scholarly journals Vitamin B12-Mediated Restoration of Defective Anaerobic Growth Leads to Reduced Biofilm Formation in Pseudomonas aeruginosa

2012 ◽  
Vol 80 (5) ◽  
pp. 1639-1649 ◽  
Author(s):  
Kang-Mu Lee ◽  
Junhyeok Go ◽  
Mi Young Yoon ◽  
Yongjin Park ◽  
Sang Cheol Kim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Replication ◽  
Cell Elongation ◽  
Biofilm Formation ◽  
Shape Change ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Vitamin B ◽  
Content Type ◽  
Cell Shape Change

ABSTRACTPseudomonas aeruginosaundergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO3−) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments againstP. aeruginosabiofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B12to be a molecule that can reinstate defective anaerobic growth ofP. aeruginosa. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B12, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B12, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth ofP. aeruginosawith normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B12, further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth ofP. aeruginosaand illustrates the ability of vitamin B12to modulate the biofilm-forming capacity ofP. aeruginosaunder such condition.

scholarly journals Acquisition and Role of Molybdate in Pseudomonas aeruginosa

2014 ◽  
Vol 80 (21) ◽  
pp. 6843-6852 ◽  
Author(s):  
Victoria G. Pederick ◽  
Bart A. Eijkelkamp ◽  
Miranda P. Ween ◽  
Stephanie L. Begg ◽  
James C. Paton ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fatty Acid Composition ◽  
Nitrate Reduction ◽  
Biofilm Formation ◽  
Energy Production ◽  
Anaerobic Growth ◽  
Membrane Composition ◽  
Content Type ◽  
High Affinity

ABSTRACTIn microaerophilic or anaerobic environments,Pseudomonas aeruginosautilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO42−). Here, we show that molybdate acquisition inP. aeruginosaoccurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of themodAgene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition ofP. aeruginosa. Collectively, these data highlight the importance of molybdate for anaerobic growth ofP. aeruginosaand reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition.

scholarly journals Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

2011 ◽  
Vol 79 (7) ◽  
pp. 2792-2800 ◽  
Author(s):  
Kang-Mu Lee ◽  
Mi Young Yoon ◽  
Yongjin Park ◽  
Joon-Hee Lee ◽  
Sang Sun Yoon
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Growth Conditions ◽  
Airway Epithelial Cells ◽  
Anaerobic Growth ◽  
Pathogenic Potential ◽  
Content Type ◽  
Airway Infections ◽  
Pai 1

ABSTRACTPseudomonas aeruginosa, an opportunistic pathogen of clinical importance, causes chronic airway infections in patients with cystic fibrosis (CF). Current literature suggests that pockets with reduced oxygen tension exist in the CF airway mucus. However, virulence features of this opportunistic pathogen under such conditions are largely unknown. Cell-free supernatant of the standard laboratoryP. aeruginosastrain PAO1 obtained from anaerobic culture, but not aerobic culture, failed to kill A549 human airway epithelial cells. Further investigation revealed that this reduced cytotoxicity upon anaerobiosis was due to the suppressed secretion of elastase, a virulence factor controlled byP. aeruginosaquorum sensing (QS). Both alacZ-reporter fusion assay and quantitative real-time PCR (RT-PCR) analysis demonstrated that transcription of the elastase-encodinglasBgene was substantially decreased during anaerobic growth compared with aerobic growth. Moreover, transcription of other genes controlled by the LasI/R QS system, such asrhlR,vqsR,mvfR, andrsaL, was also repressed under the same anaerobic growth conditions. Importantly, synthesis of 3-oxo-C12-HSL (PAI-1), an autoinducer molecule that mediates induction of the LasI/R QS system, was >22-fold decreased during anaerobic growth while C4-HSL (PAI-2), which mediates RhlI/R QS, was nondetectable under the same growth conditions. Transcription of thelasBgene was restored by exogenous supplementation with autoinducers, with PAI-2 more effective than PAI-1 orPseudomonasquinolone signal (PQS) at restoring transcription of thelasBgene. Together, these results suggest that anaerobiosis deprivesP. aeruginosaof the ability to regulate its virulence via QS and this misregulation attenuates the pathogenic potential of this important pathogen.

