以恶臭假单胞菌发酵制备D-氨基葡萄糖酸

从土样中分离到D-氨基葡萄糖酸高产菌株GNA5,综合Biolog细菌自动鉴定系统与16S rDNA序列分析结果,鉴定为恶臭假单胞菌(Pseudomonas putida). P. putida GNA5能耐受较高浓度底物D-氨基葡萄糖,可发酵积累D-氨基葡萄糖酸. 初步优化培养基为(g/L)：D-氨基葡萄糖盐酸盐30,葡萄糖5,尿素5, KH2PO4 2, MgSO4×7H2O 0.5, CaCO3 10, pH 7.0. 菌株GNA5在该培养基中发酵48 h可制备25.5 g/L的D-氨基葡萄糖酸,摩尔转化率达93.91%. 自行筛选的P. putida GNA5显示了底物转化能力强及产物分解能力弱的特点,具有进一步产业化开发的潜力.     

Development and Characterization of Indole-Responsive Whole-Cell Biosensor Based on the Inducible Gene Expression System from Pseudomonas putida KT2440

Indole is a biologically active compound naturally occurring in plants and some bacteria. It is an important specialty chemical that is used as a precursor by the pharmaceutical and chemical industries, as well as in agriculture. Recently, indole has been identified as an important signaling molecule for bacteria in the mammalian gut. The regulation of indole biosynthesis has been studied in several bacterial species. However, this has been limited by the lack of in vivo tools suitable for indole-producing species identification and monitoring. The genetically encoded biosensors have been shown to be useful for real-time quantitative metabolite analysis. This paper describes the identification and characterization of the indole-inducible system PpTrpI/P_{PP_RS00425} from Pseudomonas putida KT2440. Indole whole-cell biosensors based on Escherichia coli and Cupriavidus necator strains are developed and validated. The specificity and dynamics of biosensors in response to indole and its structurally similar derivatives are investigated. The gene expression system PpTrpI/P_{PP_RS00425} is shown to be specifically induced up to 639.6-fold by indole, exhibiting a linear response in the concentration range from approximately 0.4 to 5 mM. The results of this study form the basis for the use of whole-cell biosensors in indole metabolism-relevant bacterial species screening and characterization.     

BTEX removal from contaminated groundwater by a co‐culture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a continuous fibrous‐bed bioreactor

A fibrous‐bed bioreactor with immobilized cells of Pseudomonas putida and Pseudomonas fluorescens was used to treat groundwater contaminated with benzene, toluene, ethylbenzene, and xylenes (collectively know as BTEX). The kinetics of BTEX biodegradation in the fibrous‐bed bioreactor operated under continuous well‐mixed conditions was studied at room temperature. Aeration was not used in the process fed with groundwater samples with an average total BTEX concentration of 2.75 mg dm^?3. All BTEX compounds present in the groundwater feed were concurrently and completely biodegraded even under oxygen‐limited or hypoxic conditions. Nearly 100% removal efficiency was obtained when the retention time was greater than 1 h. BTEX removal efficiency decreased with decreasing the retention time, with p‐ and o‐xylenes showed up first in the treated groundwater, followed by benzene and then other BTEX compounds. Biodegradation rates of BTEX generally increased with increasing BTEX concentration and loading rate. The maximum BTEX biodegradation rate was 5.76 mg h^?1 dm^?3 at the loading rate of 6.54 mg dm^?3 h^?1. The bioreactor had a stable performance, maintaining its ability for efficient BTEX degradation without requiring additional nutrients for more than 1 month. The good performance of the fibrous‐bed bioreactor was attributed to the high cell density (～15 g dm^?3 reactor volume) in the fibrous matrix. © 2002 Society of Chemical Industry     

Pseudomonas putida Biofilm Depends on the vWFa-Domain of LapA in Peptides-Containing Growth Medium

The biofilm of Pseudomonas putida is complexly regulated by several intercellular and extracellular factors. The cell surface adhesin LapA of this bacterium is a central factor for the biofilm and, consequently, the regulation of lapA expression, for example, by Fis. It has been recently shown that peptides in growth media enhance the formation of P. putida biofilm, but not as a source of carbon and nitrogen. Moreover, the peptide-dependent biofilm appeared especially clearly in the fis-overexpression strain, which also has increased LapA. Therefore, we investigate here whether there is a relationship between LapA and peptide-dependent biofilm. The P. putida strains with inducible lapA expression and LapA without the vWFa domain, which is described as a domain similar to von Willebrand factor domain A, were constructed. Thereafter, the biofilm of these strains was assessed in growth media containing extracellular peptides in the shape of tryptone and without it. We show that the vWFa domain in LapA is necessary for biofilm enhancement by the extracellular peptides in the growth medium. The importance of vWFa in LapA was particularly evident for the fis-overexpression strain F15. The absence of the vWFa domain diminished the positive effect of Fis on the F15 biofilm.     

