/nitrogen assimilation under multiple environmental conditions. Nutrient availability can be perceived by a Pseudomonas biofilm which, in turn, readapts its metabolism to finally tune its own formation and dispersion. A growing number of papers is now focusing on the mechanism of nutrient perception as a possible strategy to weaken the biofilm barrier by environmental cues. One of the most important nutrients is amino acid L-arginine, a crucial metabolite sustaining bacterial growth both as a carbon and a nitrogen source. Under low-oxygen conditions, L-arginine may also serve for ATP production, thus allowing bacteria to survive in anaerobic environments. L-arginine has been associated with biofilms, virulence, and antibiotic resistance. L-arginine is also a key precursor of regulatory molecules such as polyamines, whose involvement in biofilm homeostasis is reported. Given the biomedical and biotechnological relevance of biofilm control, the state of the art on the effects mediated by the L-arginine nutrient on biofilm modulation is presented, with a special focus on the Pseudomonas biofilm. Possible biotechnological and biomedical applications are also discussed.

The type VI secretion system (T6SS) is an important translocation apparatus that is widely employed by Gram-negative bacteria to deliver toxic effectors into eukaryotic and prokaryotic target cells, causing host damage and providing competitive advantages in polymicrobial environments. The genome of Pseudomonas aeruginosa harbors three T6SS clusters (H1-T6SS, H2-T6SS, H3-T6SS). Activities of these systems are tightly regulated by a complicated signaling network which remains largely elusive. In this study, we focused on a previously characterized two-component system FleS/FleR, and performed comparative transcriptome analysis between the PAO1 wild-type strain and its isogenic Δ fleR mutant, which revealed the important role of FleS/FleR in regulating multiple physiological pathways including T6SS. Gene expression and bacterial killing assays showed that the expression and activity of H1-T6SS are repressed in the wild-type strain owing to the high intracellular c-di-GMP content. Further explorations demonstrated that c-di-GMP relies on the transcription factor FleQ to repress H1-T6SS and its synthesis is controlled by a global regulator AmrZ which is induced by the active FleS/FleR. Interestingly, repression of H1-T6SS by FleS/FleR in PAO1 is independent of RetS which is known to regulate H1-T6SS by controlling the central post-transcriptional factor RsmA. Together, our results identified a novel regulator of H1-T6SS and provided detailed mechanisms of this signaling pathway in PAO1. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen distributed widely in the environment. The genome of this pathogen contains three T6SS clusters which contribute significantly to its virulence. Understanding the complex regulatory network that controls the activity of T6SS is essential for the development of effective therapeutic treatments for P. aeruginosa infections. In this study, transcriptome analysis led to the identification of a novel regulator FleS/FleR which inversely regulates H1-T6SS and H2-T6SS in P. aeruginosa PAO1. We further revealed a detailed FleS/FleR-mediated regulatory pathway of H1-T6SS in PAO1 which involves two additional transcriptional regulators AmrZ and FleQ and the second messenger c-di-GMP, providing important implications to develop novel anti-infective strategies and antimicrobial drugs.

Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found that extracellular polysaccharide (EPS) production, regulation of motility, and induction of stress response pathways contribute to biofilm tolerance during such insults. However, little is known regarding the mechanism(s) whereby biofilm maintenance is regulated when exposed to such environmental challenges. Here, we provide evidence that the diguanylate cyclase YfiN is important for the regulation of biofilm maintenance when exposed to peroxide. We find that compared to the wild type (WT), static biofilms of the Δ yfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H 2 O 2 ); this defect can be rescued through genetic complementation. Additionally, we found that the Δ yfiN mutant biofilms produce less c-di-GMP than WT and that H 2 O 2 treatment enhanced motility of surface-associated bacteria and increased cell death for the Δ yfiN mutant grown as a biofilm compared to WT biofilms. These data provide evidence that YfiN is required for biofilm maintenance by P. aeruginosa, via c-di-GMP signaling, to limit motility and protect viability in response to peroxide stress. These findings add to the growing recognition that biofilm maintenance by P. aeruginosa is an actively regulated process that is controlled, at least in part, by the wide array of c-di-GMP metabolizing enzymes found in this microbe. IMPORTANCE We build on previous findings that suggest that Pseudomonas aeruginosa utilizes c-di-GMP metabolizing enzymes to actively maintain a mature biofilm. Here, we explore how the diguanylate cyclase YfiN contributes to the regulation of biofilm maintenance during peroxide exposure. We find that mature P. aeruginosa biofilms require YfiN to synthesize c-di-GMP, regulate motility, and ensure viability during peroxide stress. These findings provide further evidence that the modulation of c-di-GMP in response to environmental signals is an important mechanism by which biofilms are maintained.

