The Diguanylate Cyclase YfiN of Pseudomonas aeruginosa Regulates Biofilm Maintenance in Response to Peroxide

Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found extracellular polysaccharide (EPS) production, regulation of motility and induction of stress response pathways as contributing 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 ΔyfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H2O2); this defect can be rescued through genetic complementation. Additionally, we found that the ΔyfiN mutant biofilms produce less c-di-GMP than WT, and that H2O2 treatment enhanced motility of surface-associated bacteria and increased cell death for the ΔyfiN 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.

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

mBio ◽

10.1128/mbio.00183-10 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 106

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.

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c-di-GMP-linked phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein

10.1101/154005 ◽

2017 ◽

Author(s):

Gianlucca G. Nicastro ◽

Gilberto H. Kaihami ◽

André A. Pulschen ◽

Jacobo Hernandez-Montelongo ◽

Ana Laura Boechat ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Strain ◽

Wild Type Strain ◽

Fine Tuning ◽

Diguanylate Cyclase ◽

Wild Type ◽

Type Iv ◽

Major Player ◽

In The Wild ◽

Specialized Function

Summaryc-di-GMP is a major player in the decision between biofilm and motile lifestyles. Several bacteria present a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be proven in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells, but scattered in the cytoplasm of cells lacking FimV. Overexpression of DgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a AfimV background. Therefore, our findings strongly suggest that DgcP is regulated by FimV and may provide the local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

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Pseudomonas aeruginosa Uses c-di-GMP Phosphodiesterases RmcA and MorA To Regulate Biofilm Maintenance

mBio ◽

10.1128/mbio.03384-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

S. Katharios-Lanwermeyer ◽

G. B. Whitfield ◽

P. L. Howell ◽

G. A. O’Toole

Keyword(s):

Pseudomonas Aeruginosa ◽

Nutrient Limitation ◽

Regulatory System ◽

Stringent Response ◽

Extracellular Polysaccharide ◽

Discrete Control ◽

Metabolizing Enzymes ◽

Content Type ◽

Polysaccharide Biosynthesis ◽

Subsequent Loss

ABSTRACT 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 (ΔrelA ΔspoT), indicating that the ΔrmcA and ΔmorA 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 ΔrmcA and ΔmorA 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 ΔrmcA and ΔmorA mutants was also found in the stringent response-defective ΔrelA ΔspoT 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.

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Influence of OprM expression on multiple antibiotic resistance in Pseudomonas aeruginosa.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.9.2009 ◽

1997 ◽

Vol 41 (9) ◽

pp. 2009-2012 ◽

Cited By ~ 13

Author(s):

K K Wong ◽

K Poole ◽

N Gotoh ◽

R E Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Antimicrobial Agents ◽

Multiple Antibiotic Resistance ◽

Wild Type ◽

Beta Lactams ◽

Efflux System ◽

Cloned Gene

MexA-MexB-OprM is an efflux system in Pseudomonas aeruginosa. OprM overproduced from the cloned gene was able to complement OprM-deficient mutants but did not alter the resistance of a wild-type P. aeruginosa strain to the different antimicrobial agents tested. This suggests that OprM cannot function by itself to efflux antibiotics, including beta-lactams targeted to the periplasm.

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Reduced Expression of therplU-rpmARibosomal Protein Operon inmexXY-Expressing Pan-Aminoglycoside-Resistant Mutants of Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00846-12 ◽

2012 ◽

Vol 56 (10) ◽

pp. 5171-5179 ◽

Cited By ~ 21

Author(s):

Calvin Ho-Fung Lau ◽

Sebastien Fraud ◽

Marcus Jones ◽

Scott N. Peterson ◽

Keith Poole

Keyword(s):

