Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA

A solvent tolerant Pseudomonas aeruginosa PseA strain was isolated from soil. It secreted a novel alkaline protease, which was stable and active in the presence of range of organic solvents, thus potentially useful for catalysis in non-aqueous media. The protease was purified 11.6-fold with 60% recovery by combination of ion exchange and hydrophobic interaction chromatography using Q-Sepharose and Phenyl Sepharose 6 Fast Flow matrix, respectively. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 35,000 Da. The enzyme was stable in the pH range of 6.0-9.0, the optimum being 8.0. The Km and Vmax towards caseinolytic activity were found to be 2.7 mg/ml and 3 micromol/min, respectively. The protease was most active at 60 degrees C and characterized as a metalloprotease because of its sensitivity to EDTA and 1,10-phenanthroline. It was tested positive for elastase activity towards elastin-orcein, thus appears to be an elastase, which is known as pseudolysin in other strains of P. aeruginosa. The protease withstands range of detergents, surfactants and solvents. It is stable and active in all the solvents having log P above 3.2, at least up to 72 h. These two properties make it an ideal choice for applications in detergent formulations and enzymatic peptide synthesis.     

Photochemical inactivation of Pseudomonas aeruginosa

Adaptability to a broad range of environments together with relatively high resistance to antibiotics and to disinfectants makes Pseudomonas aeruginosa a concern in hospitals and in public health. We investigated whether UVA-mediated photochemical inactivation of P. aeruginosa could be accomplished with high efficiency while at the same time preserving the sensitivity of subsequent diagnostic tests. We characterized dose responses and bactericidal kinetic rates of 5-iodonaphthyl 1-azide (INA) and of amotosalen (AMO) as these substances exposed to UVA are known to inactivate germs with minimal impact to blood products or to viral antigens. Neither UVA without photochemicals nor INA or AMO in the dark inactivated bacteria. We found that AMO was ca 1000-fold more effective in inactivating P. aeruginosa cells than INA under similar conditions. Photoinactivation with either INA or AMO at conditions that abolished bacterial infectivity did not impair polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) testing. For comparison, similar titers of Bacillus atrophaeus spores (a surrogate for B. anthracis) remained unaffected at conditions that reduced the survival of P. aeruginosa below detection levels. The results presented in this study should assist in improved methods to inactivate P. aeruginosa in environmental, clinical and forensic samples without impairing subsequent nucleic acid- or immune-based analysis.     

Structure of aerugine from Pseudomonas aeruginosa

A new optically active phenolic alkaloid with the composition C₁₀H₁₁NO₂S, mp 85–88°C, [α]_D +28° (c 1.0; chloroform) (I) has been isolated from the microorganismPseudomonas aeruginosa (strain 590) and has been called aerugine. The action of diazomethane gave an O-methyl derivative (II). On the basis of the formation of ortho-cresol by the hydrogenolytic desulfuration reaction, a study of the IR, mass, and PMR spectra and (I) and its acetyl derivative (III), and also the¹³C NMR spectrum of (I), the structure of 4-hydroxymethyl-2-(o-hydroxyphenyl)-2-thiazoline has been established for aerugine. The spectral characteristics of the compounds mentioned are given.     

A complement-sensitive mutant of Pseudomonas aeruginosa

The role of complement in the bactericidal activity of human serum against a mutant strain of Pseudomonas aeruginosa used as a model was demonstrated. The involvement of complement in the bacterial destruction of P. aeruginosa, and the contribution of the alternative and classical pathways of the complement system were directly evidenced by using sera from complement-deficient patients.     

Structure of aerugine from Pseudomonas aeruginosa

A new optically active phenolic alkaloid with the composition C₁₀H₁₁NO₂S, mp 85–88°C, []_D +28° (c 1.0; chloroform) (I) has been isolated from the microorganismPseudomonas aeruginosa (strain 590) and has been called aerugine. The action of diazomethane gave an O-methyl derivative (II). On the basis of the formation of ortho-cresol by the hydrogenolytic desulfuration reaction, a study of the IR, mass, and PMR spectra and (I) and its acetyl derivative (III), and also the¹³C NMR spectrum of (I), the structure of 4-hydroxymethyl-2-(o-hydroxyphenyl)-2-thiazoline has been established for aerugine. The spectral characteristics of the compounds mentioned are given.Institute of the Chemistry of Plant Substances of the Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 553–558, July–August, 1987.     

Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa

Pseudomonas aeruginosa were used for extra-cellular biosynthesis of gold nanoparticles (Au NPs). Consequently, Au NPs were formed due to reduction of gold ion by bacterial cell supernatant of P. aeruginosa ATCC 90271, P. aeruginosa (2) and P. aeruginosa (1). The UV-vis and fluorescence spectra of the bacterial as well as chemical prepared Au NPs were recorded. Transmission electron microscopy (TEM) micrograph showed the formation of well-dispersed gold nanoparticles in the range of 15-30 nm. The process of reduction being extra-cellular and may lead to the development of an easy bioprocess for synthesis of Au NPs.     

Directed evolution and axial chirality: optimization of the enantioselectivity of Pseudomonas aeruginosa lipase towards the kinetic resolution of a racemic allene

Directed evolution of Pseudomonas aeruginosa lipase by the use of combinatorial active site saturation test (CAST) criteria provided a highly enantioselective mutant (Leu162Phe) for kinetic resolution of an axially chiral allene, p-nitrophenyl 4-cyclohexyl-2-methylbuta-2,3-dienoate (E=111); the high enantioselectivity of the Leu162Phe mutant was rationalized by pi-pi stacking.     

Asymmetrization of meso-1,3-diols utilising Pseudomonas fluorescens lipase

A convenient and enantioselective synthesis of monoacetates of meso-1,3-diols 2-substituted with an alkoxymethyl or a thiophenylmethyl group, by enzyme catalyzed acylation, is described. The absolute stereochemistries of two monoacetates were assigned by chemical correlation.     

Neutron diffraction study of Pseudomonas aeruginosa lipopolysaccharide bilayers

Lipopolysaccharides (LPSs) are a major class of macromolecules populating the surface of Gram-negative bacteria. They contribute significantly to the bacterium's surface properties and play a crucial role in regulating the permeability of its outer membrane. Here, we report on neutron diffraction studies performed on aligned, self-assembled bilayers of LPS isolated from Pseudomonas aeruginosa PAO1. This LPS system is comprised of a mixture of rough and smooth A-band and B-band LPS, similar to that naturally found in P. aeruginosa. Temperature scans were conducted at various levels of hydration, and the phases adopted by LPS, along with their corresponding transition temperatures, have been identified. Because of LPS's chemical heterogeneity, the gel-to-liquid-crystalline transition was continuous and not abrupt as commonly observed in single-component phospholipid systems. From the construction of one-dimensional scattering length density profiles, we find that water penetrates into the hydrocarbon region up to and including the center of liquid-crystalline LPS bilayers. This permeability to water also extends to bilayers in the continuous phase transition region and could have far-reaching implications as to how small molecules penetrate the outer membrane of Gram-negative bacteria.     

Electron transfer reactivity of type zero Pseudomonas aeruginosa azurin

Type zero copper is a hard-ligand analogue of the classical type 1 or blue site in copper proteins that function as electron transfer (ET) agents in photosynthesis and other biological processes. The EPR spectroscopic features of type zero Cu(II) are very similar to those of blue copper, although lacking the deep blue color, due to the absence of thiolate ligation. We have measured the rates of intramolecular ET from the pulse radiolytically generated C3-C26 disulfide radical anion to the Cu(II) in both type zero C112D/M121L and type 2 C112D Pseudomonas aeruginosa azurins in pH 7.0 aqueous solutions between 8 and 45 °C. We also have obtained rate/temperature (10-30 °C) profiles for ET reactions between these mutants and the wild-type azurin. Analysis of the rates and activation parameters for both intramolecular and intermolecular ET reactions indicates that the type zero copper reorganization energy falls in a range (0.9-1.1 eV) slightly above that for type 1 (0.7-0.8 eV), but substantially smaller than that for type 2 (>2 eV), consistent with XAS and EXAFS data that reveal minimal type zero site reorientation during redox cycling.     

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.     

