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PubMed1982

Inhibition of Photosynthetic Energy Conversion by Cupric Ion : EVIDENCE FOR Cu-COUPLING FACTOR 1 INTERACTION.

This study describes a specific Cu(2+) and light-dependent inhibition of spinach (Spinacia oleracea L.) chloroplast reactions involving coupling factor 1 function. A primary effect is an inhibition of photophosphorylation induced by illumination of Class II chloroplasts with micromolar Cu(2+) and pyocyanine in the absence of ADP, Mg(2+), and HPO(4) (2-). The inhibition, which is dependent on free Cu(2+) as indicated by protection by ethylene diamine tetraacetic acid and dithiothreitol, requires illumination (electron flow) for establishment of the specific inhibition to be noted. Protection is also afforded by uncouplers and some partial protection is provided by micromolar concentrations of ADP and ATP. The data strongly suggest that Cu(2+) causes an O(2)-independent oxidation of sulfhydryl groups on coupling factor 1, which are essential to catalytic function. This conclusion is supported by the reduction of energy-dependent (3)H-N-ethylmaleimide labeling of the gamma subunit of coupling factor 1 by the Cu(2+)-light pretreatment.

Journal Article↗

PubMed1984

Temperature dependence of energy-transducing functions and inhibitor sensitivity in chloroplasts.

A comparative analysis of the temperature dependence of energy-transducing reactions in spinach (Spinacia oleracea) chloroplasts and their sensitivity for uncouplers and energy-transfer inhibitors at different temperatures is presented. Arrhenius plots reveal two groups of transitions, around 19 degrees C and around 12 degrees C. Activities that show transitions around 19 degrees C include linear electron flow from water to ferricyanide, its coupled photophosphorylation, the dark-release of the fluorescent probe atebrin, and the slow component of the 515 nm (carotenoid) absorbance decay after a flash. The transitions around 12 degrees C are observed with pyocyanine-mediated cyclic photophosphorylation, light- and dithioerythritol-activated ATP hydrolysis, the dark-release of protons, and the fast 515 nm decay component. It is suggested that both groups of temperature transitions are determined by proton displacements in different domains of the exposed thylakoid membranes. The effects of various uncouplers and an energy-transfer inhibitor are temperature dependent. Some uncouplers also show a different relative inhibition of proton uptake and ATP synthesis at lower temperatures. The efficiency of energy transduction (ATP/e(2)) varied with temperature and was optimal around 10 degrees C.

Journal Article↗

PubMed1986

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase.

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl(2), carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg(2+) at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K(act)(CO(2)) of rubisco activation in the reconstituted system was 4 micromolar CO(2), compared to a K(act)(CO(2)) of 25 to 30 micromolar CO(2) for the previously reported spontaneous CO(2)/Mg(2+) activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO(2) and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.

Journal Article↗

PubMed1995

Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the auto-inducer molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), the synthesis of which is directed by LasI. Previously we have shown that in PAN067, an elastase-negative mutant of PAO1, elastase production can be restored to some extent by addition of exogenous N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Here we report that PAN067 is also defective in the production of alkaline protease, haemolysin, cyanide, pyocyanin and autoinducer(s). As neither addition of exogenous OdDHL nor introduction of lasR restored PAN067 to the parental phenotype, we sought to complement PAN067 with PAO1 DNA. From a cosmid library, a 2 kb DNA fragment was identified which re-established production of autoinducer(s) and exoproducts in PAN067. From the nucleotide sequence of this fragment, two genes termed rhIR and rhII were identified. RhII is responsible for autoinducer synthesis and shares 31% homology with LasI; RhIR has been previously identified in P. aeruginosa strain DSM2659 as a regulator of rhamnolipid biosynthesis and shares 28% identity with LasR. These data provide clear evidence that multiple families of quorum-sensing modulons interactively regulate gene expression in P. aeruginosa.

Amino Acid Sequence↗

PubMed2006

MvfR, a key Pseudomonas aeruginosa pathogenicity LTTR-class regulatory protein, has dual ligands.

