phoU Inactivation in Pseudomonas aeruginosa Enhances Accumulation of ppGpp and Polyphosphate (original) (raw)
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phoU inactivation in Pseudomonas aeruginosa enhances ppGpp and polyphosphate accumulation
Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In P. 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. Here we show 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.
Two different mechanisms mediate chemotaxis to inorganic phosphate in Pseudomonas aeruginosa
Scientific Reports, 2016
Inorganic phosphate (Pi) is a central signaling molecule that modulates virulence in various pathogens. In Pseudomonas aeruginosa, low Pi concentrations induce transcriptional alterations that increase virulence. Also, under low Pi levels, P. aeruginosa exhibits Pi chemotaxis—a process mediated by the two non-paralogous receptors CtpH and CtpL. Here we show that the two receptors operate via different mechanisms. We demonstrate that the ligand binding domain (LBD) of CtpH but not CtpL binds Pi directly. We identify the periplasmic ligand binding protein PstS as the protein that binds in its Pi loaded state to CtpL, resulting in receptor stimulation. PstS forms part of the Pi transporter and has thus a double function in Pi transport and chemotaxis. The affinity of Pi for CtpH was modest whereas that for PstS very high, which may explain why CtpH and CtpL mediate chemotaxis to high and low Pi concentrations, respectively. The pstS/ctpH double mutant was almost devoid of Pi taxis, ind...
Microbiology, 2004
Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 49-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.
Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Enzyme Research, 2015
Pseudomonas aeruginosaexopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1plus inorganic phosphate(Pi). In a recent work we have shown thatpaPpx is involved in the pathogenesis ofP. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described forE. coliPpx (ecPpx) but we also discovered new and original characteristics ofpaPpx: (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) Zn2+is also an activator ofpaPpx and may substitute Mg2+in the catalytic site; and (iv)paPpx also has phosphotransferase activity, dependent on Mg2+and capable of producing ATP regardless of the presence or absence of K+orNH4+ions. In addition, we detected that the active site responsible for the phosphatase activity is als...
Microbiology, 2005
Pseudomonas aeruginosais a Gram-negative bacterium associated with nosocomial infections and cystic fibrosis. Chronic bacterial infections are increasingly associated with the biofilm lifestyle in which microcolonies are embedded in an extracellular matrix. Screening procedures for identifying biofilm-deficient strains have allowed the characterization of several key determinants involved in this process. Biofilm-deficientP. aeruginosaPAK strains affected in a seven-gene cluster calledpelwere characterized. Thepelgenes encode proteins with similarity to components involved in polysaccharide biogenesis, of which PelF is a putative glycosyltransferase. PelG was also identified as a putative component of the polysaccharide transporter (PST) family. Thepelgenes were previously identified in theP. aeruginosaPA14 strain as required for the production of a glucose-rich matrix material involved in the formation of a thick pellicle and resistant biofilm. However, in PA14, thepelmutants have ...
THE EFFECT OF KNOCKING OUT OF PVDP GENE IN THE VIRULENCE OF PSEUDOMONAS AERUGINOSA
International Journal of Applied Pharmaceutics, 2024
The aim of the study is to investigate the role of PvdP enzyme in the virulence of Pseudomonas aeruginosa both in vitro and in vivo. Methods: In this study, we investigate the effect of deletion of pvdP gene on P. aeruginosa by observing its phenotypes. The observed phenotypes are the growth, pyoverdine production, motilities, 3-oxo-C12-Homo Serine Lactone (3-oxo-C12-HSL) accumulation and biofilm formation. The growth and pyoverdine production were investigated under a low iron condition, while the motilities of the mutant were investigated in a semisolid media. The accumulation 3-oxo-C12-HSL was facilitated by a biosensor strain and biofilm formation was investigated using a spectrophotometer through a crystal violet staining method. The in vivo study was performed to Galleria mellonella larvae as an infection model. Results: The deletion of the pvdP gene does not affect the growth of the P. aeruginosa but significantly reduces the production of pyoverdine. The motility properties of the bacteria were not affected by the deletion of the pvdP gene. The P. aeruginosa PvdP knockout mutant also showed a reduction in the biofilm formation and the accumulation of 3-oxo-C12-HSL at low iron concentrations. In an in vivo experiment, the PvdP knockout mutant caused a significantly reduced death rate of G. mellonella larvae infection model compared to the control group. Conclusion: The findings underscore the major role of PvdP in pyoverdine production, its contribution to biofilm formation, and the motility of P. aeruginosa. Those results confirm the important role of PvdP in the virulence of P. aeruginosa in vitro and in vivo.
mBio, 2011
Gram-negative outer membrane (OM) integrity is maintained in part by Mg2+ cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose...
Biochemical Journal, 2018
A quantitative Pseudomonas aeruginosa proteomics approach revealed increased abundance of the so-far uncharacterized protein PA3911 in anaerobic biofilms grown under conditions of the cystic fibrosis lung. Physiological relevance of ORF PA3911 was demonstrated, inter alia, using phenotype microarray experiments. The mutant strain showed increased susceptibility in the presence of antimicrobials (minocycline, nafcillin, oxacillin, chloramphenicol and thiamphenicol), enhanced twitching motility and significantly impaired biofilm formation. PA3911 is a soluble, cytoplasmic protein in P. aeruginosa. In protein–lipid overlay experiments, purified PA3911 bound specifically to phosphatidic acid (PA), the central hub of phospholipid metabolism. Structure-guided site-directed mutagenesis was used to explore the proposed ligand-binding cavity of PA3911. Protein variants of Leu56, Leu58, Val69 and Leu114 were shown to impair PA interaction. A comparative shotgun lipidomics approach demonstrate...