SadC Reciprocally Influences Biofilm Formation and Swarming Motility via Modulation of Exopolysaccharide Production and Flagellar Function

ABSTRACT Pseudomonas aeruginosa has served as an important organism in the study of biofilm formation; however, we still lack an understanding of the mechanisms by which this microbe transitions to a surface lifestyle. A recent study of the early stages of biofilm formation implicated the control of flagellar reversals and production of an exopolysaccharide (EPS) as factors in the establishment of a stable association with the substratum and swarming motility. Here we present evidence that SadC (PA4332), an inner membrane-localized diguanylate cyclase, plays a role in controlling these cellular functions. Deletion of the sadC gene results in a strain that is defective in biofilm formation and a hyperswarmer, while multicopy expression of this gene promotes sessility. A ΔsadC mutant was additionally found to be deficient in EPS production and display altered reversal behavior while swimming in high-viscosity medium, two behaviors proposed to influence biofilm formation and swarming motility. Epistasis analysis suggests that the sadC gene is part of a genetic pathway that allows for the concomitant regulation of these aspects of P. aeruginosa surface behavior. We propose that SadC and the phosphodiesterase BifA (S. L. Kuchma et al., J. Bacteriol. 189:8165-8178, 2007), via modulating levels of the signaling molecule cyclic-di-GMP, coregulate swarming motility and biofilm formation as P. aeruginosa transitions from a planktonic to a surface-associated lifestyle.

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Cyclic-di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa: the pilY1 Gene and Its Impact on Surface-Associated Behaviors

Journal of Bacteriology ◽

10.1128/jb.01642-09 ◽

2010 ◽

Vol 192 (12) ◽

pp. 2950-2964 ◽

Cited By ~ 97

Author(s):

S. L. Kuchma ◽

A. E. Ballok ◽

J. H. Merritt ◽

J. H. Hammond ◽

W. Lu ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Intracellular Signaling ◽

Biofilm Formation ◽

Diguanylate Cyclase ◽

Swarming Motility ◽

Signaling Molecule ◽

Single Mutant ◽

Epistasis Analysis ◽

Pilin Subunit ◽

Enhanced Expression

ABSTRACT The intracellular signaling molecule cyclic-di-GMP (c-di-GMP) has been shown to influence surface-associated behaviors of Pseudomonas aeruginosa, including biofilm formation and swarming motility. Previously, we reported a role for the bifA gene in the inverse regulation of biofilm formation and swarming motility. The bifA gene encodes a c-di-GMP-degrading phosphodiesterase (PDE), and the ΔbifA mutant exhibits increased cellular pools of c-di-GMP, forms hyperbiofilms, and is unable to swarm. In this study, we isolated suppressors of the ΔbifA swarming defect. Strains with mutations in the pilY1 gene, but not in the pilin subunit pilA gene, show robust suppression of the swarming defect of the ΔbifA mutant, as well as its hyperbiofilm phenotype. Despite the ability of the pilY1 mutation to suppress all the c-di-GMP-related phenotypes, the global pools of c-di-GMP are not detectably altered in the ΔbifA ΔpilY1 mutant relative to the ΔbifA single mutant. We also show that enhanced expression of the pilY1 gene inhibits swarming motility, and we identify residues in the putative VWA domain of PilY1 that are important for this phenotype. Furthermore, swarming repression by PilY1 specifically requires the diguanylate cyclase (DGC) SadC, and epistasis analysis indicates that PilY1 functions upstream of SadC. Our data indicate that PilY1 participates in multiple surface behaviors of P. aeruginosa, and we propose that PilY1 may act via regulation of SadC DGC activity but independently of altering global c-di-GMP levels.

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The SagS sensory protein modulates biofilm formation and c-di-GMP levels by Pseudomonas aeruginosa in response to glucose-6-phosphate.

10.5194/biofilms9-122 ◽

2020 ◽

Author(s):

Soyoung Park ◽

Jozef Dingemans ◽

Madison Gowett ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Genetic Analysis ◽

Signaling Pathway ◽

Biofilm Formation ◽

Phosphate Concentration ◽

Dependent Manner ◽

Diguanylate Cyclase ◽

Swarming Motility ◽

Two Component ◽

Insight Into

<p>In <em>Pseudomonas aeruginosa</em>, the orphan two-component sensor SagS contributes to both, the 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 as glucose-6-phosphate likewise enhanced biofilm formation. We show that exposure to glucose-6-phosphate results in decreased swarming motility but increased cellular c-di-GMP levels in biofilms. Genetic analysis indicated that the diguanylate cyclase NicD is an activator of biofilm formation and is not only required for enhanced biofilm formation in response to glucose-6-phosphate but also interacts with SagS. Our findings indicate glucose-6-phosphate to likely mimic a signal or conditions sensed by SagS to activate its motile-sessile switch function. Additionally, our findings provide new insight into the interfaces between the ligand-mediated TCS signaling pathway and c-di-GMP levels.</p>

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

10.1101/483339 ◽

2018 ◽

Cited By ~ 1

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.

