scholarly journals Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Judith H. Merritt ◽  
Dae-Gon Ha ◽  
Kimberly N. Cowles ◽  
Wenyun Lu ◽  
Diana K. Morales ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Critical Question ◽  
Cyclic Diguanylate ◽  
Critical Signal ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

scholarly journals A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Erin B. Purcell ◽  
Robert W. McKee ◽  
David S. Courson ◽  
Elizabeth M. Garrett ◽  
Shonna M. McBride ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stationary Phase ◽  
Nutrient Availability ◽  
Biofilm Formation ◽  
Biochemical Characterization ◽  
Toxin Production ◽  
Physiological Adaptation ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

scholarly journals Thermoregulation of Pseudomonas aeruginosa Biofilm Formation

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Suran Kim ◽  
Xi-Hui Li ◽  
Hyeon-Ji Hwang ◽  
Joon-Hee Lee
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Effect Of Temperature ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Protection Mechanism ◽  
Formation Pattern ◽  
Temperature Ranges ◽  
Different Strains

ABSTRACT 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.

scholarly journals A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Nathan Feirer ◽  
Jing Xu ◽  
Kylie D. Allen ◽  
Benjamin J. Koestler ◽  
Eric L. Bruger ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Signaling Pathway ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Dominant Role ◽  
Second Messenger ◽  
Dual Function ◽  
Diguanylate Cyclase ◽  
Cyclic Diguanylate ◽  
Content Type

ABSTRACTThe motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogenAgrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP inA. tumefaciensare controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins.IMPORTANCEPathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have characterized a novel signaling pathway that plays a dominant role in the regulation of biofilm formation in the model pathogenAgrobacterium tumefaciens. This control pathway involves small metabolites called pterins, well studied in eukaryotes, but this is the first example of pterin-dependent signaling in bacteria. The described pathway controls levels of an important intracellular second messenger (cyclic diguanylate monophosphate) that regulates key bacterial processes such as biofilm formation, motility, and virulence. Pterins control the balance of activity for an enzyme that both synthesizes and degrades the second messenger. These findings reveal a complex, multistep pathway that modulates this enzyme, possibly identifying new targets for antibacterial intervention.

scholarly journals Flagellar Stators Activate a Diguanylate Cyclase To Inhibit Flagellar Stators

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Daniel B. Kearns
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Secondary Metabolite ◽  
Positive Feedback ◽  
Biofilm Formation ◽  
Feedback Loop ◽  
Diguanylate Cyclase ◽  
Positive Feedback Loop ◽  
Content Type ◽  
Dependent Inhibition

ABSTRACT The bacterial secondary metabolite cyclic di-GMP is a widespread, cytoplasmic signal that promotes a physiological transition in which motility is inhibited and biofilm formation is activated. A paper published in this issue (A. E. Baker, S. S. Webster, A. Diepold, S. L. Kuchma, E. Bordeleau, et al., J Bacteriol 201:e00741-18, 2019, https://doi.org/10.1128/JB.00741-18) makes an important connection between cyclic di-GMP and flagellar components. They show that stator units, which normally interact with the flagellum to power rotation, can alternatively interact with and activate an enzyme that synthesizes cyclic di-GMP in Pseudomonas aeruginosa. Moreover, the same stator units are also the target of cyclic-di-GMP-dependent inhibition such that the more the stators are inhibited, the more cyclic di-GMP is made. The resulting positive-feedback loop not only inhibits motility but also may initiate and stabilize biofilm formation.

scholarly journals Deletion Mutant Library for Investigation of Functional Outputs of Cyclic Diguanylate Metabolism in Pseudomonas aeruginosa PA14

2014 ◽  
Vol 80 (11) ◽  
pp. 3384-3393 ◽  
Author(s):  
Dae-Gon Ha ◽  
Megan E. Richman ◽  
George A. O'Toole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Deletion Mutant ◽  
Deletion Mutants ◽  
Mutant Library ◽  
Potential Utility ◽  
Cyclic Diguanylate ◽  
Swarming Motility ◽  
Content Type ◽  
Swimming Motility

ABSTRACTWe constructed a library of in-frame deletion mutants targeting each gene inPseudomonas aeruginosaPA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.

