Recognition of intestinal epithelial HIF-1alpha activation by Pseudomonas aeruginosa

Human intestinal epithelial cell monolayers (Caco-2) subjected to hypoxia and reoxygenation release soluble factors into the apical medium that activate the virulence of the opportunistic pathogen Pseudomonas aeruginosa to express the potent barrier-dysregulating protein PA-I lectin/adhesin. In this study, we defined the role of hypoxia-inducible factor (HIF)-1alpha in this response. We tested the ability of medium from Caco-2 cells with forced expression of HIF-1alpha to increase PA-I expression in P. aeruginosa and found that medium from Caco-2 cells overexpressing HIF-1alpha increased PA-I expression compared with medium from control cells (P < 0.001, ANOVA). To identify the components responsible for this response, medium was fractionated by molecular weight and subjected to mass spectroscopy, which identified adenosine as the possible mediator. Both adenosine and its immediate downstream metabolite inosine induced PA-I expression in P. aeruginosa in a dose-dependent fashion. Because inosine was not detectable in the medium of Caco-2 cells exposed to hypoxia or overexpressing HIF-1alpha, we hypothesized that P. aeruginosa itself might metabolize adenosine to inosine. Using mutant and parental strains of P. aeruginosa, we demonstrated that P. aeruginosa metabolized adenosine to inosine via adenosine deaminase and that the conditioned medium enhanced the extracellular accumulation of inosine. Together, these results provide evidence that P. aeruginosa can recognize and respond to extracellular end products of intestinal hypoxia that are released after activation of HIF-1alpha. The ability of P. aeruginosa to metabolize adenosine to inosine may represent a subversive microbial virulence strategy that deprives the epithelium of the cytoprotective actions of adenosine.     

Lipids of Pseudomonas aeruginosa Cells Grown on Hydrocarbons and on Trypticase Soy Broth

Lipids were extracted from cells of Pseudomonas aeruginosa grown on a pure hydrocarbon (tridecane), mixed hydrocarbons (JP-4 jet fuel), and on Trypticase Soy Broth. Total lipids produced from each substrate represented from 7.1 to 8.2% of cellular dry weight, of which 5.0 to 6.4% were obtained before cellular hydrolysis (free lipids) and 1.7 to 2.0% were extracted after cellular hydrolysis (bound lipids). Free lipids from cells grown on each medium were separated into four fractions by thin-layer chromatography. All fractions were present in cells from each type of medium, and the "neutral fraction" constituted the largest fraction. The fatty acid composition of free lipids was determined by gas-liquid chromatography. Cells grown on each medium contained saturated and unsaturated C(14) to C(20) fatty acids. Trace amounts of C(13) fatty acids were found in tridecane-grown cells. Saturated C(16) and C(18) were the major acids present in all cells. Quantitative differences were found in fatty acids produced on the three media, but specific correlations between substrate carbon sources and fatty acid content of cells were not evident. Tridecane-grown cells contained only traces of C(13) acid and small amounts of C(15) and C(17) acids, suggesting that the organism's fatty acids were derived from de novo synthesis rather than by direct incorporation of the hydrocarbon.     

Ultrastructure of two oil-degrading bacteria isolated from the tropical soil environment

Two oil-degrading bacteria identified as Pseudomonas aeruginosa and Micrococcus luteus were isolated from crude-oil-polluted soils in Nigeria. The organisms were grown on n-hexadecane and sodium succinate and then examined for the presence of hydrocarbon inclusions. Inclusion bodies were found in n-hexadecane-grown cells and were absent in succinate-grown cells. Formation of hydrocarbon inclusion bodies appears to be a general phenomenon among hydrocarbon utilizers.     

The enhancement of conjugal plasmid pBHR1 transfer between bacteria in the presence of extracellular metabolic products produced by Microcystis aeruginosa

Conjugal plasmid transfer from Escherichia coli S17-1 (pBHR1) to Pseudomonas stutzeri was investigated in the presence of a cyanophyta Microcystis aeruginosa. The plasmid transfer frequency increased with higher densities of M. aeruginosa. The extracellular metabolic products (EMPs) from M. aeruginosa were found to enhance the plasmid transfer between bacteria. Furthermore, the plasmid transfer frequency in medium containing EMPs was significantly higher than that in culture medium with or without glucose. These results suggest that M. aeruginosa enhances conjugal plasmid transfer between bacteria through its EMPs, and that identity of the carbon source is an important factor affecting conjugal plasmid transfer in aquatic environments.     

Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon alpha,omega-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

铜绿微囊藻对萼花臂尾轮虫与大型溞竞争关系的影响

为探究铜绿微囊藻(Microcystis aeruginosa)浓度变化对浮游动物竞争关系的影响,通过控制实验法,评估了在3个铜绿微囊藻浓度梯度下,萼花臂尾轮虫(Brachionus calyciflorus)和大型溞(Daphnia magna)之间的种间竞争关系。结果表明不同浓度铜绿微囊藻对萼花臂尾轮虫、大型溞的增长及二者种间竞争影响具有差异,并且在3种铜绿微囊藻浓度下均以大型溞为主要优势类群。低浓度(5×10⁴ cells/m L)铜绿微囊藻仅促进大型溞种群增长(P<0.01),大型溞占据主要优势地位;中浓度(1×10⁵ cells/m L)铜绿微囊藻对萼花臂尾轮虫和大型溞增长均有显著影响(P<0.01),在此浓度下大型溞在种群竞争中依旧占优势地位,使得萼花臂尾轮虫种群衰亡;在高浓度铜绿微囊藻(5×10⁵ cells/m L)环境中种群生长均受到抑制(P<0.01),在共培养体系中仅大型溞种群存活。在无其他外在影响因素存在时,实验结果显示在不同浓度的铜绿微囊藻下,大型溞均占优势,说明铜绿微囊藻的浓...     

Adaptation of Pseudomonas aeruginosa ATCC 15442 to didecyldimethylammonium bromide induces changes in membrane fatty acid composition and in resistance of cells

The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of didecyldimethyl ammonium bromide (DDAB) was investigated. In this study, the strain was sub-cultured in a medium with increasing DDAB concentrations. After adaptation, Ps. aeruginosa was able to grow until the DDAB concentration in the medium was about five times greater than the Minimal Inhibitory Concentration. Resistance of cells to the bactericidal activity of DDAB also increased gradually during adaptation. This resistance was dependent on the presence of the biocide, as it quickly decreased when the cells were transferred to medium without biocide. Adapted cells showed changes in membrane fatty acid composition. The modifications mainly affected lauric, beta-hydroxylauric and palmitic acids, and they underlined the implication of the membranes in the cell response to the presence of the biocide. Simple linear regression analysis showed that the membrane fatty acid composition of Ps. aeruginosa played an important part in the resistance mechanisms of cells to the bactericidal activity of DDAB.     

Swarming of Pseudomonas aeruginosa PAO1 without differentiation into elongated hyperflagellates on hard agar minimal medium

Polar flagellated Pseudomonas aeruginosa PAO1 demonstrated extensive spreading growth in 2 days on 1.5% agar medium. Such spreading growth of P. aeruginosa PAO1 strains was absent on Luria-Bertani 1.5% agar medium, but remarkable on Davis minimal synthetic agar medium (especially that containing 0.8% sodium citrate and 1.5% Eiken agar) under aerobic 37 degrees C conditions. Analyses using isogenic mutants and complementation transformants showed that bacterial flagella and rhamnolipid contributed to the surface-spreading behavior. On the other hand, a type IV pilus-deficient pilA mutant did not lose the spreading growth activity. Flagella staining of PAO1 T cells from the frontal edge of a spreading colony showed unipolar and normal-sized rods with one or two flagella. Thus, the polar flagellate P. aeruginosa PAO1 T appears to swarm on high-agar medium by producing biosurfactant rhamnolipid and without differentiation into an elongated peritrichous hyperflagellate.     

Comparative study of membrane filtration and enrichment media for the isolation and enumeration of Pseudomonas aeruginosa from sewage, surface water, and swimming pools

The recovery of Pseudomonas aeruginosa on several selective culture media was tested using raw sewage and secondary sewage effluent samples as well as spiked chlorinated imitation swimming water and samples from whirlpools. mPA-medium B gave good recovery of both vital and chlorine-injured P. aeruginosa and selectivity was greater than 90% when analysing whirlpool samples. It is therefore the medium recommended for examination of chlorinated swimming pools. When analysing sewage polluted water with the mPA-B medium, reduced selectivity was noted from low verification rates and from overgrowth by competitive flora. A modified medium (mPA-D; addition of cetrimide, omission of sulphapyridine and actidione) was more selective and sufficiently recovered noninjured cells. Chlorine-injured cells were completely inhibited, however. C-390 (9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan) was confirmed to be highly selective for P. aeruginosa when used in spread plates at a concentration of 30 micrograms/mL; P. aeruginosa was slightly inhibited. However, the medium could not be used with conventional membrane filtration techniques, because cellulose ester filters interfered with the selective action of C-390. Selectivity could be improved by using Gelman Tuffryn (polysulphone) filters and increasing the C-390 concentration to 120 micrograms/mL. At this concentration, however, the medium was strongly inhibitory to P. aeruginosa; resuscitation only partially improved recovery. Two other membrane filtration media were tested. Both cetrimide - nalidixic acid agar and Drake's medium No. 19 were inhibitory to chlorine-injured cells. Several types of membrane filters were tested and there was little difference between them.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenazine-1-carboxylic acid,a secondary metabolite of Pseudomonas aeruginosa,alters expression of immunomodulatory proteins by human airway epithelial cells