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The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense

Research in Microbiology ◽

10.1016/j.resmic.2015.12.004 ◽

2016 ◽

Vol 167 (3) ◽

pp. 190-201 ◽

Cited By ~ 16

Author(s):

Alberto Ramírez-Mata ◽

Lilia I. López-Lara ◽

Ma. Luisa Xiqui-Vázquez ◽

Saúl Jijón-Moreno ◽

Angelica Romero-Osorio ◽

...

Keyword(s):

Biofilm Formation ◽

Azospirillum Brasilense ◽

Diguanylate Cyclase ◽

Exopolysaccharide Production

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Coordination of Swarming Motility, Biosurfactant Synthesis, and Biofilm Matrix Exopolysaccharide Production in Pseudomonas aeruginosa

Applied and Environmental Microbiology ◽

10.1128/aem.01237-14 ◽

2014 ◽

Vol 80 (21) ◽

pp. 6724-6732 ◽

Cited By ~ 42

Author(s):

Shiwei Wang ◽

Shan Yu ◽

Zhenyin Zhang ◽

Qing Wei ◽

Lu Yan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Bacterial Survival ◽

Swarming Motility ◽

Content Type ◽

Exopolysaccharide Production ◽

Complex Process ◽

Exopolysaccharide Biosynthesis ◽

Biofilm Matrix

ABSTRACTBiofilm formation is a complex process in which many factors are involved. Bacterial swarming motility and exopolysaccharides both contribute to biofilm formation, yet it is unclear how bacteria coordinate swarming motility and exopolysaccharide production. Psl and Pel are two key biofilm matrix exopolysaccharides inPseudomonas aeruginosa. This opportunistic pathogen has three types of motility, swimming, twitching, and swarming. In this study, we found that elevated Psl and/or Pel production reduced the swarming motility ofP. aeruginosabut had little effect on swimming and twitching. The reduction was due to decreased rhamnolipid production with no relation to the transcription ofrhlAB, two key genes involved in the biosynthesis of rhamnolipids. Rhamnolipid-negativerhlRandrhlABmutants synthesized more Psl, whereas exopolysaccharide-deficient strains exhibited a hyperswarming phenotype. These results suggest that competition for common sugar precursors catalyzed by AlgC could be a tactic forP. aeruginosato balance the synthesis of exopolysaccharides and rhamnolipids and to control bacterial motility and biofilm formation inversely because the biosynthesis of rhamnolipids, Psl, and Pel requires AlgC to provide the sugar precursors and an additionalalgCgene enhances the biosynthesis of Psl and rhamnolipids. In addition, our data indicate that the increase in RhlI/RhlR expression attenuated Psl production. This implied that the quorum-sensing signals could regulate exopolysaccharide biosynthesis indirectly in bacterial communities. In summary, this study represents a mechanism that bacteria utilize to coordinate swarming motility, biosurfactant synthesis, and biofilm matrix exopolysaccharide production, which is critical for biofilm formation and bacterial survival in the environment.

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Inverse Regulation of Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14

Journal of Bacteriology ◽

10.1128/jb.01685-06 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3603-3612 ◽

Cited By ~ 182

Author(s):

Nicky C. Caiazza ◽

Judith H. Merritt ◽

Kimberly M. Brothers ◽

George A. O'Toole

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Opportunistic Pathogen ◽

Wild Type ◽

Regulatory Pathway ◽

Early Step ◽

Swarming Motility ◽

Epistasis Analysis

ABSTRACT We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.

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BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14

Journal of Bacteriology ◽

10.1128/jb.00586-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8165-8178 ◽

Cited By ~ 227

Author(s):

Sherry L. Kuchma ◽

Kimberly M. Brothers ◽

Judith H. Merritt ◽

Nicole T. Liberati ◽

Frederick M. Ausubel ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Intracellular Signaling ◽

Biofilm Formation ◽

Cyclase Activity ◽

Diguanylate Cyclase ◽

Phosphodiesterase Activity ◽

Swarming Motility ◽

Ggdef Domain

ABSTRACT The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01049-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 9

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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