Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa ΔplaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-Å-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the PlaF mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.

APEOs, the reader is referred to other books and reviews. [24][25][26][27][28][29] II. Analysis A. Sampling and StorageAnalytical results significantly depend on the homogeneity of the samples and accurate storage procedures which guarantee that no changes take place in the composition of the samples. The main problem of surfactants in general is their tendency to adsorb on all phase boundaries due to their amphiphilic nature. Consequently losses to surfaces or suspended solids from aqueous solutions are commonplace. Especially for matrices like sewage sludge, sediment, or soils, quantitative recovery of analytes turns out to be very difficult. Therefore, internal standards are added to the samples to correct for nonquantitative recovery during isolation and quantification of the analytes. Giger et al. used n-nonylbenzene 11,30 and 2,4,6-tribromophenol 8 in gas chromatographic determinations of APs/APEOs from sludge and water, respectively. 4-n-Nonylphenol, which is not included in technical NP, is applied to the quantification of NP in soils by GC. 31 For water analysis by HPLC 2,4,6-trimethylphenol is well suited. 32 This approach, however, is useless for nonspecific methods since they cannot discriminate analytes initially present from added internal standards.Environmental samples have to be preserved immediately upon collection with chemical biocides to minimize and prevent microbial degradation of the surfactant present. Water samples from sewage treatment plants, rivers, or seas are generally collected in glass bottles, preserved with 1% formaldehyde, and stored at 4 °C. [33][34][35][36][37] A less common preservation method for aqueous samples is the addition of methylene chloride and acidification to pH 2 with hydrochloric acid. 38 Kubeck et al. showed that refrigeration alone was sufficient to stabilize river water samples for up to 4 weeks. 35 Due to diurnal variations of APEO concentrations in the influents and effluents of sewage treatment plants, 24-h and 2-h composite samples should be collected, ideally, with automatic sampling devices. 39 Sewage sludges are dealt with in the same way as water samples, i.e. preservation with 1% formaldehyde and storage at 4 °C. 39,40 Jobst et al. preferred aluminium vessels to store the sludge samples. 41 Sediment samples are collected from the upper 2 cm using a grab sampler and frozen at -20 °C until analysis. 33,[42][43][44] In the laboratory, samples are freezedried 44 or air-dried at 21 °C43 .The application of sewage sludges to agricultural land has resulted in the need to monitor concentrations of detergents in sludge-amended soils. Soil samples are collected from the upper 5 cm with a stainless steel corer, dried at 60 °C, pulverized, and stored in the dark at 4 °C. 40 Biological matrices represent a difficult problem with regard to a representative sampling and a unchanged composition of the samples during storage. The Environmental Specimen Bank (ESB) of Germany has developed a method for collection and preparation of fresh biological materials. 45,46 ...

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The biotrophic fungus Ustilago maydis causes smut disease on maize (Zea mays), which is characterized by immense plant tumours. To establish disease and reprogram organ primordia to tumours, U. maydis deploys effector proteins in an organ-specific manner. However, the cellular contribution to leaf tumours remains unknown. We investigated leaf tumour formation at the tissue- and cell type-specific levels. Cytology and metabolite analysis were deployed to understand the cellular basis for tumourigenesis. Laser-capture microdissection was performed to gain a cell type-specific transcriptome of U. maydis during tumour formation. In vivo visualization of plant DNA synthesis identified bundle sheath cells as the origin of hyperplasic tumour cells, while mesophyll cells become hypertrophic tumour cells. Cell type-specific transcriptome profiling of U. maydis revealed tailored expression of fungal effector genes. Moreover, U. maydis See1 was identified as the first cell type-specific fungal effector, being required for induction of cell cycle reactivation in bundle sheath cells. Identification of distinct cellular mechanisms in two different leaf cell types and of See1 as an effector for induction of proliferation of bundle sheath cells are major steps in understanding U. maydis-induced tumour formation. Moreover, the cell type-specific U. maydis transcriptome data are a valuable resource to the scientific community.

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A method has been developed for quantification of 20 amino acids as well as 13 (15)N-labeled amino acids in barley plants. The amino acids were extracted from plant tissues using aqueous HCl-ethanol and directly analyzed without further purification. Analysis of the underivatized amino acids was performed by liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) in the positive ESI mode. Separation was achieved on a strong cation exchange column (Luna 5micro SCX 100A) with 30 mM ammonium acetate in water (solvent A) and 5% acetic acid in water (solvent B). Quantification was accomplished using d (2)-Phe as an internal standard. Calibration curves were linear over the range 0.5-50 microM, and limits of detection were estimated to be 0.1-3.0 microM. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 degrees C uniform root temperature (RT) and 20-10 degrees C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20-10 degrees C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. (15)NO(3) (-) labeling experiments showed lower (15)N/(14)N ratios for Glu, Ser, Ala and Val in plants grown at 20-10 degrees C RTG compared with those grown at 15 degrees C RT.

b Sulfadiazine (SDZ)-degrading bacterial cultures were enriched from the topsoil layer of lysimeters that were formerly treated with manure from pigs medicated with 14 C-labeled SDZ. The loss of about 35% of the applied radioactivity after an incubation period of 3 years was attributed to CO 2 release due to mineralization processes in the lysimeters. Microcosm experiments with moist soil and soil slurries originating from these lysimeters confirmed the presumed mineralization potential, and an SDZ-degrading bacterium was isolated. It was identified as Microbacterium lacus, denoted strain SDZm4. During degradation studies with M. lacus strain SDZm4 using pyrimidine-ring labeled SDZ, SDZ disappeared completely but no 14 CO 2 was released during 10 days of incubation. The entire applied radioactivity (AR) remained in solution and could be assigned to 2-aminopyrimidine. In contrast, for parallel incubations but with phenyl ring-labeled SDZ, 56% of the AR was released as 14 CO 2 , 16% was linked to biomass, and 21% remained as dissolved, not yet identified 14 C. Thus, it was shown that M. lacus extensively mineralized and partly assimilated the phenyl moiety of the SDZ molecule while forming equimolar amounts of 2-aminopyrimidine. This partial degradation might be an important step in the complete mineralization of SDZ by soil microorganisms.

At the end of the annual horticultural production cycle of greenhouse-grown crops, large quantities of residual biomass are discarded. Here, we propose a new value chain to utilize horticultural leaf biomass for the extraction of secondary metabolites. To increase the secondary metabolite content of leaves, greenhouse-grown crop plants were exposed to low-cost abiotic stress treatments after the last fruit harvest. As proof of concept, we evaluated the production of the flavonoid rutin in tomato plants subjected to nitrogen deficiency. In an interdisciplinary approach, we observed the steady accumulation of rutin in young plants under nitrogen deficiency, tested the applicability of nitrogen deficiency in a commercial-like greenhouse, developed a high efficiency extraction for rutin, and evaluated the acceptance of the proposed value chain by its key actors economically. On the basis of the positive interdisciplinary evaluation, we identified opportunities and challenges for the successful establishment of horticultural leaf biomass as a novel source for secondary metabolites.