Pseudomonas Aeruginosa ◽

Promoter Region ◽

Ribosomal Proteins ◽

Antimicrobial Agents ◽

Resistant Mutant ◽

Wild Type ◽

Efflux System ◽

Aminoglycoside Resistance ◽

Gene Encoding ◽

Content Type

ABSTRACTPan-aminoglycoside-resistantPseudomonas aeruginosamutants expressing themexXYcomponents of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in themexZgene encoding a repressor of this efflux system and in themexXYpromoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068–1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of therplU-rpmAoperon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-typeP. aeruginosaPAO1. Moreover, correction of the mutation restoredrplU-rpmAexpression and, significantly, reversed the elevatedmexXYexpression and pan-aminoglycoside resistance of the mutant. ReducedrplU-rpmAexpression was also observed in a secondmexXY-expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in therplU-rpmApromoter region. Restoration ofrplU-rpmAexpression in the K2968 mutant following chromosomal integration of therplU-rpmAoperon derived from wild-typeP. aeruginosafailed, however, to reverse the elevatedmexXYexpression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impactingrplU-rpmAexpression in K2968 also impacts othermexXY-related genes. IncreasedmexXYexpression owing to reducedrplU-rpmAexpression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment ofmexXYand does so via PA5471, reminiscent ofmexXYinduction by ribosome-disrupting antimicrobial agents. Interestingly, reducedrplU-rpmAexpression was also observed in amexXY-expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.

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The Diguanylate Cyclase GcbA Facilitates Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA

Journal of Bacteriology ◽

10.1128/jb.02244-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 174-187 ◽

Cited By ~ 43

Author(s):

Olga E. Petrova ◽

Kathryn E. Cherny ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Posttranslational Modifications ◽

Opportunistic Pathogen ◽

Diguanylate Cyclase ◽

Wild Type ◽

Biofilm Growth ◽

Content Type ◽

Chemosensory Protein ◽

Biofilm Dispersion ◽

Specific Regulation

Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogenPseudomonas aeruginosais known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype ofgcbAmutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion.

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rpoN Gene of Pseudomonas aeruginosa Alters Its Susceptibility to Quinolones and Carbapenems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00348-06 ◽

2007 ◽

Vol 51 (4) ◽

pp. 1455-1462 ◽

Cited By ~ 25

Author(s):

Darija Viducic ◽

Tsuneko Ono ◽

Keiji Murakami ◽

Mikiko Katakami ◽

Heni Susilowati ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Agent ◽

Stationary Phase ◽

Antimicrobial Agents ◽

Wild Type Strain ◽

Fluorescent Protein ◽

Wild Type ◽

Green Fluorescent Protein Reporter ◽

Green Fluorescent ◽

Fluorescent Protein Reporter

ABSTRACT The alternative sigma factor σ54 has been implicated in diverse functions within the cells. In this study, we have constructed an rpoN mutant of Pseudomonas aeruginosa and investigated its importance as a target for antimicrobial agents, such as quinolones and carbapenems. The stationary-phase cells of the rpoN mutant displayed a survival rate approximately 15 times higher than that of the wild-type cells in the presence of quinolones and carbapenems. The stationary phase led to substantial production of pyoverdine by the P. aeruginosa rpoN mutant. Pyoverdine synthesis correlated with decreased susceptibility to antimicrobial agents. Quantitative real-time PCR revealed that stationary-phase cells of the rpoN mutant grown without an antimicrobial agent had approximately 4- to 140- and 2- to 14-fold-higher levels of transcripts of the pvdS and vqsR genes, respectively, than the wild-type strain. In the presence of an antimicrobial agent, levels of pvdS and vqsR transcripts were elevated 400- and 5-fold, respectively, in comparison to the wild-type levels. Flow cytometry assays using a green fluorescent protein reporter demonstrated increased expression of the vqsR gene in the rpoN mutant throughout growth. A pvdS mutant of P. aeruginosa, deficient in pyoverdine production, was shown to be susceptible to biapenem. These findings suggest that rpoN is involved in tolerance to antimicrobial agents in P. aeruginosa and that its tolerant effect is partly dependent on increased pyoverdine production and vqsR gene expression.

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