Studies on the biosorption of Pb(II) ions using Pseudomonas putida

ABSTRACT

The complete removal of Pb(II) was achieved by intact Pseudomonas putida cells. The biosorption isotherm exhibited Langmuirian behaviour and followed pseudo-second-order rate kinetics. The standard Gibbs free energy change (?G°) for the biosorption of Pb(II) ions was found to be ?26.4 kJ mol^?1, attesting to a chemisorption process. Thermolysis of P. putida cells improved the Pb(II) binding capacity by around 27%. All the four components tested, namely DNA, protein, polysaccharide and lipid, were found to contribute to the uptake of Pb(II) ions. The possible mechanisms of Pb(II) binding by P. putida have been delineated.     

Production of salicylate hydroxylase from Pseudomonas putida UUC-1 and its application in the construction of a biosensor

The enzyme salicylate hydroxylase was produced from Pseudomonas putida UUC-1 in an 80 dm³ 16 h batch fermentation process yielding, ～ 9 K units per 60 dm³. This enzyme preparation has been studied for its application in the construction of biosensor systems, e.g. carbon past electrodes, screen-printed carbon electrodes, and disposable carbon enzyme electrodes, which will be used for the rapid estimation of salicylate in blood sample. The linear range of the disposable carbon enzyme electrode in response to salicylate was achieved to 1·8 mmol dm^?3.     

Pseudomonas putida KT2442制备中长链聚羟基烷酸酯

初步探讨了利用PseudomonasputidaKT2442制备中长链聚羟基烷酸酯(MCL PHAs)的工艺,考察了不同碳源对菌体生长和产物合成的影响。在2.5L发酵罐上采用高密度细胞培养制备:以油酸为碳源,细胞干重达120g L,MCL PHAs质量浓度达40g L,产物质量分数为34%,生产速率为0.965g·L-1·h-1;以玉米油水解物为碳源,细胞干重达105g L,MCL PHAs达28g L,产物质量分数为26.5%,生产速率为0.667g·L-1·h-1。以玉米油水解物为碳源所得细胞浓度、产物浓度与以油酸为碳源所得结果比较接近,有利于降低生产成本。     

磁场作用下Pseudomonas sp.WQ-03降解苯酚的试验研究

在磁场作用下,对能够用苯酚作为唯一碳源和能源的Pseudomonas sp.WQ-03菌株进行了在不同磁感应强度以及不同苯酚质量浓度下的生长情况以及苯酚的降解试验,结果表明该菌株生长的最佳磁感应强度为50Gs,外加磁场对菌体的生长以及苯酚的降解都有一定的促进作用。在磁感应强度为50Gs的条件下,完全降解1100mg/L的苯酚需要49h,而没有磁场作用下完全降解1100mg/L的苯酚则需要56h。     

Physiological comparison of Pseudomonas putida between two growth phases during cometabolism of 4‐chlorophenol in presence of phenol and glutamate: a proteomics approach

BACKGROUND: In cometabolic transformation of 4‐chlorophenol (4‐cp) in the presence of phenol and sodium glutamate (SG), a new biphasic growth pattern has been reported. This study investigates the physiological changes of Pseudomonas putida P8 during the biphasic growth by means of 2‐dimensional gel electrophoresis (2‐DE) and MALDI‐TOF analyses. RESULTS: A total of 49 protein spots were selected and identified in the 2‐DE gels from P. putida P8 grown on a mixed substrate containing 200 mg L^?1 of phenol, 200 mg L^?1 of 4‐cp and 1000 mg L^?1 SG. Among them, 16 protein spots were found differentially expressed in the two exponential growth phases during the biphasic growth, including six catabolic enzymes (DmpC, DmpD, DmpE, DmpF, DmpG and AspA) for substrate utilization. The expression of other proteins involved in detoxification and stress responses, carbohydrate and energy metabolism, and environmental information processing as well as a multifunctional xenobiotic reductase (XenA) was quantitatively analyzed and discussed. CONCLUSION: The expression levels of the identified catabolic enzymes during growth in the two growth phases correlated well with the substrate utilization patterns observed in previous kinetics studies. Furthermore, the results show that cells growing on a mixture of aromatic substrates undergo significant physiological changes. Copyright © 2009 Society of Chemical Industry     

