Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

PhoU inactivation in Pseudomonas aeruginosa enhances accumulation of ppGpp and polyphosphate.

ABSTRACT: Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In Pseudomonas aeruginosa, Pi is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced Pi removal from the medium and polyP accumulation. The phoU mutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition, phoU inactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by the phoU mutation.

SUBMITTER: de Almeida LG

PROVIDER: S-EPMC4393453 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

phoU inactivation in Pseudomonas aeruginosa enhances accumulation of ppGpp and polyphosphate.

de Almeida Luiz Gustavo LG Ortiz Julia Helena JH Schneider René P RP Spira Beny B

Applied and environmental microbiology 20150220 9

Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In Pseudomonas aeruginosa, Pi is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced Pi ...[more]

PMID: 25710363

Similar Datasets

Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase.

Project description:Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn-1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4 (+) is an activator of the enzyme and may function at concentrations lower than those of K(+); (iii) Zn(2+) is also an activator of paPpx and may substitute Mg(2+) in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg(2+) and capable of producing ATP regardless of the presence or absence of K(+) or NH4 (+) ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity.

| S-EPMC4631893 | biostudies-literature

Inorganic Polyphosphate Accumulation in Escherichia coli Is Regulated by DksA but Not by (p)ppGpp.

Project description:Production of inorganic polyphosphate (polyP) by bacteria is triggered by a variety of different stress conditions. polyP is required for stress survival and virulence in diverse pathogenic microbes. Previous studies have hypothesized a model for regulation of polyP synthesis in which production of the stringent-response second messenger (p)ppGpp directly stimulates polyP accumulation. In this work, I have now shown that this model is incorrect, and (p)ppGpp is not required for polyP synthesis in Escherichia coli However, stringent mutations of RNA polymerase that frequently arise spontaneously in strains defective in (p)ppGpp synthesis and null mutations of the stringent-response-associated transcription factor DksA both strongly inhibit polyP accumulation. The loss of polyP synthesis in a mutant lacking DksA was reversed by deletion of the transcription elongation factor GreA, suggesting that competition between these proteins for binding to the secondary channel of RNA polymerase plays an important role in controlling polyP activation. These results provide new insights into the poorly understood regulation of polyP synthesis in bacteria and indicate that the relationship between polyP and the stringent response is more complex than previously suspected.IMPORTANCE Production of polyP in bacteria is required for virulence and stress response, but little is known about how bacteria regulate polyP levels in response to changes in their environments. Understanding this regulation is important for understanding how pathogenic microbes resist killing by disinfectants, antibiotics, and the immune system. In this work, I have clarified the connections between polyP regulation and the stringent response to starvation stress in Escherichia coli and demonstrated an important and previously unknown role for the transcription factor DksA in controlling polyP levels.

| S-EPMC6456864 | biostudies-literature

Bioenergetics of the polyphosphates accumulation in Pseudomonas aeruginosa via polyphosphate kinase activation by choline in a lung colonization model.

Project description:Pseudomonas aeruginosa is an ubiquitous and opportunistic bacteria found in water, soil, plants, and immunocompromised humans. Cystic fibrosis (CF) patients are the most vulnerable population to lung colonization by these bacteria. Upon infection, choline and succinate are released from the CF lungs and are catabolized by P. aeruginosa. The bacteria accumulate inorganic polyphosphates, rather than succinate, when choline is catabolized, producing physiological and morphological changes leading to ineradicable infection. Thus, we sought to quantify the enzymes responsible for polyphosphate accumulation and to determine how choline catabolism affects energy flow and storage. Subcellular fractions showed that exo-polyphosphate phosphatase (PPX) activity resides mainly in the periplasm, and three isoenzymes of 24, 70, and 200 KD were found. The PPX activity in the periplasm of bacteria grown with choline was inhibited in an anti-competitive manner from Km 0.5 to 1 μM, and their Vmax increased from 50 to 100 nmol PO4≡ /min/g of protein in succinate medium. Since PPX inhibition by choline did not explain the 3.8-fold increase in polyphosphates, we quantified the polyphosphate kinase activity, and its significant 2.4-fold increase was consistent with the accumulation. Furthermore, intracellular ATP concentration directly correlated with the energetic yield of the carbon source and was significantly higher for succinate, suggesting that the restriction of energy caused by choline catabolism may induce morphological and physiological changes to the swarm form thus facilitating their migration and tissue colonization.

| S-EPMC9929196 | biostudies-literature

Pyocyanin biosynthesis protects Pseudomonas aeruginosa from nonthermal plasma inactivation.