MvfR (PqsR), a Pseudomonas aeruginosa LysR-type transcriptional regulator, plays a critical role in the virulence of this pathogen. MvfR modulates the expression of multiple quorum sensing (QS)-regulated virulence factors; and the expression of the phnAB and pqsA-E genes that encode functions mediating 4-hydroxy-2-alkylquinolines (HAQs) signalling compounds biosynthesis, including 3,4-dihydroxy-2heptylquinoline (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ). PQS enhances the in vitro DNA-binding affinity of MvfR to the pqsA-E promoter, to suggest it might function as the in vivo MvfR ligand. Here we identify a novel MvfR ligand, as we show that HHQ binds to the MvfR ligand-binding-domain and potentiates MvfR binding to the pqsA-E promoter leading to transcriptional activation of pqsA-E genes. We show that HHQ is highly produced in vivo, where it is not fully converted into PQS, and demonstrate that it is required for MvfR-dependent gene expression and pathogenicity; PQS is fully dispensable, as pqsH-mutant cells, which produce HHI but completely lack PQS, display normal MvfR-dependent gene expression and virulence. Conversely, PQS is required for full production of pyocyanin. These results uncover a novel biological role for HHQ; and provide novel insights on MvfR activation that may aid in the development of therapies that prevent or treat P. aeruginosa infections in humans.

Animals↗

PubMed1984

Kinetics of reduction by substrate or dithionite and heme-heme electron transfer in the multiheme hydroxylamine oxidoreductase.

Hydroxylamine oxidoreductase of Nitrosomonas catalyzes the dehydrogenation of NH2OH. It contains hemes c553, c559 and P460 in the ratio 5:2:1. At equilibrium four or five c hemes are reduced by NH2OH or NH2NH2, respectively. Heme P460 is the site of electron entry into the enzyme; electrons exit via P460 to O2 or H2O2 with rate constants of 30s-1. We report that hydroxylamine oxidoreductase has two categories of electron-accepting sites: (a) heme P460, an H2O2-sensitive site, which is reactive with NH2OH (2.2 hemes c557 and 2 hemes c559 are reduced) or NH2NH2 (3.3 heme c 553 and 2 heme c559 are reduced) and (b) an H2O2-insensitive site(s) which is reactive with H2O2 (approximately 0.15 heme c553 is reduced); hydroquinone, pyrogallol, N-methyl hydroxylamine, pyocyanine, and ascorbate (approximately 0.8 heme c553 is reduced); or Na2S2O4 or EDTA-photoreduction with proflavin, deazalumiflavin or acridine orange and methylviologen (all hemes are reduced). The rate constants at 19 degrees C for reduction by dithionite were: 0.7 heme c553 (7s-1), 4.3 hemes c553 (0.07 s-1), 0.7 heme c559 (0.8s-1), 1.3 hemes c559 (0.1s-1), P460 (0.013s-1). At 2 degrees C the rate constant for 0.8 heme c559 was 1.7s-1. The data indicate that one heme c552 is reduced by dithionite at the same rate as mammalian cytochrome c; other hemes are reduced much more slowly and are possibly inaccessible to the solvent. The rate constants at 2 degrees C for reduction by NH2OH were: 1.8 hemes c553 (30s-1), 0.2 heme c553 (2.4s-1), 1.7 hemes c559 (19s-1), 0.3 heme c559 (1.4s-1). For reduction by NH2NH2 the values were: 2.6 hemes c553 (23s-1), 0.7 heme c553 (1.6s-1), 1.3 hemes c559 (22s-1), 0.7 heme c559 (4.2s-1). Thus reduction by NH2OH at the substrate site was at least an order of magnitude faster than reduction of hydroxylamine oxidoreductase heme by Na2S2O4. Comparison of rates of heme-heme electron transfer on the enzyme during reoxidation by O2 or H2O2, reduction by Na2S2O4 and reduction by NH2OH or NH2NH2 indicates that the enzyme can exist in distinct states which result in different rates of heme-heme electron transfer. Comparison of the rate of substrate reduction of c hemes of hydroxylamine oxidoreductase (HAO) with the turnover of the enzyme in vivo is consistent with the electron path NH2OH----HAO P460----HAO c hemes----biological electron acceptor.

Anaerobiosis↗

PubMed1986

Regulation of the chloroplast H+-ATPase by light. The involvement of Mg2+ ions.

