The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa.
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SUBMITTER: Vo CD
PROVIDER: S-EPMC7335794 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.
| S-EPMC94946 | biostudies-literature
Project description:UnlabelledOxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO(3-)? NO(2-)? NO ? N2O ? N2 Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment.ImportanceThe processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen.
| S-EPMC4836231 | biostudies-literature
Project description:The most efficient means of generating cellular energy is through aerobic respiration. Under anaerobic conditions, several prokaryotes can replace oxygen by nitrate as final electron acceptor. During denitrification, nitrate is reduced via nitrite, NO and N2 O to molecular nitrogen (N2 ) by four membrane-localized reductases with the simultaneous formation of an ion gradient for ATP synthesis. These four multisubunit enzyme complexes are coupled in four electron transport chains to electron donating primary dehydrogenases and intermediate electron transfer proteins. Many components require membrane transport and insertion, complex assembly and cofactor incorporation. All these processes are mediated by fine-tuned stable and transient protein-protein interactions. Recently, an interactomic approach was used to determine the exact protein-protein interactions involved in the assembly of the denitrification apparatus of Pseudomonas aeruginosa. Both subunits of the NO reductase NorBC, combined with the flavoprotein NosR, serve as a membrane-localized assembly platform for the attachment of the nitrate reductase NarGHI, the periplasmic nitrite reductase NirS via its maturation factor NirF and the N2 O reductase NosZ through NosR. A nitrate transporter (NarK2), the corresponding regulatory system NarXL, various nitrite (NirEJMNQ) and N2 O reductase (NosFL) maturation proteins are also part of the complex. Primary dehydrogenases, ATP synthase, most enzymes of the TCA cycle, and the SEC protein export system, as well as a number of other proteins, were found to interact with the denitrification complex. Finally, a protein complex composed of the flagella protein FliC, nitrite reductase NirS and the chaperone DnaK required for flagella formation was found in the periplasm of P. aeruginosa. This work demonstrated that the interactomic approach allows for the identification and characterization of stable and transient protein-protein complexes and interactions involved in the assembly and function of multi-enzyme complexes.
| S-EPMC5658584 | biostudies-literature
Project description:Most bacteria can generate ATP by respiratory metabolism, in which electrons are shuttled from reduced substrates to terminal electron acceptors, via quinone molecules like ubiquinone. Dioxygen (O2) is the terminal electron acceptor of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone. Here, we characterize a novel, O2-independent pathway for the biosynthesis of ubiquinone. This pathway relies on three proteins, UbiT (YhbT), UbiU (YhbU), and UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory factor of the biosynthetic pathway, while UbiU and UbiV (UbiU-UbiV) are involved in hydroxylation reactions and represent a novel class of O2-independent hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activity. The UbiT, -U, and -V proteins are found in alpha-, beta-, and gammaproteobacterial clades, including several human pathogens, supporting the widespread distribution of a previously unrecognized capacity to synthesize ubiquinone in the absence of O2 Together, the O2-dependent and O2-independent ubiquinone biosynthesis pathways contribute to optimizing bacterial metabolism over the entire O2 range.IMPORTANCE In order to colonize environments with large O2 gradients or fluctuating O2 levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence, and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes of Escherichia coli to this pathway and show that the pathway functions independently from O2 In contrast, the long-described pathway for ubiquinone biosynthesis requires O2 as a substrate. In fact, we find that many proteobacteria are equipped with the O2-dependent and O2-independent pathways, supporting that they are able to synthesize ubiquinone over the entire O2 range. Overall, we propose that the novel O2-independent pathway is part of the metabolic plasticity developed by proteobacteria to face various environmental O2 levels.
| S-EPMC6747719 | biostudies-literature
Project description:Microbes in biofilms face the challenge of substrate limitation. In particular, oxygen often becomes limited for cells in Pseudomonas aeruginosa biofilms growing in the laboratory or during host colonization. Previously we found that phenazines, antibiotics produced by P. aeruginosa, balance the intracellular redox state of cells in biofilms. Here, we show that genes involved in denitrification are induced in phenazine-null (?phz) mutant biofilms grown under an aerobic atmosphere, even in the absence of nitrate. This finding suggests that resident cells employ a bet-hedging strategy to anticipate the potential availability of nitrate and counterbalance their highly reduced redox state. Consistent with our previous characterization of aerobically grown colonies supplemented with nitrate, we found that the pathway that is induced in ?phz mutant colonies combines the nitrate reductase activity of the periplasmic enzyme Nap with the downstream reduction of nitrite to nitrogen gas catalyzed by the enzymes Nir, Nor, and Nos. This regulatory relationship differs from the denitrification pathway that functions under anaerobic growth, with nitrate as the terminal electron acceptor, which depends on the membrane-associated nitrate reductase Nar. We identified the sequences in the promoter regions of the nap and nir operons that are required for the effects of phenazines on expression. We also show that specific phenazines have differential effects on nap gene expression. Finally, we provide evidence that individual steps of the denitrification pathway are catalyzed at different depths within aerobically grown biofilms, suggesting metabolic cross-feeding between community subpopulations.IMPORTANCE An understanding of the unique physiology of cells in biofilms is critical to our ability to treat fungal and bacterial infections. Colony biofilms of the opportunistic pathogen Pseudomonas aeruginosa grown under an aerobic atmosphere but without nitrate express a denitrification pathway that differs from that used for anaerobic growth. We report that the components of this pathway are induced by electron acceptor limitation and that they are differentially expressed over the biofilm depth. These observations suggest that (i) P. aeruginosa exhibits "bet hedging," in that it expends energy and resources to prepare for nitrate availability when other electron acceptors are absent, and (ii) cells in distinct biofilm microniches may be able to exchange substrates to catalyze full denitrification.
