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 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 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.

scholarly journals Immediate activation of chemosensory neuron gene expression by bacterial metabolites is selectively induced by distinct cyclic GMP-dependent pathways in C. elegans

2019 ◽  
Author(s):  
Jaeseok Park ◽  
Joshua D Meisel ◽  
Dennis H Kim
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Sensory Neurons ◽  
Cyclic Gmp ◽  
Bacterial Load ◽  
Transcriptional Responses ◽  
Nervous Systems ◽  
Chemosensory Neurons ◽  
C Elegans ◽  
Calcium Dependent

AbstractDynamic gene expression in neurons shapes fundamental processes of the nervous systems of animals. But how different stimuli that activate the same neuron can lead to distinct transcriptional responses remains unclear. We have been studying how microbial metabolites modulate gene expression in chemosensory neurons of Caenorhabditis elegans. Considering the diverse environmental stimuli that can activate chemosensory neurons of C. elegans, we have sought to understand how specific transcriptional responses can be generated in these neurons in response to distinct cues. We have focused on the mechanism of rapid (<6 min) and selective transcriptional induction of daf-7, a gene encoding a TGF-β ligand that promotes bacterial lawn avoidance, in the ASJ chemosensory neurons in response to the pathogenic bacterium Pseudomonas aeruginosa. Here, we define the involvement of two distinct cyclic GMP (cGMP)-dependent pathways that are required for daf-7 expression in the ASJ neuron pair in response to P. aeruginosa. We show that a calcium-independent pathway dependent on the cGMP-dependent protein kinase G (PKG) EGL-4, and a canonical calcium-dependent signaling pathway dependent on the activity of a cyclic nucleotide-gated channel subunit CNG-2, function in parallel to activate rapid, selective transcription of daf-7 in response to P. aeruginosa metabolites. Our data suggest a requirement for PKG in promoting the fast, selective early transcription of neuronal genes in shaping responses to distinct microbial stimuli in a pair of chemosensory neurons of C. elegans.Author SummaryThe nervous systems of animals carry out the crucial roles of sensing and interpreting the external environment. When the free-living microscopic roundworm C. elegans is exposed to the pathogenic bacteria Pseudomonas aeruginosa, sensory neurons detect metabolites produced by the pathogen and induce expression of the gene for a neuroendocrine ligand called DAF-7. In turn, activity of DAF-7 is required for the full avoidance response to the P. aeruginosa, allowing the animals to reduce bacterial load and survive longer. Here, we systematically dissect the molecular pathway between the sensation of P. aeruginosa metabolites and the expression of daf-7 in a pair of C. elegans sensory neurons. We show that the intracellular signaling molecule cyclic GMP is a key signaling intermediate. In addition, we show that there are calcium-dependent and calcium-independent pathways that are both required to engage daf-7 expression, highlighting an organizational principle that allows neurons to distinguish between various stimuli.

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

&lt;p&gt;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].&lt;em&gt; Pseudomonas aeruginosa&lt;/em&gt;, 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].&lt;br /&gt;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&lt;sub&gt;2&lt;/sub&gt; [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.&lt;br /&gt;To establish these methods, the two known phosphodiesterases, PdeH from &lt;em&gt;Escherichia coli&lt;/em&gt; [5] and RocR from &lt;em&gt;P. aeruginosa&lt;/em&gt; [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 &lt;em&gt;E. coli&lt;/em&gt; phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity &lt;em&gt;in vivo&lt;/em&gt;.&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt; &lt;p&gt;[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.&lt;/p&gt; &lt;p&gt;[2] R&amp;#246;mling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629&amp;#8211;639.&lt;/p&gt; &lt;p&gt;[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547&amp;#8211;12555.&lt;/p&gt; &lt;p&gt;[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17&amp;#8211;23.&lt;/p&gt; &lt;p&gt;[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434&amp;#8211;2446.&lt;/p&gt; &lt;p&gt;[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.&amp;#160; (2012). Signaling. J. Bacteriol. 194: 4837&amp;#8211;4846&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt;

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.

Sign in / Sign up

Export Citation Format

Share Document