Project description:Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time-dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP-treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain-derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5-methyl phenazine-1-carboxylic acid (5-Me-PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.

| S-EPMC9151332 | biostudies-literature

Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830.

Project description:Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P. aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of P. aeruginosa 8830. Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that ppk is transcribed from a sigmaE-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.

| S-EPMC91299 | biostudies-literature

Structural insights into PA3488-mediated inactivation of Pseudomonas aeruginosa PldA.

Project description:PldA, a phospholipase D (PLD) effector, catalyzes hydrolysis of the phosphodiester bonds of glycerophospholipids-the main component of cell membranes-and assists the invasion of the opportunistic pathogen Pseudomonas aeruginosa. As a cognate immunity protein, PA3488 can inhibit the activity of PldA to avoid self-toxicity. However, the precise inhibitory mechanism remains elusive. We determine the crystal structures of full-length and truncated PldA and the cryogenic electron microscopy structure of the PldA-PA3488 complex. Structural analysis reveals that there are different intermediates of PldA between the "open" and "closed" states of the catalytic pocket, accompanied by significant conformational changes in the "lid" region and the peripheral helical domain. Through structure-based mutational analysis, we identify the key residues responsible for the enzymatic activity of PldA. Together, these data provide an insight into the molecular mechanisms of PldA invasion and its neutralization by PA3488, aiding future design of PLD-targeted inhibitors and drugs.

| S-EPMC9550806 | biostudies-literature

ppGpp and polyphosphate modulate cell cycle progression in Caulobacter crescentus.

Project description:Caulobacter crescentus differentiates from a motile, foraging swarmer cell into a sessile, replication-competent stalked cell during its cell cycle. This developmental transition is inhibited by nutrient deprivation to favor the motile swarmer state. We identify two cell cycle regulatory signals, ppGpp and polyphosphate (polyP), that inhibit the swarmer-to-stalked transition in both complex and glucose-exhausted media, thereby increasing the proportion of swarmer cells in mixed culture. Upon depletion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or polyP improperly initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell fate determinant DivJ, and develop polar stalks. Furthermore, we show that swarmer cells produce more ppGpp than stalked cells upon starvation. These results provide evidence that ppGpp and polyP are cell-type-specific developmental regulators.

| S-EPMC3256613 | biostudies-literature

Pseudomonas aeruginosa kills Staphylococcus aureus in a polyphosphate-dependent manner.

Project description:Due to their frequent coexistence in many polymicrobial infections, including in patients with burn or chronic wounds or cystic fibrosis, recent studies have started to investigate the mechanistic details of the interaction between the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa rapidly outcompetes S. aureus under in vitro co-cultivation conditions, which is mediated by several of P. aeruginosa's virulence factors. Here, we report that polyphosphate (polyP), an efficient stress defense system and virulence factor in P. aeruginosa, plays a role for the pathogen's ability to inhibit and kill S. aureus in a contact-independent manner. We show that P. aeruginosa cells characterized by low polyP level are less detrimental to S. aureus growth and survival while the gram-positive pathogen is significantly more compromised by the presence of P. aeruginosa cells that produce high level of polyP. We show that the polyP-dependent phenotype could be a direct effect by the biopolymer, as polyP is present in the spent media and causes significant damage to the S. aureus cell envelope. However, more likely is that polyP's effects are indirect through the regulation of one of P. aeruginosa's virulence factors, pyocyanin. We show that pyocyanin production in P. aeruginosa occurs polyP-dependent and harms S. aureus through membrane damage and the generation of reactive oxygen species, resulting in increased expression of antioxidant enzymes. In summary, our study adds a new component to the list of biomolecules that the gram-negative pathogen P. aeruginosa generates to compete with S. aureus for resources.

| S-EPMC10723280 | biostudies-literature

Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa.

Project description:Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.

| S-EPMC6166797 | biostudies-literature

A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects.

Project description:Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the beta-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.

| S-EPMC1803759 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data