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 Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

2012 ◽  
Vol 78 (15) ◽  
pp. 5060-5069 ◽  
Author(s):  
Morten T. Rybtke ◽  
Bradley R. Borlee ◽  
Keiji Murakami ◽  
Yasuhiko Irie ◽  
Morten Hentzer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Subsequent Development ◽  
Cellular Level ◽  
Host Immune System ◽  
Chronic Infections ◽  
Content Type ◽  
Genes Encoding ◽  
Green Fluorescent

ABSTRACTThe increased tolerance toward the host immune system and antibiotics displayed by biofilm-formingPseudomonas aeruginosaand other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsivecdrApromoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP inP. aeruginosastrains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment ofP. aeruginosawith the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology ofP. aeruginosa.

scholarly journals Effect of Nitroxides on Swarming Motility and Biofilm Formation, Multicellular Behaviors in Pseudomonas aeruginosa

2013 ◽  
Vol 57 (10) ◽  
pp. 4877-4881 ◽  
Author(s):  
César de la Fuente-Núñez ◽  
Fany Reffuveille ◽  
Kathryn E. Fairfull-Smith ◽  
Robert E. W. Hancock
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Wild Type ◽  
Planktonic Cells ◽  
Swarming Motility ◽  
Content Type ◽  
Exogenous Addition ◽  
Biofilm Dispersion ◽  
Dispersal Activity

ABSTRACTThe ability of nitric oxide (NO) to induce biofilm dispersion has been well established. Here, we investigated the effect of nitroxides (sterically hindered nitric oxide analogues) on biofilm formation and swarming motility inPseudomonas aeruginosa. A transposon mutant unable to produce nitric oxide endogenously (nirS) was deficient in swarming motility relative to the wild type and the complemented strain. Moreover, expression of thenirSgene was upregulated by 9.65-fold in wild-type swarming cells compared to planktonic cells. Wild-type swarming levels were substantially restored upon the exogenous addition of nitroxide containing compounds, a finding consistent with the hypothesis that NO is necessary for swarming motility. Here, we showed that nitroxides not only mimicked the dispersal activity of NO but also prevented biofilms from forming in flow cell chambers. In addition, anirStransposon mutant was deficient in biofilm formation relative to the wild type and the complemented strain, thus implicating NO in the formation of biofilms. Intriguingly, despite its stand-alone action in inhibiting biofilm formation and promoting dispersal, a nitroxide partially restored the ability of anirSmutant to form biofilms.

scholarly journals Contribution of Cell Elongation to the Biofilm Formation of Pseudomonas aeruginosa during Anaerobic Respiration

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e16105 ◽  
Author(s):  
Mi Young Yoon ◽  
Kang-Mu Lee ◽  
Yongjin Park ◽  
Sang Sun Yoon
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Elongation ◽  
Biofilm Formation ◽  
Anaerobic Respiration

scholarly journals The Anaerobic Regulatory Network Required for Pseudomonas aeruginosa Nitrate Respiration

2007 ◽  
Vol 189 (11) ◽  
pp. 4310-4314 ◽  
Author(s):  
Kerstin Schreiber ◽  
Robert Krieger ◽  
Beatrice Benkert ◽  
Martin Eschbach ◽  
Hiroyuki Arai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Bending ◽  
Regulatory Gene ◽  
Regulatory System ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Nitrate Respiration ◽  
Genetic Response ◽  
Anaerobic Conditions ◽  
Regulatory Model

ABSTRACT In Pseudomonas aeruginosa, the narK 1 K 2 GHJI operon encodes two nitrate/nitrite transporters and the dissimilatory nitrate reductase. The narK 1 promoter is anaerobically induced in the presence of nitrate by the dual activity of the oxygen regulator Anr and the N-oxide regulator Dnr in cooperation with the nitrate-responsive two-component regulatory system NarXL. The DNA bending protein IHF is essential for this process. Similarly, narXL gene transcription is enhanced under anaerobic conditions by Anr and Dnr. Furthermore, Anr and NarXL induce expression of the N-oxide regulator gene dnr. Finally, NarXL in cooperation with Dnr is required for anaerobic nitrite reductase regulatory gene nirQ transcription. A cascade regulatory model for the fine-tuned genetic response of P. aeruginosa to anaerobic growth conditions in the presence of nitrate was deduced.