| S-EPMC5892114 | biostudies-literature
Project description:Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P. aeruginosa can perform (complete) denitrification, a respiratory process of dissimilatory nitrate reduction to nitrogen gas via nitrite (NO2), nitric oxide (NO) and nitrous oxide (N2O). This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P. aeruginosa. To our knowledge, this is the first mathematical model of denitrification for this bacterium. Analysis of the long-term behavior of the network under changing concentration levels of oxygen (O2), nitrate (NO3), and phosphate (PO4) suggests that PO4 concentration strongly affects denitrification performance. The model provides three predictions on denitrification activity of P. aeruginosa under various environmental conditions, and these predictions are either experimentally validated or supported by pertinent biological literature. One motivation for this study is to capture the effect of PO4 on a denitrification metabolic network of P. aeruginosa in order to shed light on mechanisms for greenhouse gas N2O accumulation during seasonal oxygen depletion in aquatic environments such as Lake Erie (Laurentian Great Lakes, USA). Simulating the microbial production of greenhouse gases in anaerobic aquatic systems such as Lake Erie allows a deeper understanding of the contributing environmental effects that will inform studies on, and remediation strategies for, other hypoxic sites worldwide.
| S-EPMC4338189 | biostudies-literature
Project description:Pseudomonas aeruginosa is an opportunistic pathogen that can thrive by anaerobic respiration in the lungs of cystic fibrosis patients using nitrate as terminal electron acceptor. Here, we report the identification and characterization of the small RNA PaiI in the P. aeruginosa strain 14 (PA14). PaiI is anaerobically induced in the presence of nitrate and depends on the two-component system NarXL. Our studies revealed that PaiI is required for efficient denitrification affecting the conversion of nitrite to nitric oxide. In the absence of PaiI anaerobic growth was impaired on glucose, which can be reconciled with a decreased uptake of the carbon source under these conditions. The importance of PaiI for anaerobic growth is further underlined by the observation that a paiI deletion mutant was impaired in growth in murine tumors.
| S-EPMC5703892 | biostudies-literature
Project description:Many pseudomonads produce redox active compounds called phenazines that function in a variety of biological processes. Phenazines are well known for their toxicity against non-phenazine-producing organisms, which allows them to serve as crucial biocontrol agents and virulence factors during infection. As for other secondary metabolites, conditions of nutritional stress or limitation stimulate the production of phenazines, but little is known of the molecular details underlying this phenomenon. Using a combination of microarray and metabolite analyses, we demonstrate that the assimilation of glycine as a carbon source and the biosynthesis of pyocyanin in Pseudomonas aeruginosa PAO1 are both dependent on the PA2449 gene. The inactivation of the PA2449 gene was found to influence the transcription of a core set of genes encoding a glycine cleavage system, serine hydroxymethyltransferase, and serine dehydratase. PA2449 also affected the transcription of several genes that are integral in cell signaling and pyocyanin biosynthesis in P. aeruginosa PAO1. This study sheds light on the unexpected relationship between the utilization of an unfavorable carbon source and the production of pyocyanin. PA2449 is conserved among pseudomonads and might be universally involved in the assimilation of glycine among this metabolically diverse group of bacteria.
| S-EPMC3624589 | biostudies-literature
Project description:Bacterial pathogenesis frequently requires metal acquisition by specialized, small-molecule metallophores. We hypothesized that the Gram-negative Pseudomonas aeruginosa encodes the enzymes nicotianamine synthase (NAS) and opine dehydrogenase (ODH), biosynthesizing a new class of opine metallophore, previously characterized only in the unrelated Gram-positive organism Staphylococcus aureus. The identity of this metallophore, herein named pseudopaline, was determined through measurements of binding affinity, the in vitro reconstitution of the biosynthetic pathway to screen potential substrates, and the confirmation of product formation by mass spectrometry. Pseudopaline and the S. aureus metallophore staphylopine exhibit opposite stereochemistry for the histidine moiety, indicating unique recognition by NAS. Additionally, we demonstrate SaODH catalysis in the presence of pyruvate, as previously shown, but also oxaloacetate, suggesting the potential for the production of a variant form of staphylopine, while PaODH specifically recognizes ?-ketoglutarate. Both the staphylopine and pseudopaline operons have been implicated in the pathogenesis of key infectious disease states and warrant further study.
| S-EPMC5699221 | biostudies-literature
Project description:Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ?treYZ?treS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ?treYZ?treS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ?treYZ?treS in trans. Surprisingly, the growth defect of the double ?treYZ?treS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved "house-keeping" anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf.
| S-EPMC3591346 | biostudies-literature