The involvement of Mg2+ ions in the light-dependent regulation of the chloroplast H+-ATPase was studied in both type C and osmotically shocked type A chloroplasts. The following results were obtained. ATPase activity measured under dark, partially uncoupling conditions, following light activation with dithiothreitol and pyocyanine, was markedly enhanced by the presence of Mg2+ in the activation stage. This Mg2+ effect required concentrations in the millimolar range, was rather slow (time range of minutes), reversible, rather unspecific and did not involve changes in the affinity to dithiothreitol. Dark deactivation of the ATPase in the absence of substrate was accelerated by Mg2+. The dark effect of Mg2+ also required millimolar concentrations, but was fast (time range of seconds), highly specific for Mg2+, and did not involve thiol oxidation. The major effect of the absence of Mg2+ from the light-activation stage or of its presence in the dark interval between activation and assay was the induction of an 'abnormal' sensitivity to uncouplers: after these treatments ATP hydrolysis was not stimulated but rather inhibited by NH4Cl or other uncouplers. The pretreatments in the light without Mg2+ or dark with Mg2+ did not affect the membrane proton permeability, nor the proton pumping coupled to ATPase activity. The results are discussed in terms of Mg2+-dependent regulation of the enzyme complex at the level of subunit interaction and its effect on the affinity to protons.

Adenosine Triphosphate↗

PubMed2001

Isolation and phenotypic characterization of Pseudomonas aeruginosa pseudorevertants containing suppressors of the catabolite repression control-defective crc-10 allele.

The amiE gene encodes an aliphatic amidase capable of converting fluoroacetamide to the toxic compound fluoroacetate and is one of many genes whose expression is subject to catabolite repression control in Pseudomonas aeruginosa. The protein product of the crc gene, Crc, is required for repression of amiE and most other genes subject to catabolite repression control in this bacterium. When grown in a carbon source such as succinate, wild-type P. aeruginosa is insensitive to fluoroacetamide (due to repression of amiE expression). In contrast, mutants harboring the crc-10 null allele cannot grow in the presence of fluoroacetamide (due to lack of repression of amiE). Selection for succinate-dependent, fluoroacetamide-resistant derivatives of the crc-10 mutant yielded three independent pseudorevertants containing suppressors that restored a degree of catabolite repression control. Synthesis of Crc protein was not reestablished in these pseudorevertants. All three suppressors of crc-10 were extragenic, and all three also suppressed a Delta crc::tetA allele. In each of the three pseudorevertants, catabolite repression control of amidase expression was restored. Catabolite repression control of mannitol dehydrogenase production was also restored in two of the three isolates. None of the suppressors restored repression of glucose-6-phosphate dehydrogenase or pyocyanin production.

Amidohydrolases↗

PubMed1992

[Growth inhibitory activity of clinical isolates of Pseudomonas aeruginosa against Staphylococcus aureus (MRSA and MSSA)].

Anti staphylococcal activity by clinical isolates of Pseudomonas aeruginosa was tested by the reversed agar plate and the filter paper stamp methods. Almost 40% of Pseudomonas aeruginosa inhibited the growth of both Methicillin resistant Staphylococcus aureus (MRSA) and Methicillin sensitive Staphylococcus aureus (MSSA). Green pigment (Pyocyanin) produced strains showed a strong inhibitory effect against MRSA and MSSA respectively. But some other pigment (Yellow, Red) strains also showed anti staphylococcal activity. These data suggest the colonization of Pseudomonas aeruginosa with anti staphylococcal activity may not be eradicated by the anti pseudomonic antibiotics.

Antibiosis↗

PubMed2006

Novel Pseudomonas aeruginosa quorum-sensing inhibitors identified in an ultra-high-throughput screen.