Pseudomonas aeruginosa, an opportunistic bacterial pathogen, can synthesize and catabolize several small cationic molecules known as polyamines. In several clades of bacteria, polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, l-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or occurs through a metabolic derivative. Here, we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa. Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that l-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation but did so by a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and l-arginine induced a significant increase in the intracellular level of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor, arginine, promote biofilm and c-di-GMP synthesis in P. aeruginosa. IMPORTANCE Biofilm formation allows bacteria to physically attach to a surface, confer tolerance to antimicrobial agents, and promote resistance to host immune responses. As a result, the regulation of biofilm formation is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switch that favors chronic infection.

One of the hallmarks of the environmental bacterium Pseudomonas aeruginosa is its excellent ecological flexibility, which can thrive in diverse ecological niches. In different ecosystems, P. aeruginosa may use different strategies to survive, such as forming biofilms in crude oil environment, converting to mucoid phenotype in the cystic fibrosis (CF) lung, or becoming persisters when treated with antibiotics. Rugose small colony variants (RSCVs) are the adaptive mutants of P. aeruginosa, which can be frequently isolated from chronic infections. During the past years, there has been a renewed interest in using P. aeruginosa as a model organism to investigate the RSCVs formation, persistence and pathogenesis, as RSCVs represent a hyper-biofilm formation, high adaptability, high-tolerance sub-population in biofilms. This review will briefly summarize recent advances regarding the phenotypic, genetic and host interaction associated with RSCVs, with an emphasis on P. aeruginosa. Meanwhile, some non-pathogenic bacteria such as Pseudomonas fluorescence, Pseudomonas putida and Bacillus subtilis will be also included. Remarkable emphasis is given on intrinsic functions of such hyper-biofilm formation characteristic as well as its potential applications in several biocatalytic transformations including wastewater treatment, microbial fermentation, and plastic degradation. Hopefully, this review will attract the interest of researchers in various fields and shape future research focused not only on evolutionary biology but also on biotechnological applications related to RSCVs., (Copyright © 2021. Published by Elsevier Inc.)

The opportunistic pathogen Pseudomonas aeruginosa is a significant cause of infection in immunocompromised individuals, cystic fibrosis patients, and burn victims. To benefit its survival, the bacterium adapt to either a motile or sessile lifestyle when infecting the host. The motile bacterium has an often activated type III secretion system (T3SS), which is virulent to the host, whereas the sessile bacterium harbors an active T6SS and lives in biofilms. Regulatory pathways involving Gac-Rsm or secondary messengers such as c-di-GMP determine which lifestyle is favorable for P. aeruginosa . Here, we introduce the RNA binding protein RtcB as a modulator of the switch between motile and sessile bacterial lifestyles. Using the wild-type P. aeruginosa PAO1, and a retS mutant PAO1(∆ retS ) in which T3SS is repressed and T6SS active, we show that deleting rtcB led to simultaneous expression of T3SS and T6SS in both PAO1(∆ rtcB ) and PAO1(∆ rtcB ∆ retS ). The deletion of rtcB also increased biofilm formation in PAO1(∆ rtcB ) and restored the motility of PAO1(∆ rtcB ∆ retS ). RNA-sequencing data suggested RtcB as a global modulator affecting multiple virulence factors, including bacterial secretion systems. Competitive killing and infection assays showed that the three T6SS systems (H1, H2, and H3) in PAO1(∆ rtcB ) were activated into a functional syringe, and could compete with Escherichia coli and effectively infect lettuce. Western blotting and RT-PCR results showed that RtcB probably exerted its function through RsmA in PAO1(∆ rtcB ∆ retS ). Quantification of c-di-GMP showed an elevated intracellular levels in PAO1(∆ rtcB ), which likely drove the switch between T6SS and T3SS, and contributed to the altered phenotypes and characteristics observed. Our data demonstrate a pivotal role of RtcB in the virulence of P. aeruginosa by controlling multiple virulence determinants, such as biofilm formation, motility, pyocyanin production, T3SS, and T6SS secretion systems towards eukaryotic and prokaryotic cells. These findings suggest RtcB as a potential target for controlling P. aeruginosa colonization, establishment, and pathogenicity.