Computer simulations of enantioselective ester hydrolyses catalyzed by Pseudomonas cepacia lipase

On the basis of the X-ray crystal structure of the lipase from Pseudomonas cepacia (PcL)-an enzyme representative for a whole family of Pseudomonas lipases (lipase PS, SAM-2, AK 10, and others with a high degree of homology with PcL)-a computational study was performed to rationalize both the enantioselectivity and substrate specificity (tolerance) displayed by this lipase in the enantioselective hydrolysis of racemic esters 1a-12a from various secondary aromatic alcohols. The major goal of this project was the development of a binding model for PcL which is able to rationalize the experimental findings to predict "a priori the enantioselective behavior of PcL toward a wider range of substrates. A two-step modeling procedure, namely, docking experiments followed by construction of tetrahedral intermediates, was used for the simulation of the involved enzyme-substrate recognition/hydrolysis processes. The study of the recognition process (docking experiments) led to unambiguous identification of the binding geometry for the two enantiomeric series of substrates, but did not suggest a definitive interpretation of the behavior of PcL. Taking into consideration the stereoelectronic requirements of the enzymatic hydrolysis reaction, both the enantioselectivity and tolerance of the enzyme were then explained through the study of the tetrahedral intermediates, in turn constructed from the calculated docking geometries of 1a-12a.     

Biosurfactants production by Pseudomonas aeruginosa FR using palm oil

Biosurfactants production by a strain of Pseudomonas aeruginosa using palm oil as a sole carbon source was investigated. The experiments were carried out in 500-mL conical flasks containing 100 mL of mineral media supplemented with palm oil as the sole carbon source. The P. aeruginosa FR strain was able to reduce surface tension of three tested inorganic media. Rotation velocities from 100 to 150 rpm provided free-cell fermented media with the lowest surface tension of approx 33 mN/m. Emulsification index results of even 100% were achieved when diesel was used as oil phase. Eight surface-active compounds produced by the bacterium were identified by mass spectrometry.     

Pulsed-field gel electrophoresis analysis of a Pseudomonas aeruginosa pathovar.

This paper presents the genome organization and mobility of Pseudomonas aeruginosa strains that had been isolated in half-year intervals from 30 patients with cystic fibrosis since the onset of colonization over a 2- to 8-year period. The chromosomes were digested with DraI or SpeI, separated by pulsed-field gel electrophoresis, blotted and hybridized with probes encoding housekeeping or virulence genes. Strains were differentiated by relatedness of macrorestriction fingerprints. After some turnover of strains during the first two years of colonization, each patient had acquired a set of strains that diversified during the course of the disease. In the majority of patients, two clonal lineages were found to account for colonization in the air passages but each lung habitat was characterized by some specific signature of bands in the macrorestriction fragment pattern.     

The Detection of Pseudomonas aeruginosa Using the Quartz Crystal Microbalance

Abstract

The development of a piezoelectric immunosensor for the detection of Pseudomonas aeruginosa in milk and dairy samples was undertaken here. This was achieved primarily by optimising the system using ELISA, investigating capture, competitive and displacement assays. Results from ELISA supplied information on detection limits and linear ranges obtained with each assay. A displacement assay was chosen to be transferred to the piezoelectric system and the reduction in mass on the surface of the crystal due to antigen displacement was measured by recording the frequency changes of the quartz crystal microbalance. The linear range obtained was from 2x10⁶ cell/ml to 1x10⁸ cell/ml and the limit of detection was 100,000 cells. The system was also tested for cross reactivity with a non-specific antigen, Pseudomonas fluorescens.     

Fast exopolysaccharide secretion of Pseudomonas aeruginosa on polar polymer surfaces

The influence of the surface chemical structure and related physicochemical properties on the adhesion of P. aeruginosa has been studied for moderately hydrophobic polymers and for hydrophilic surfaces obtained by O2-plasma treatments and 50 keV Ar+ beam irradiation of poly(hydroxymethylsiloxane) and poly(ethyleneterephthalate). The surface chemical structure has been obtained by X-ray photoelectron spectroscopy, the roughness was measured by atomic force microscopy, and the surface free energy was evaluated from contact angle measurements for all the polymer substrates before and after the irradiation treatments. It is shown that a massive and unusually fast secretion of exopolysaccharides onto highly polar surfaces, corresponding to the formation of complex three-dimensional multilayers (i.e., biofilm-like structures), occurs already after 2 h of incubation. It is suggested that such highly polar surfaces can operate either by promoting, by means of a still unknown biomolecular mechanism, an early gene expression process or by mimicking the P. aeruginosa cellular walls.