The opportunistic pathogen Pseudomonas aeruginosa has two complete acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-LasI and RhlR-RhlI. LasI catalyzes the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12-HSL), and LasR is a transcription factor that requires 3OC12-HSL as a ligand. RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (C4), and RhlR is a transcription factor that responds to C4. LasR and RhlR control the transcription of hundreds of P. aeruginosa genes, many of which are critical virulence determinants, and LasR is required for RhlR function. We developed an ultra-high-throughput cell-based assay to screen a library of approximately 200,000 compounds for inhibitors of LasR-dependent gene expression. Although the library contained a large variety of chemical structures, the two best inhibitors resembled the acyl-homoserine lactone molecule that normally binds to LasR. One compound, a tetrazole with a 12-carbon alkyl tail designated PD12, had a 50% inhibitory concentration (IC50) of 30 nM. The second compound, V-06-018, had an IC50 of 10 microM and is a phenyl ring with a 12-carbon alkyl tail. A microarray analysis showed that both compounds were general inhibitors of quorum sensing, i.e., the expression levels of most LasR-dependent genes were affected. Both compounds also inhibited the production of two quorum-sensing-dependent virulence factors, elastase and pyocyanin. These compounds should be useful for studies of LasR-dependent gene regulation and might serve as scaffolds for the identification of new quorum-sensing modulators.

Anti-Bacterial Agents↗

PubMed2005

Pseudomonas aeruginosa AmpR is a global transcriptional factor that regulates expression of AmpC and PoxB beta-lactamases, proteases, quorum sensing, and other virulence factors.

In members of the family Enterobacteriaceae, ampC, which encodes a beta-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR+ parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal beta-lactamase levels. However, this increase was not followed by a concomitant increase in the P(ampC) promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated beta-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the beta-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive P(ampR) promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the P(lasA), P(lasI), and P(lasR) expression. The reduction in the LasB activity was positively correlated with the P(rhlR) expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

Bacterial Proteins↗

PubMed1983

Isolation of a Pseudomonas sp. Which Utilizes the Phosphonate Herbicide Glyphosate.

A strain of bacteria has been isolated which rapidly and efficiently utilizes the herbicide glyphosate (N-phosphonomethylglycine) as its sole phosphorus source in a synthetic medium. The strain (PG2982) was isolated by subculturing Pseudomonas aeruginosa ATCC 9027 in a synthetic broth medium containing glyphosate as the sole phosphorus source. Strain PG2982 differs from the culture of P. aeruginosa in that it is nonflagellated, does not produce pyocyanin, and has an absolute requirement for thiamine. Strain PG2982 has been tentatively identified as a Pseudomonas sp. strain by its biochemical activities and moles percent guanine plus cytosine. Measurements of glyphosate with an amino acid analyzer show that glyphosate rapidly disappears from the medium during exponential growth of strain PG2982. In batch culture at 30 degrees C, this isolate completely utilized 1.0 mM glyphosate in 96 h and yielded a cell density equal to that obtained with 1.0 mM phosphate as the phosphorus source. However, a longer lag phase and greater generation time were noted in the glyphosate-containing medium. Strain PG2982 can efficiently utilize glyphosate as an alternate phosphorus source.

Journal Article↗

PubMed2004

Biofuel cells select for microbial consortia that self-mediate electron transfer.

Microbial fuel cells hold great promise as a sustainable biotechnological solution to future energy needs. Current efforts to improve the efficiency of such fuel cells are limited by the lack of knowledge about the microbial ecology of these systems. The purposes of this study were (i) to elucidate whether a bacterial community, either suspended or attached to an electrode, can evolve in a microbial fuel cell to bring about higher power output, and (ii) to identify species responsible for the electricity generation. Enrichment by repeated transfer of a bacterial consortium harvested from the anode compartment of a biofuel cell in which glucose was used increased the output from an initial level of 0.6 W m(-2) of electrode surface to a maximal level of 4.31 W m(-2) (664 mV, 30.9 mA) when plain graphite electrodes were used. This result was obtained with an average loading rate of 1 g of glucose liter(-1) day(-1) and corresponded to 81% efficiency for electron transfer from glucose to electricity. Cyclic voltammetry indicated that the enhanced microbial consortium had either membrane-bound or excreted redox components that were not initially detected in the community. Dominant species of the enhanced culture were identified by denaturing gradient gel electrophoresis and culturing. The community consisted mainly of facultative anaerobic bacteria, such as Alcaligenes faecalis and Enterococcus gallinarum, which are capable of hydrogen production. Pseudomonas aeruginosa and other Pseudomonas species were also isolated. For several isolates, electrochemical activity was mainly due to excreted redox mediators, and one of these mediators, pyocyanin produced by P. aeruginosa, could be characterized. Overall, the enrichment procedure, irrespective of whether only attached or suspended bacteria were examined, selected for organisms capable of mediating the electron transfer either by direct bacterial transfer or by excretion of redox components.