Pseudomonas aeruginosa is an opportunistic human pathogen and is the primary cause of nosocomial infections. Biofilm formation by this organism results in chronic and hard to eradicate infections. The intracellular signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a secondary messenger in bacterial cells crucial for motile to sessile transition. The signalling pathway components encompass two classes of enzymes with antagonistic activities, the diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that regulate the cellular levels of c-di-GMP at distinct stages of biofilm initiation, maturation and dispersion. This review summarizes the structural analysis and functional studies of the DGCs and PDEs involved in biofilm regulation in P. aeruginosa. In addition, we also describe the effector proteins that sense the perturbations in c-di-GMP levels to elicit a functional output. Finally, we discuss possible mechanisms that allow the dynamic levels of c-di-GMP to regulate cognate cellular response. Uncovering the details of the regulation of the c-di-GMP signalling pathway is vital for understanding the behaviour of the pathogen and characterization of novel targets for anti-biofilm interventions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation., Results: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain., Conclusions: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation., (© 2021. The Author(s).)

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa . ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA + strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa , we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement., Competing Interests: The authors declare no competing interest.

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We previously presented evidence that the ectopic expression of the Escherichia coli phosphodiesterase YhjH in Pseudomonas aeruginosa results in biofilm dispersal. More recently, however, evidence has been presented that the induction of native c-di-GMP phosphodiesterases does not lead to a dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for the development of antibiofilm drugs. However, here, we demonstrate that the induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed results in the dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and a flow cell biofilm system., (Copyright © 2021 American Society for Microbiology.)

During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second-messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact are still poorly understood, although mechanosensing is often invoked, with few data supporting this claim. Using a combination of molecular genetics and single-cell analysis, with biophysical, biochemical, and genomics techniques, we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that TFP/PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain of PilY1 results in modestly lower surface adhesion forces, reduced sustained conformational changes, and increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate a role for TFP in twitching motility from surface-sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains and its absence in the ∼720 sequenced strains of P. aeruginosa PAO1 may contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1. IMPORTANCE Most bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second-messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade is unclear. Here, we provide key insight into this question; we show that the eukaryote-like vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives the production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that may account for differing surface colonization strategies.

In Pseudomonas aeruginosa , the orphan two-component sensor SagS contributes both to transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug-tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment since glucose-6-phosphate likewise enhanced biofilm formation and also enhanced the expression of select biofilm marker genes. Moreover, exposure to glucose-6-phosphate coincided with decreased swarming motility but increased cellular cyclic-di-GMP (c-di-GMP) levels in biofilms. No such response was noted for compounds modulating attachment and biofilm formation in a manner independent of SagS. Modulation of c-di-GMP in response to glucose-6-phosphate was due to the diguanylate cyclase NicD, with NicD also being required for enhanced biofilm formation. The latter was independent of the sensory domain of NicD but dependent on NicD activity, SagS, and the interaction between NicD and SagS. Our findings indicate that glucose-6-phosphate likely mimics a signal or conditions sensed by SagS to activate its motile-sessile switch function. In addition, our findings provide new insight into the interfaces between the ligand-mediated two-component system signaling pathway and c-di-GMP levels. IMPORTANCE Pathogens sense and respond to signals and cues present in their environment, including host-derived small molecules to modulate the expression of their virulence repertoire. Here, we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa responds to glucose-6-phosphate. Since glucose-6-phosphate is primarily made available due to cell lysis, it is likely that glucose-6-phosphate represents a cross-kingdom cell-to-cell signal that enables P. aeruginosa to adapt to the (nutrient-poor) host environment by enhancing biofilm formation, cyclic-di-GMP, and the expression of genes linked to biofilm formation in a concentration- and SagS-dependent manner., (Copyright © 2021 Park et al.)