Simultaneous degradation of nitroaromatic compounds TNT,RDX, atrazine,and simazine by Pseudomonas putida HK‐6 in bench‐scale bioreactors

BACKGROUND: Environmental contamination by nitroaromatic compounds such as 2,4,6‐trinitrotoluene (TNT), hexahydro‐1,3,5‐trinitro‐1,3,5‐s‐triazine (RDX), atrazine, and/or simazine (TRAS) generated as waste from military and agricultural activities is a serious worldwide problem. Microbiological treatment of these compounds is an attractive method because many explosives and herbicides are biodegradable and the process can be made cost‐effective. We explored the feasibility of using cultures of Pseudomonas putida HK‐6 for simultaneous degradation of TRAS with the aim of microbial application in wastewater treatment in bench‐scale bioreactors. RESULTS: Experiments were conducted to study the effects of supplemental carbons, nitrogens, and Tween‐80 on the degradation of Ps. putida HK‐6 in media containing TRAS as target substrate(s). The most effective TRAS degradation was shown in the presence of molasses. Addition of nitrogen sources produced a delayed effect for the target substrate(s). Tween‐80 enhanced the degradation of target substrate(s). Simultaneous degradation of these compounds proceeded to completion within the given period. CONCLUSIONS: Ps. putida HK‐6 was capable of growth with TRAS, and the effects of supplements on TRAS degradation and simultaneous TRAS degradation were evaluated in bench‐scale bioreactors. The results of this study have practical applications in the processes of industrial waste stream treatment where the disposal of TRAS may be problematic. Copyright © 2008 Society of Chemical Industry     

The Connection between Czc and Cad Systems Involved in Cadmium Resistance in Pseudomonas putida

Heavy metal pollution is widespread and persistent, and causes serious harm to the environment. Pseudomonas putida, a representative environmental microorganism, has strong resistance to heavy metals due to its multiple efflux systems. Although the functions of many efflux systems have been well-studied, the relationship between them remains unclear. Here, the relationship between the Czc and Cad systems that are predominantly responsible for cadmium efflux in P. putida KT2440 is identified. The results demonstrated that CzcR3, the response regulator of two-component system CzcRS3 in the Czc system, activates the expression of efflux pump genes czcCBA1 and czcCBA2 by directly binding to their promoters, thereby helping the strain resist cadmium stress. CzcR3 can also bind to its own promoter, but it has only a weak regulatory effect. The high-level expression of czcRS3 needs to be induced by Cd²⁺, and this relies on the regulation of CadR, a key regulator in the Cad system, which showed affinity to czcRS3 promoter. Our study indicates that the Cad system is involved in the regulation of the Czc system, and this relationship is important for maintaining the considerable resistance to cadmium in P. putida.     

Production of D-p-Hydroxyphenylglycine From DL-5-p-Hydroxyphenyl Hydantoin by Immobilized Pseudomonas Putida Cells in a Hollow Fiber Memgrane Bioreactor

1 INTRODUCTION D-p-Hydroxyphenylglycine(D-pHPG) is one of the valuable intermediates in the production of semisynthetic β -lactam antibiotics. It can be used for the production of semisynthetic penicillin and cephalosporin. With the increasing demand for semisynthetic antibiotics, the production of D-pHPG from DL-5-p-hydroxyphenyl hydantoin (DL-pHPH) has received more and more attention. Compared with the chemo-enzymatic route developed by Yamada et al. [1,2], the direct enzymatic production of D-pHPG has advantages of low environmental pollution, less equipment needed and mild reaction conditions.     