Animals↗

PubMed1972

Evaluation of Pseudosel agar as an aid in the identification of Pseudomonas aeruginosa.

Growth of Pseudomonas aeruginosa and thirty-five other species of gramnegative bacilli was observed on 0.03% cetrimide in heart infusion agar medium and Pseudosel agar (BBL). The 0.03% cetrimide agar was more selective for growth of P. aeruginosa than was Pseudosel agar; however, certain bacteria other than P. aeruginosa also grew on the former medium. Although Pseudosel agar was not a highly selective medium for P. aeruginosa, it was preferable to technicolor agar for detection of the pyocyanin and pyorubin pigments produced by P. aeruginosa.

Agar↗

PubMed1973

Incidence and identification of Pseudomonas fluorescens and Pseudomonas putida in the clinical laboratory.

Strains of Pseudomonas producing fluorescin but no pyocyanin or pyorubrin were studied by biochemical and antibiotic sensitivity testing. A rapid nitrate test was found to be useful in distinguishing P. aeruginosa (positive) from P. fluorescens and P. putida (both negative). A shortened gelatin test differentiated P. fluorescens (positive) from P. putida (negative). P. fluorescens and P. putida were very sensitive to low levels of kanamycin and resistant to carbenicillin, a pattern just the opposite of that obtained with P. aeruginosa.

Anti-Bacterial Agents↗

PubMed1986

Effect of ciliostatic factors from Pseudomonas aeruginosa on rabbit respiratory cilia.

Heat-stable factors released by Pseudomonas aeruginosa in culture supernatants inhibit functional cilia of rabbit tracheal epithelium. Chloroform extraction removed heat-stable factors from stationary-phase culture supernatants. The extracts contained at least seven components separable by thin-layer chromatography (TLC). Cilioinhibitory components were identified as a phenazine derivative, pyo compounds (2-alkyl-4-hydroxyquinolines), and a rhamnolipid, also known as a hemolysin. Fluorescence and absorption spectra, relative migration on TLC, staining characteristics, and gas chromatography were the basis for identification. Inhibitory concentrations of each active component were established by quantitative measures of percent motility and beat frequency. Corresponding damage to ciliary ultrastructure was examined by electron microscopy. The pyo compounds produced ciliostasis at concentrations of 50 micrograms/ml, but without obvious ultrastructural lesions. The phenazine derivative also inhibited ciliary motility and caused some membrane disruption, although at substantially greater concentrations of 400 micrograms/ml. Limited exposure of tracheal explants to the rhamnolipid resulted in ciliostasis which was associated with altered ciliary membranes. More extensive exposure to rhamnolipid was associated with removal of dynein arms from axonemes. Pyocyanin at a concentration of 0.5 mg/ml did not inhibit ciliary beating under our conditions. The data suggest that the pyo compounds are the most effective per weight ciliostatic factors released by P. aeruginosa and rhamnolipid is the most destructive of cilia ultrastructure. By interfering with normal ciliary function, these ciliostatic factors may enable P. aeruginosa to more easily colonize the respiratory tract.

Animals↗

PubMed1988

Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium.

The phenazine pigments pyocyanin and 1-hydroxyphenazine were resolved by high-pressure liquid chromatography from the sputum sol phase from 9 of 13 patients with cystic fibrosis or bronchiectasis colonized by Pseudomonas aeruginosa. The concentrations measured were each sufficient to inhibit ciliary beating in vitro and contributed a significant proportion of sol phase toxicity for respiratory epithelium.

Cilia↗

PubMed1991

Production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human neutrophils is inhibited by Pseudomonas aeruginosa phenazine derivatives.

Pyocyanin, a phenazine pigment produced by Pseudomonas aeruginosa, and its metabolite 1-hydroxyphenazine inhibited leukotriene B4 and 5-hydroxyeicosatetraenoic acid production by up to 70% in human neutrophils stimulated with the calcium ionophore A23187 (5 microM). This potential anti-inflammatory effect was dose dependent and occurred at low concentrations (10 to 50 microM) that did not inhibit neutrophil viability.

Calcimycin↗