While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa -phage interaction to identify rmcA and morA , two genes which encode bis-(3',5')-cyclic dimeric GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide, and/or biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, as well as subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant (Δ relA Δ spoT ), indicating that the Δ rmcA and Δ morA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the Δ rmcA and Δ morA mutant strains in an established biofilm under nutrient limitation. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, suggesting that a wide variety of c-di-GMP metabolizing enzymes in organisms such as P. aeruginosa allows for discrete control over the formation, maintenance or dispersion of biofilms. IMPORTANCE Recent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how biofilms initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in Δ rmcA and Δ morA mutants was also found in the stringent response-defective Δ relA Δ spoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, extracellular polysaccharide biosynthesis, and the nutrient limitation response is important for biofilm persistence. We uncover components of an important regulatory system needed for P. aeruginosa biofilms to persist in nutrient-poor conditions and provide some of the first evidence that maintaining a mature biofilm is an active process., (Copyright © 2021 Katharios-Lanwermeyer et al.)

Introduction: Actively dispersed Pseudomonas aeruginosa biofilm cells differ from planktonic cells, as they have a lower intracellular cyclic di-guanosine monophosphate (c-di-GMP) concentration and show increased virulence. In addition, the nature of the dispersion trigger has been shown to influence the antibiotic susceptibility of dispersed cells. However, properties of passively-dispersed cells, in which the dispersion trigger directly releases cells from the biofilm, have not been described. The present study determined c-di-GMP concentration, virulence in Galleria mellonella and antibiotic susceptibility of P. aeruginosa cells dispersed from biofilm using various triggers., Materials and Methods: P. aeruginosa biofilms grown in flow-cells were dispersed actively [exposure to the nitric oxide (NO)-donor sodium nitroprusside (SNP) or to glutamate] or passively [by stopping and restarting the flow or exposure to laser-induced vapor nanobubbles (VNB)], and properties of these dispersed cells were compared to those of spontaneously-dispersed cells., Results: The passively dispersed P. aeruginosa biofilm cells had significantly lower intracellular c-di-GMP levels than actively-dispersed cells. However, this did not result in differences in virulence in Galleria mellonella, nor in tobramycin and ciprofloxacin susceptibility. Passively-dispersed cells were more susceptible to colistin than actively- and spontaneously-dispersed cells. In cells dispersed by interrupting the flow, increased susceptibility to colistin was immediate, whereas this was delayed for VNB-dispersed cells., Conclusion: Passively-dispersed P. aeruginosa biofilm cells have a decreased intracellular c-di-GMP concentration and an increased colistin susceptibility compared to actively-dispersed cells. No differences in virulence or susceptibility to tobramycin or colistin were observed., (Copyright © 2020. Published by Elsevier Ltd.)

We investigated the effect of temperature on the biofilm formation of Pseudomonas aeruginosa and revealed that the biofilm formation increased rapidly at temperatures lower than 25°C. P. aeruginosa formed the most robust biofilm of a conspicuous mushroom-like structure at 20°C. However, when the temperature increased to 25°C, the biofilm formation rapidly decreased. Above 25°C, as the temperature rose, the biofilm formation increased again little by little despite its less-structured form, indicating that 25°C is the low point of biofilm formation. The intracellular 3',5'-cyclic diguanylate (c-di-GMP) levels also decreased rapidly as the temperature rose from 20 to 25°C. The expression levels of pelA , algD , and pslA encoding Pel, alginate, and Psl, respectively, were also dramatically affected by temperature, with pelA being regulated in a pattern similar to that of the intracellular c-di-GMP levels, and the pattern seen for algD regulation was the most similar to the actual biofilm formation pattern. Total exopolysaccharide production was thermoregulated and followed the regulation pattern of c-di-GMP. Interestingly, the thermoregulation patterns in biofilm formation were different depending on the strain of P. aeruginosa Unlike PAO1, another strain, PA14, showed a gradual decrease in biofilm formation and c-di-GMP in the range of 20 to 37°C, and P. aeruginosa clinical isolates also showed slightly different patterns in biofilm formation in conjunction with temperature change, suggesting that different strains may sense different temperature ranges for biofilm formation. However, it is obvious that P. aeruginosa forms more biofilms at lower temperatures and that temperature is an important factor in determining the biofilm formation. IMPORTANCE Biofilm formation is an important protection mechanism used by most microorganisms and provides cells with many advantages, like high infectivity, antibiotic resistance, and strong survivability. Since most persistent bacterial infections are believed to be associated with biofilms, biofilm control is an important issue in medicine, environmental engineering, and industry. Biofilm formation is influenced by various environmental factors. Temperature is the most direct environmental cue encountered by microorganisms. Here, we investigated the effect of temperature on the biofilm formation of P. aeruginosa , a notorious pathogen, and found that temperature is an important factor determining the amount and structure of biofilms. Low temperatures greatly increase biofilm formation and give biofilms a highly conspicuous structure. Although thermoregulation of biofilm formation is mainly mediated by c-di-GMP, some c-di-GMP-independent regulations were also observed. This study shows how biofilms are formed at various temperatures and provides new insights to control biofilms using temperature., (Copyright © 2020 American Society for Microbiology.)

