Distinct functions for diguanylate cyclases in regulating Pseudomonas aeruginosa PA14 surface-associated behaviors.

2009 ◽  
Author(s):  
Judith H. Merritt
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Diguanylate Cyclases

scholarly journals Oligoribonuclease is the primary degradative enzyme for pGpG inPseudomonas aeruginosathat is required for cyclic-di-GMP turnover

2015 ◽  
Vol 112 (36) ◽  
pp. E5048-E5057 ◽  
Author(s):  
Mona W. Orr ◽  
Gregory P. Donaldson ◽  
Geoffrey B. Severin ◽  
Jingxin Wang ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Half Life ◽  
Biofilm Formation ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Capillary Action ◽  
Diguanylate Cyclases ◽  
Active Site Mutants ◽  
Degradative Enzyme

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A fromPseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆ornstrains ofP. aeruginosaPA14 for pGpG stability. The lysates from ∆ornshowed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆ornstrain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulatedpelpromoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆ornstrain in apel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆ornstrain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

scholarly journals Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence

2006 ◽  
Vol 103 (8) ◽  
pp. 2839-2844 ◽  
Author(s):  
H. Kulasakara ◽  
V. Lee ◽  
A. Brencic ◽  
N. Liberati ◽  
J. Urbach ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cyclic Gmp ◽  
Diguanylate Cyclases

scholarly journals Induction of native c-di-GMP phosphodiesterases leads to dispersal of Pseudomonas aeruginosa biofilms

2021 ◽  
Author(s):  
Jens Bo Andersen ◽  
Kasper Nørskov Kragh ◽  
Louise Dahl Hultqvist ◽  
Morten Rybtke ◽  
Martin Nilsson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Single Cell ◽  
Biofilm Formation ◽  
Ectopic Expression ◽  
Second Messenger ◽  
Flow Cell ◽  
Diguanylate Cyclases ◽  
Biofilm Dispersal ◽  
High Level

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 have previously presented evidence that ectopic expression in Pseudomonas aeruginosa of the Escherichia coli phosphodiesterase YhjH results in biofilm dispersal. More recently, however, evidence has been presented that induction of native c-di-GMP phosphodiesterases does not lead to dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for development of anti-biofilm drugs. However, here we demonstrate that induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed does result in dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and in a flow-cell biofilm system.

Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Anna Scherhag ◽  
Martina Rüger ◽  
Katrin Gerbracht ◽  
Jaqueline Rehner ◽  
Susanne Zehner ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Model Organism ◽  
Functional Complementation ◽  
Full Length ◽  
Protein Activity ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Ggdef Domain

<p>The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2].<em> Pseudomonas aeruginosa</em>, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].<br />One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O<sub>2</sub> [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.<br />To establish these methods, the two known phosphodiesterases, PdeH from <em>Escherichia coli</em> [5] and RocR from <em>P. aeruginosa</em> [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an <em>E. coli</em> phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity <em>in vivo</em>.</p> <p> </p> <p> </p> <p>[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.</p> <p>[2] Römling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629–639.</p> <p>[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.</p> <p>[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17–23.</p> <p>[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434–2446.</p> <p>[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.  (2012). Signaling. J. Bacteriol. 194: 4837–4846</p> <p> </p>

scholarly journals Ethanol decreases Pseudomonas aeruginosa flagellar motility through a cyclic-di-GMP- and stator-dependent pathway

2018 ◽  
Author(s):  
Kimberley A. Lewis ◽  
Amy E. Baker ◽  
Annie I. Chen ◽  
Colleen E. Harty ◽  
Sherry L. Kuchma ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Microscopic Analysis ◽  
Fold Increase ◽  
Bioactive Metabolites ◽  
Flagellar Motility ◽  
Planktonic Cells ◽  
Motile Cells ◽  
Diguanylate Cyclases ◽  
Surface Sensing ◽  
In The Wild

AbstractPseudomonas aeruginosa frequently encounters microbes that produce bioactive metabolites including ethanol. At concentrations that do not affect growth, we found that ethanol reduces P. aeruginosa motility by 30% in a swim agar assay and this decrease is accompanied by a 2.5-fold increase in levels of cyclic diguanylate (c-di-GMP), a second messenger that represses motility, in planktonic cells. A screen of mutants lacking genes involved in c-di-GMP metabolism identified SadC and GcbA as diguanylate cyclases involved in swim zone reduction by ethanol and ethanol-induced c-di-GMP production. The reduction of swimming in response to ethanol also required the stator set, MotAB, two PilZ-domain proteins (FlgZ and PilZ), PilY1-a proposed surface-sensing protein, and PilMNOP, which comprises the pilus alignment complex and these proteins have been previously implicated in the control of motility on agar surfaces. Microscopic analysis of the fraction of quiescent cells in swim medium showed that ethanol decreased the portion of motile cells in the wild type, but had opposite effects in the ∆pilY1, ∆pilMNOP, ∆motAB, and ∆pilZ∆flgZ mutants. Together, these data indicate ethanol induces a regulated change in motility in planktonic cells at concentrations similar to those produced by other microbes. We propose that this ethanol-responsiveness may contribute to the co-localization of P. aeruginosa with ethanol-producing microbes.

scholarly journals rpoS-mutation variants are selected in Pseudomonas aeruginosa biofilms under imipenem pressure

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiangke Duan ◽  
Yanrong Pan ◽  
Zhao Cai ◽  
Yumei Liu ◽  
Yingdan Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Treatment ◽  
Point Mutations ◽  
Opportunistic Pathogen ◽  
Antimicrobial Treatment ◽  
High Dose ◽  
Sequencing Analysis ◽  
Adverse Conditions ◽  
Diguanylate Cyclases ◽  
Cyclic Treatment

Abstract Background Pseudomonas aeruginosa is a notorious opportunistic pathogen causing various types of biofilm-related infections. Biofilm formation is a unique microbial strategy that allows P. aeruginosa to survive adverse conditions such as antibiotic treatment and human immune clearance. Results In this study, we experimentally evolved P. aeruginosa PAO1 biofilms for cyclic treatment in the presence of high dose of imipenem, and enriched hyperbiofilm mutants within six cycles in two independent lineages. The competition assay showed that the evolved hyperbiofilm mutants can outcompete the ancestral strain within biofilms but not in planktonic cultures. Whole-genome sequencing analysis revealed the hyperbiofilm phenotype is caused by point mutations in rpoS gene in all independently evolved mutants and the same mutation was found in P. aeruginosa clinical isolates. We further showed that mutation in rpoS gene increased the intracellular c-di-GMP level by turning on the expression of the diguanylate cyclases. Mutation in rpoS increased pyocyanin production and virulence in hyperbiofilm variants. Conclusion Here, our study revealed that antibiotic treatment of biofilm-related P. aeruginosa infections might induce a hyperbiofilm phenotype via rpoS mutation, which might partially explain antimicrobial treatment failure of many P. aeruginosa biofilm-related infections.

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