Pseudomonas aeruginosa is a gram-negative bacterium that causes both acute and chronic lung disease in susceptible patient populations. P. aeruginosa secretes numerous proteins and secondary metabolites, many of which have biological effects that likely contribute to disease pathogenesis. An unidentified small-molecular-weight factor was previously reported to increase IL-8 release both in vitro and in vivo. To identify this factor, we subjected the <3-kDa fraction from P. aeruginosa-conditioned medium to HPLC analysis. A peak fraction that stimulated IL-8 release was found by mass spectrometry to have a molecular mass (MM) of 224 Da. On the basis of this MM and other biochemical properties, we hypothesized that the factor was phenazine-1-carboxylic acid (PCA). Subsequent studies and comparison with purified PCA confirmed this hypothesis. Purified PCA exhibited a number of biological effects in human airway epithelial cells, including increasing IL-8 release and ICAM-1 expression, as well as decreasing RANTES and monocyte chemoattractant protein-1 (MCP-1) release. PCA also increased intracellular oxidant formation as measured by electron paramagnetic resonance and by an intracellular oxidant-sensitive probe. Antioxidants inhibited PCA-dependent increases in IL-8 and ICAM-1, suggesting that oxidants contributed to these effects. However, in contrast to the related phenazine compound pyocyanin, PCA did not oxidize NAD(P)H at physiologically relevant pH, providing preliminary evidence that PCA and pyocyanin may have distinct redox chemistries within the cell. Thus PCA is a biologically active factor secreted by P. aeruginosa that has several activities that could alter the host immune and inflammatory response and thereby contribute to bacterial disease pathogenesis.     

Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa

A gamma ray-induced mutant of Pseudomonas aeruginosa strain S8, capable of hyperproduction of biosurfactant from hydrocarbons, was isolated and named as EBN-8. The mutant showed 3–4 times more hydrocarbon emulsification/conversion as compared to the parent when grown on Khaskheli crude oil in minimal medium. Enhanced biosurfactant production and hydrocarbon utilization by the mutant was also observed during growth on heptadecane in minimal medium as indicated by emulsion index and surface tension of cell-free culture broth. Using heptadecane as carbon and energy source, time course for the growth (cfu ml^-1) and biosurfactant production were compared for both parent and mutant. These studies were carried out for 24 d at 30 ± 2°C and for 20 d at 37°C. Growth of EBN-8 was much faster compared to the parent as well as being 2–3 times more hyperproductive.     

The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.     

The metal dependence of pyoverdine interactions with its outer membrane receptor FpvA

To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 microM AlCl(3) to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.     

Chemical composition and role of Pseudomonas aeruginosa peptidoglycolipid in hydrocarbon assimilation

Pseudomonas aeruginosa P-20 releases a lipophilic compound during growth in a medium with hexadecane. The compound was shown to be a peptidoglycolipid. The peptide moiety consists of 7 amino acids: lysine, aspartic and glutamic acids, serine, proline, valine and leucine. The carbohydrate component is ramnose. The lipid moiety is represented by a mixture of fatty acids with the number of carbon atoms from 11 to 18 among which C11:1, C16:0, C18:1 and C17:3 predominate. The content of unsaturated acids is 64.62%. The peptidoglycolipid stimulates the process of hydrocarbon assimilation.     

Effect of Pseudomonas aeruginosa melanin on antibiotic activity]

The properties of microbial melanines are very diverse. Melanine of P. aeruginosa is little studied. The pigment was isolated from a strain of P. aeruginosa possessing all characteristic properties of the species. Interaction of P. aeruginosa melanine with various antibiotics was determined by the method of serial dilutions in beaf-peptone broth, using Staph. aureus 209 as a test-microbe, which was added to the medium in an amount of 10(6) cells to each tube. It was found that P. aeruginosa melanine differed from DOPA-melanine in a concentration of 1 mg/ml and did not change the activity of penicillin, tetracycline, oleandomycin, kanamycin and gentamicin with respect to Staph. aureus.     

Effects of Cultural Conditions on Mononucleotide Phosphorylating Activity of Yeast Cells

It was found that the AMP phosphorylating activity of Candida sp. N–25–2 (a hydrocarbon assimilating yeast) was affected extremely by the liquid volume of cultural medium and the concentration of inorganic salts in medium. The yeast cells having no fermentative activity showed a strong activity of AMP phosphorylation when they were cultured under relative anaerobic conditions. It was observed that the glucose consumption of yeast cells was promoted by the addition of Mg²⁺ ion and AMP into the reaction system, and that the AMP phosphorylation was promoted in the presence of F-1,6-DP or phosphaenolpyruvate.

The cells of Candida sp. N–25–2 grown on glucose medium had a remarkable fermentative activity, while the cells grown on acetate or ethanol medium had a weak activity. On the other hand, it was found that the cells grown at strong aeration on glucose medium were able to produce remarkably the phosphorylated substances from mononucleotides, when F-1,6-DP was added as a phosphate donor. Similar phenomenon was observed in case of the cells grown on the carbon sources such as acetate, ethanol and hydrocarbon.     

Enterobactin-mediated iron transport in Pseudomonas aeruginosa.

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin. Apparently, at least two uptake systems for ferrienterobactin exist in P. aeruginosa: one of higher affinity which is specifically inducible by enterobactin under iron-limiting conditions and the second, of lower affinity, which is also inducible under iron-limiting conditions but is independent of enterobactin for induction.