The Pel polysaccharide is a structural component of the extracellular matrix of Pseudomonas aeruginosa biofilms. Recent analyses suggest that Pel production proceeds via a synthase-dependent polysaccharide secretion pathway, which in Gram-negative bacteria is defined by an outer membrane β-barrel porin, a periplasmic tetratricopeptide repeat-containing scaffold protein, and an inner membrane-embedded synthase. Polymerization is catalyzed by the glycosyltransferase domain of the synthase component of these systems, which is allosterically regulated by cyclic 3',5'-dimeric GMP (c-di-GMP). However, while the outer membrane and periplasmic components of the Pel system have been characterized, the inner membrane complex required for Pel polymerization has yet to be defined. To address this, we examined over 500 pel gene clusters from diverse species of Proteobacteria This analysis identified an invariant set of four syntenic genes, three of which, pelD , pelE , and pelG , are predicted to reside within the inner membrane, while the fourth, pelF , encodes a glycosyltransferase domain. Using a combination of gene deletion analysis, subcellular fractionation, coimmunoprecipitation, and bacterial two-hybrid assays, we provide evidence for the existence of an inner membrane complex of PelD, PelE, and PelG. Furthermore, we show that this complex interacts with PelF in order to facilitate its localization to the inner membrane. Mutations that abolish c-di-GMP binding to the known receptor domain of PelD had no effect on complex formation, suggesting that c-di-GMP binding stimulates Pel production through quaternary structural rearrangements. Together, these data provide the first experimental evidence of an inner membrane complex involved in Pel polysaccharide production. IMPORTANCE The exopolysaccharide Pel plays an important role in bacterial cell-cell interactions, surface adhesion, and protection against certain antibiotics. We identified invariant pelDEFG gene clusters in over 500 diverse proteobacterial species. Using Pseudomonas aeruginosa , we demonstrate that PelD, PelE, PelF, and PelG form a complex at the inner membrane and propose that this complex represents the previously unidentified Pel polysaccharide synthase, which is responsible for Pel polymerization and transport across the cytoplasmic membrane. We show that the formation of this complex is independent of cyclic 3',5'-dimeric GMP (c-di-GMP) binding to the receptor PelD. Collectively, these data establish the widespread Pel apparatus as a member of the synthase-dependent pathway of polysaccharide biosynthetic systems and broaden the architectural diversity of already-established bacterial polysaccharide synthases., (Copyright © 2020 American Society for Microbiology.)

Flagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in Pseudomonas aeruginosa and other microbes is cyclic diguanylate (c-di-GMP). High levels of this second messenger repress motility and stimulate biofilm formation. c-di-GMP levels regulate motility in P. aeruginosa in part by influencing the localization of its two flagellar stator sets, MotAB and MotCD. Here, we show that while c-di-GMP can influence stator localization, stators can in turn impact c-di-GMP levels. We demonstrate that the swarming motility-driving stator MotC physically interacts with the transmembrane region of the diguanylate cyclase SadC. Furthermore, we demonstrate that this interaction is capable of stimulating SadC activity. We propose a model by which the MotCD stator set interacts with SadC to stimulate c-di-GMP production under conditions not permissive to motility. This regulation implies a positive-feedback loop in which c-di-GMP signaling events cause MotCD stators to disengage from the motor; then disengaged stators stimulate c-di-GMP production to reinforce a biofilm mode of growth. Our studies help to define the bidirectional interactions between c-di-GMP and the flagellar machinery. IMPORTANCE The ability of bacterial cells to control motility during early steps in biofilm formation is critical for the transition to a nonmotile, biofilm lifestyle. Recent studies have clearly demonstrated the ability of c-di-GMP to control motility via a number of mechanisms, including through controlling transcription of motility-related genes and modulating motor function. Here, we provide evidence that motor components can in turn impact c-di-GMP levels. We propose that communication between motor components and the c-di-GMP synthesis machinery allows the cell to have a robust and sensitive switching mechanism to control motility during early events in biofilm formation., (Copyright © 2019 American Society for Microbiology.)

Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in Δ motAB or Δ sadC Δ gcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol. IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa , here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings., (Copyright © 2019 American Society for Microbiology.)

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration., Competing Interests: CA, CL, JP, Jd, AX, KZ, BT, LH, FJ, CH, GW, MP No competing interests declared, (© 2019, Armbruster et al.)

The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr, and involved a reduction of the level of c-di-GMP, as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa effectively colonizes host epithelia using pili as primary adhesins. Here we uncover a surface-specific asymmetric virulence program that enhances P. aeruginosa host colonization. We show that when P. aeruginosa encounters surfaces, the concentration of the second messenger c-di-GMP increases within a few seconds. This leads to surface adherence and virulence induction by stimulating pili assembly through activation of the c-di-GMP receptor FimW. Surface-attached bacteria divide asymmetrically to generate a piliated, surface-committed progeny (striker) and a flagellated, motile offspring that leaves the surface to colonize distant sites (spreader). Cell differentiation is driven by a phosphodiesterase that asymmetrically positions to the flagellated pole, thereby maintaining c-di-GMP levels low in the motile offspring. Infection experiments demonstrate that cellular asymmetry strongly boosts infection spread and tissue damage. Thus, P. aeruginosa promotes surface colonization and infection transmission through a cooperative virulence program that we termed Touch-Seed-and-Go., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bacterial cyclic-di-GMP (c-di-GMP) is an important messenger molecule that influences diverse cellular processes including motility, virulence and cytotoxicity systems, polysaccharide synthesis and biofilm formation. The YfiBNR tripartite signalling system in P. aeruginosa modulates the cellular c-di-GMP levels in response to signals received from the periplasm. In this study, we analyse the structures of activating mutants of the outer membrane protein YfiB that give rise to increased surface attachment and biofilm formation. The F48S and W55L mutants of YfiB(27-168) crystallize in the same dimeric arrangement as our previously reported YfiB structures that preclude complex formation with YfiR. The L43P mutant of YfiB(27-168) is monomeric and forms a stable complex with YfiR. The YfiB(L43P)-YfiR crystal structure reveals a dramatic rearrangement of the N-terminal fragment, which is implicated in increased YfiB activation and membrane attachment, upon YfiR binding. Comparison with our previous complex structure between YfiB(59-168) and YfiR reveals extensive interactions between the N-terminal fragment of YfiB (residues 35-55) and YfiR., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Toxin-antitoxin (TA) systems play important roles in bacteria persister formation. Increasing evidence demonstrate the roles of TA systems in regulating virulence factors in pathogenic bacteria. The toxin HigB in Pseudomonas aeruginosa contributes to persister formation and regulates the expression of multiple virulence factors and biofilm formation. However, the regulatory mechanism remains elusive. In this study, we explored the HigB mediated regulatory pathways. We demonstrate that HigB decreases the intracellular level of c-di-GMP, which is responsible for the increased expression of the type III secretion system (T3SS) genes and repression of biofilm formation. By analyzing the expression levels of the known c-di-GMP metabolism genes, we find that three c-di-GMP hydrolysis genes are up regulated by HigB, namely PA2133, PA2200 and PA3825. Deletion of the three genes individually or simultaneously diminishes the HigB mediated regulation on the expression of T3SS genes and biofilm formation. Therefore, our results reveal novel functions of HigB in P. aeruginosa .