Efficiency of naphthalene biodegradation by Pseudomonas putida G7 in soil

The efficiency of naphthalene degradation by Pseudomonas putida G7 in soil was assessed using a mathematical model. The number of microorganisms and the concentration of naphthalene in soil samples were monitored. The feasibility of a spectrofluorometric method for naphthalene assay in soil samples was compared with high pressure liquid chromatography. A proposed mathematical model described the growth of the naphthalene‐degrading strains and the consumption of substrates (naphthalene, naphthalene degradation intermediates and soil organic substances) in soil. To describe the growth kinetics of microorganisms having high affinity to substrates with low solubility, two differential equations with substrate exponent 2/3 were proposed. These equations were used to describe utilization of soil organic matter. The model parameters characterize the growth rates for different substrates and respective yield coefficients, specific bacterial death and adaptation rates, and also the rates of PAHs degradation and evaporation. These characteristics can be used in choosing the bacterial strains for biopreparations and efficient clean‐up biotechnology of polluted soils. Copyright © 2004 Society of Chemical Industry     

CRISPR/Cas9–Mediated Genome Editing for Pseudomonas fulva,a Novel Pseudomonas Species with Clinical,Animal, and Plant–Associated Isolates

As one of the most widespread groups of Gram–negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant–associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time–consuming and laborious endeavor. Here, by using the CRISPR–Cas system, a versatile gene–editing tool with a two–plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic–related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked–in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.     

Production of D-p-Hydroxyphenylglycine From DL-5-p-Hydroxyphenyl Hydantoin by Immobilized Pseudomonas Putida Cells in a Hollow Fiber Memgrane Bioreactor

1 INTRODUCTIOND-p-Hydroxyphenylglycine(D-pHPG) is one of the valuable intermediates in the production ofsemisynthetic β-lactam antibiotics.It can be used for the production of semisynthetic penicillinand cephalosporin.With the increasing demand for semisynthetic antibiotics,the productionof D-pHPG from DL-5-P-hydroxyphenyl hydantoin(DL-pHPH)has received more and more at-tention.Compared with the chemo-enzymatic route developed by Yamada et al.,the direct     

Improved mass transfer and biodegradation rates of naphthalene particles using a novel bead mill bioreactor

One of the main challenges in the treatment of polycyclic aromatic hydrocarbons (PAHs) in controlled bioreactors is the hydrophobicity and low solubility of these compounds in the aqueous phase, resulting in appreciable mass transfer limitations within the bioreactor. To address this challenge, we have developed a modified roller bioreactor (Bead Mill Bioreactor) in which inert particles are used to improve mass transfer from the solid phase to the aqueous phase. Experimental results with naphthalene as a model PAH and Pseudomonas putida as a candidate bacterium indicate that both the mass transfer rate from the solid phase to liquid phase and the biodegradation rate in the Bead Mill Bioreactor (BMB) were significantly higher than those in a conventional roller bioreactor (20‐fold and 5.5‐fold, respectively). The enhancement of mass transfer was dependent on the type, size and volumetric loading of the inert particles, as well as concentration of particulate naphthalene. The highest mass transfer coefficient (k_La = 2.1 h^?1) was achieved with 3 mm glass beads at a volumetric loading of 50% (particle volume/working volume) with 10 000 mg dm^?3 particulate naphthalene. The maximum biodegradation rate of naphthalene attained in the bead mill bioreactor (59.2 mg dm^?3 h^?1 based on the working volume and 118.4 mg dm^?3 h^?1 based on the liquid volume) surpasses most other rates published in the literature and is equivalent to values reported for more complex bioreaction systems. The bead mill bioreactor developed in the present work not only enjoys a simple design but shows excellent performance for treatment of PAHs suspended in an aqueous phase. This fundamental information will be of significant value for future studies involving soil‐bound PAHs. Copyright © 2005 Society of Chemical Industry     

Pseudomonas Phage MD8: Genetic Mosaicism and Challenges of Taxonomic Classification of Lambdoid Bacteriophages

Pseudomonas phage MD8 is a temperate phage isolated from the freshwater lake Baikal. The organisation of the MD8 genome resembles the genomes of lambdoid bacteriophages. However, MD8 gene and protein sequences have little in common with classified representatives of lambda-like phages. Analysis of phage genomes revealed a group of other Pseudomonas phages related to phage MD8 and the genomic layout of MD8-like phages indicated extensive gene exchange involving even the most conservative proteins and leading to a high degree of genomic mosaicism. Multiple horizontal transfers and mosaicism of the genome of MD8, related phages and other λ-like phages raise questions about the principles of taxonomic classification of the representatives of this voluminous phage group. Comparison and analysis of various bioinformatic approaches applied to λ-like phage genomes demonstrated different efficiency and contradictory results in the estimation of genomic similarity and relatedness. However, we were able to make suggestions for the possible origin of the MD8 genome and the basic principles for the taxonomic classification of lambdoid phages. The group comprising 26 MD8-related phages was proposed to classify as two close genera belonging to a big family of λ-like phages.