scholarly journals Flagellar stators stimulate c-di-GMP production by Pseudomonas aeruginosa

2018 ◽  
Author(s):  
Amy E. Baker ◽  
Shanice S. Webster ◽  
Andreas Diepold ◽  
Sherry L. Kuchma ◽  
Eric Bordeleau ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Feedback Loop ◽  
Bacterial Cells ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Swarming Motility ◽  
Surface Attachment ◽  
Bidirectional Interactions ◽  
Gmp Production

AbstractFlagellar 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 just as c-di-GMP can influence the stators, stators can 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 in 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 define the bidirectional interactions between c-di-GMP and the motility machinery.Importance.The ability of bacterial cells to control motility during early steps in biofilm formation is critical for the transition to a non-motile, 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 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.

scholarly journals Anthranilate Deteriorates the Structure of Pseudomonas aeruginosa Biofilms and Antagonizes the Biofilm-Enhancing Indole Effect

2015 ◽  
Vol 81 (7) ◽  
pp. 2328-2338 ◽  
Author(s):  
Soo-Kyoung Kim ◽  
Ha-Young Park ◽  
Joon-Hee Lee
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Shear Force ◽  
Degradation Products ◽  
Early Stage ◽  
Bacterial Species ◽  
Extracellular Polysaccharide ◽  
Content Type ◽  
Tryptophan Degradation ◽  
Biofilm Development

ABSTRACTAnthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation ofPseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product ofP. aeruginosa, had an opposite effect onP. aeruginosabiofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment ofP. aeruginosaat the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole onP. aeruginosabiofilm formation.

scholarly journals Flagellar Stators Stimulate c-di-GMP Production by Pseudomonas aeruginosa

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Amy E. Baker ◽  
Shanice S. Webster ◽  
Andreas Diepold ◽  
Sherry L. Kuchma ◽  
Eric Bordeleau ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Feedback Loop ◽  
Bacterial Cells ◽  
Flagellar Motility ◽  
Swarming Motility ◽  
Surface Attachment ◽  
Content Type ◽  
Bidirectional Interactions ◽  
Gmp Production

ABSTRACT 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.

scholarly journals The PA3177 Gene Encodes an Active Diguanylate Cyclase That Contributes to Biofilm Antimicrobial Tolerance but Not Biofilm Formation by Pseudomonas aeruginosa

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Bandita Poudyal ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Drug Tolerance ◽  
Diguanylate Cyclase ◽  
Wild Type ◽  
Planktonic Cells ◽  
Swarming Motility ◽  
Content Type ◽  
First Time

ABSTRACT A hallmark of biofilms is their heightened resistance to antimicrobial agents. Recent findings suggested a role for bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) in the susceptibility of bacteria to antimicrobial agents; however, no c-di-GMP modulating enzyme(s) contributing to the drug tolerance phenotype of biofilms has been identified. The goal of this study was to determine whether c-di-GMP modulating enzyme(s) specifically contributes to the biofilm drug tolerance of Pseudomonas aeruginosa. Using transcriptome sequencing combined with biofilm susceptibility assays, we identified PA3177 encoding a probable diguanylate cyclase. PA3177 was confirmed to be an active diguanylate cyclase, with overexpression affecting swimming and swarming motility, and inactivation affecting cellular c-di-GMP levels of biofilm but not planktonic cells. Inactivation of PA3177 rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin and hydrogen peroxide. Inactivation of PA3177 also eliminated the recalcitrance of biofilms to killing by tobramycin, with multicopy expression of PA3177 but not PA3177_GGAAF harboring substitutions in the active site, restoring tolerance to wild-type levels. Susceptibility was linked to BrlR, a previously described transcriptional regulator contributing to biofilm tolerance, with inactivation of PA3177 negatively impacting BrlR levels and BrlR-DNA binding. While PA3177 contributed to biofilm drug tolerance, inactivation of PA3177 had no effect on attachment and biofilm formation. Our findings demonstrate for the first time that biofilm drug tolerance by P. aeruginosa is linked to a specific c-di-GMP modulating enzyme, PA3177, with the pool of PA3177-generated c-di-GMP only contributing to biofilm drug tolerance but not to biofilm formation.

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