scholarly journals Mapping of the Denitrification Pathway in Burkholderia thailandensis by Genome-Wide Mutant Profiling

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Alessandra Vitale ◽  
Sarah Paszti ◽  
Kohei Takahashi ◽  
Masanori Toyofuku ◽  
Gabriella Pessi ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Anaerobic Growth ◽  
Gene Encoding ◽  
Terminal Electron Acceptor ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Sufficient Energy ◽  
Genes Encoding ◽  
Human Infections

ABSTRACT Burkholderia thailandensis is a soil saprophyte that is closely related to the pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of B. thailandensis. Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon narIJHGK2K1, the aniA gene encoding a previously unknown nitrite reductase, and the petABC genes encoding a cytochrome bc1, as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of B. thailandensis and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic Burkholderia species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates N2O, which may play a role in the dispersal of B. thailandensis biofilms. IMPORTANCE Burkholderia thailandensis is a soil-dwelling saprophyte that is often used as surrogate of the closely related pathogen Burkholderia pseudomallei, the causative agent of melioidosis and a classified biowarfare agent. Both organisms are adapted to grow under oxygen-limited conditions in rice fields by generating energy through denitrification. Microoxic growth of B. pseudomallei is also considered essential for human infections. Here, we have used a Tn-Seq approach to identify the genes encoding the enzymes and regulators required for growth under denitrifying conditions. We show that a mutant that is defective in the conversion of N2O to N2, the last step in the denitrification process, is unaffected in microoxic growth but is severely impaired in biofilm formation, suggesting that N2O may play a role in biofilm dispersal. Our study identified novel targets for the development of therapeutic agents to treat meliodiosis.

scholarly journals Pseudomonas aeruginosa Requires the DNA-Specific Endonuclease EndA To Degrade Extracellular Genomic DNA To Disperse from the Biofilm

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Kathryn E. Cherny ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Genomic Dna ◽  
Early Stage ◽  
Content Type ◽  
Biofilm Dispersion ◽  
Biofilm Structure ◽  
First Time ◽  
Biofilm Matrix ◽  
Dispersed Cells

ABSTRACT The dispersion of biofilms is an active process resulting in the release of planktonic cells from the biofilm structure. While much is known about the process of dispersion cue perception and the subsequent modulation of the c-di-GMP pool, little is known about subsequent events resulting in the release of cells from the biofilm. Given that dispersion coincides with void formation and an overall erosion of the biofilm structure, we asked whether dispersion involves degradation of the biofilm matrix. Here, we focused on extracellular genomic DNA (eDNA) due to its almost universal presence in the matrix of biofilm-forming species. We identified two probable nucleases, endA and eddB, and eddA encoding a phosphatase that were significantly increased in transcript abundance in dispersed cells. However, only inactivation of endA but not eddA or eddB impaired dispersion by Pseudomonas aeruginosa biofilms in response to glutamate and nitric oxide (NO). Heterologously produced EndA was found to be secreted and active in degrading genomic DNA. While endA inactivation had little effect on biofilm formation and the presence of eDNA in biofilms, eDNA degradation upon induction of dispersion was impaired. In contrast, induction of endA expression coincided with eDNA degradation and resulted in biofilm dispersion. Thus, released cells demonstrated a hyperattaching phenotype but remained as resistant to tobramycin as biofilm cells from which they egress, indicating EndA-dispersed cells adopted some but not all of the phenotypes associated with dispersed cells. Our findings indicate for the first time a role of DNase EndA in dispersion and suggest weakening of the biofilm matrix is a requisite for biofilm dispersion. IMPORTANCE The finding that exposure to DNase I impairs biofilm formation or leads to the dispersal of early stage biofilms has led to the realization of extracellular genomic DNA (eDNA) as a structural component of the biofilm matrix. However, little is known about the contribution of intrinsic DNases to the weakening of the biofilm matrix and dispersion of established biofilms. Here, we demonstrate for the first time that nucleases are induced in dispersed Pseudomonas aeruginosa cells and are essential to the dispersion response and that degradation of matrix eDNA by endogenously produced/secreted EndA is required for P. aeruginosa biofilm dispersion. Our findings suggest that dispersing cells mediate their active release from the biofilm matrix via the induction of nucleases.

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