Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography–mass spectrophotometry (MS) and by high-performance liquid chromatography–MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l⁻¹. Under these conditions, the surface tension of the culture decreased to 30 mN m⁻¹, and the critical micelle concentration was 26.5 mg l⁻¹. It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.     

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.     

低温氧等离子体杀灭铜绿假单胞菌的效果及机理

【目的】本文采用低温氧等离子体,在自行设计的远程等离子体反应装置中,对位于不同放电区域（放电区,余辉区,远程区）的模拟染菌载体聚对苯二甲酸乙二醇酯（PET）表面的铜绿假单胞菌(Pseudomonas aeruginosa)的杀灭效果和机理进行了研究。【方法】采用扫描电子显微镜观察了等离子体处理前后细菌细胞的形貌变化,采用考马斯亮蓝法测定了等离子体处理后细菌蛋白质泄漏量,采用双悬浮探针对氧等子体的电子温度和离子浓度以及电子自旋共振波谱对自由基浓度进行了测定。【结果】强放电区、余辉区和远程区处理30s后的灭菌效果分别为4.2 ,3.8和2.6;扫描电镜观察结果和蛋白泄漏量测定结果证明细菌细胞被损毁,在强放电区是电子、离子、自由基和紫外光子的协同作用,而在余辉区和远程区的灭杀作用主要因自由基所为。【结论】证明该反应装置可有效实现活性粒子的分离,在远程等离子体场中揭示了等离子体灭菌的规律和机理。     

GacA对假单胞菌株M18两个phz基因簇和菌群传感的调控

摘要:【目的】假单胞菌株M18（Pseudomonas sp. M18）是从甜瓜根际土壤中分离获得的一株对多种植物病原菌具有显著拮抗作用的菌株,在菌群传感（quorum sensing）系统的调控下,能分泌吩嗪-1-羧酸（PCA）以及多种吩嗪（phz）类衍生物的抗真菌物质。全局性因子GacA是M18菌株吩嗪类物质的合成与菌群传感系统的重要调控因子,本文将就GacA对上述两者的调控做进一步研究。【方法】PCR基因扩增和测序研究M18菌株中PCA合成基因簇,运用RT-PCR及构建phzA-lacZ转录融合手段     

Characterization of zinc-binding properties of a novel imidase from Pseudomonas putida YZ-26

The imidase from Pseudomonas putida YZ-26 consisting of 293-amino acid residues is a novel imidase with four subunits as the holo-enzyme and low molecular weight which is significantly different from known mammalian imidase. This study measured the zinc-binding properties of the imidase using inductively coupled plasma-atomic emission spectrometry and competition assay combined with activity determinations. Results show that each subunit of the imidase binds the zinc ion by 1:1 stoichiometry with apparent binding constant of 9.5 × 10⁸ M⁻¹. The activity of the apo-imidase (20 μM) was recovered with the addition of zinc in the lower concentration (0-20 μM), whereas the enzymatic activity is decreased in the presence of high concentration of zinc (above 100 μM). The site-directed mutagenesis of His₂₄₇, His₈₆ or Cys₇, Cys₁₀₈ in imidase resulted in loss of activity and zinc-binding abilities at different degrees, showing that these residues may critically affect both enzymatic activity and conformation.     

Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonasaeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21 nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21 nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.     

Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis

Biofilms are known to be robust biocatalysts. Conventionally, they have been mainly applied for wastewater treatment, however recent reports about their employment for chemical synthesis are increasingly attracting attention. Engineered Pseudomonas sp. strain VLB120ΔC biofilm growing in a tubular membrane reactor was utilized for the continuous production of (S)‐styrene oxide. A biofilm specific morphotype appeared in the effluent during cultivation, accounting for 60–80% of the total biofilm irrespective of inoculation conditions but with similar specific activities as the original morphotype. Mass transfer of the substrate styrene and the product styrene oxide was found to be dependent on the flow rate but was not limiting the epoxidation rate. Oxygen was identified as one of the main parameters influencing the biotransformation rate. Productivity was linearly dependent on the specific membrane area and on the tube wall thickness. On average volumetric productivities of 24 g L day^?1 with a maximum of 70 g L day^?1 and biomass concentrations of 45 g_BDW L have been achieved over long continuous process periods (≥50 days) without reactor downtimes. Biotechnol. Bioeng. 2010. 105: 705–717. © 2009 Wiley Periodicals, Inc.     

Monovalent and bivalent N-fucosyl amides as high affinity ligands for Pseudomonas aeruginosa PA-IIL lectin

The adhesion of bacteria to human glycoconjugates can be inhibited by soluble glycomimetics that compete with the natural target. Four monovalent and one divalent α-fucosyl amides have been tested for their affinity for a fucose-binding lectin from Pseudomonas aeruginosa. Isothermal calorimetric titrations demonstrated that they bind to the lectin in the micromolar range, with highest affinity for the divalent ligand. Molecular modelling established that, compared to Ο-fucoside compounds, the glycomimetic amide group resulted in the loss of water-bridged hydrogen bonds that could be partially compensated by additional contact of the aglycone with the protein surface.