Watasemycin biosynthesis in Streptomyces venezuelae : thiazoline C-methylation by a type B radical-SAM methylase homologue

2-Hydroxyphenylthiazolines are a family of iron-chelating nonribosomal peptide natural products that function as virulence-conferring siderophores in various Gram-negative bacteria. They have also been reported as metabolites of Gram-positive Streptomyces species. Transcriptional analyses of Streptomyces venezuelae ATCC 10712 revealed that its genome contains a putative 2-hydroxyphenylthiazoline biosynthetic gene cluster. Heterologous expression of the gene cluster in Streptomyces coelicolor M1152 showed that the mono- and dimethylated derivatives, thiazostatin and watasemycin, respectively, of the 2-hydroxyphenylthiazoline enantiopyochelin are two of its metabolic products. In addition, isopyochelin, a novel isomer of pyochelin containing a C-methylated thiazolidine, was identified as a third metabolic product of the cluster. Metabolites with molecular formulae corresponding to aerugine and pulicatins A/B were also detected. The structure and stereochemistry of isopyochelin were confirmed by comparison with synthetic standards. The role of two genes in the cluster encoding homologues of PchK, which is proposed to catalyse thiazoline reduction in the biosynthesis of enantiopyochelin in Pseudomonas protegens, was investigated. One was required for the production of all the metabolic products of the cluster, whereas the other appears not to be involved in the biosynthesis of any of them. Deletion of a gene in the cluster encoding a type B radical-SAM methylase homologue abolished the production of watasemycin, but not thiazostatin or isopyochelin. Feeding of thiazostatin to the mutant lacking the functional PchK homologue resulted in complete conversion to watasemycin, demonstrating that thiazoline C-methylation by the type B radical-SAM methylase homologue is the final step in watasemycin biosynthesis

Modification of GLEAMS for Modeling Movement of Organic Contaminants from Land-Applied Biosolids

Municipal biosolids are commonly applied to agricultural lands as fertilizer, but this also poses potential risks to groundwater and surface water quality from constituents that may be mobilized during storm events. In the present study, an existing model, Groundwater Loading Effects of Agricultural Management Systems (GLEAMS), is modified to predict the fate and transport of organic contaminants from land-applied biosolids, primarily via addition of a labile biosolids organic carbon phase distinct from soil organic carbon. While capable of simulating contaminant transport in runoff and via percolation, only the runoff portion of the model was able to be calibrated using existing experimental data, and showed good agreement with field runoff data for acetaminophen, ibuprofen, triclosan, triclocarban, and estrone, but substantially under-predicted concentrations for carbamazepine, androstenedione, and progesterone. The model is applied to various scenarios using varied chemical properties, application date in the arid west, and application method (i.e., surface spreading vs. incorporation). Chemicals with longer half-lives and lower KOCs exhibited higher losses in runoff than chemicals with shorter half-lives and higher KOCs. For short half-life chemicals (i.e., ≤100 days), application at the beginning of the dry season resulted in the lowest losses. However, for long half-life chemicals (∼1000 days) with high KOC (10,000–100,000), application during the rainy season resulted in the lowest losses, because this caused organic carbon to be high during the period of highest runoff. While further work is necessary to calibrate the percolation and subsurface transport portion, the model can help predict environmental risk from land-application of biosolids, highlight gaps in our knowledge about how chemicals are mobilized and transported from biosolids, and help identify management practices that result in minimal impacts to water quality

Development of a synthetic human thigh impact surrogate for sports personal protective equipment testing

© IMechE 2015. Synthetic impact surrogates are widely used in the sporting goods industry in the evaluation of personal protective equipment. Existing surrogates, exemplified by those used in safety standards, have many shortcomings, primarily relating to their mass, stiffness, geometries and levels of constraint which limit their biofidelity and subsequent usefulness in personal protective equipment evaluations. In sports, absence from competition is a primary severity measure for injuries; consequently, blunt trauma injuries, such as contusions and lacerations, become pertinent and serious concerns. It is important, therefore, that synthetic surrogates provide an adequate description of these soft tissues to effectively evaluate injury risk. A novel, multi-material human thigh surrogate has been presented with consideration to the tissue structures, geometries and simulant materials used. This study presents the detailed development stages undertaken to fabricate a multi-material synthetic soft tissue surrogate with skin, subcutaneous adipose and muscle tissue components. The resultant surrogate demonstrates the successful use of sequential moulding techniques to construct a full-scale anatomical human impact surrogate which can be used in personal protective equipment testing

Accurate measurements of Optical Turbulence with Sonic-anemometers

The minimization of optical turbulence in and around the dome is key to reach optimum performance on large telescopes equipped with adaptive optics. We present the method and preliminary results of in-situ measurements of optical measurements made using sonic-anemometers. We show the impact of correcting the raw data for aliasing, path averaging, pulse sequence delays and Taylors' hypothesis. Finally, we highlight the occurrence of non-Kolmogorov turbulence which complicates the quantitative impact of the measurements on the telescope's resolution

Microbial engineering of new streptomyces sp. from extreme environments for novel antibiotics and anticancer drugs

Today there is a tremendous need for new antibiotics and novel cytotoxic compounds against cancer cells to develop efficient alternative treatment to chemotherapy. We have searched for highly active Streptomyces strains in the driest desert in the world, the Atacama desert in northern Chile. We have identified several new strains and found many novel antibiotics and anticancer agents (“Chaxamycins”, “Chaxalactins” and “Atacamycins”) from Streptomyces C34 and C38. A genome scale model of the metabolism of Streptomyces leeuwenhoekii C34 has been developed from its genome sequence. The model, iVR1007, has 1726 reactions including 239 for transport, reactions for secondary metabolite biosynthesis, 1463 metabolites and 1007 genes. The model was validated with experimental data of growth in 89, 54 and 23 sole carbon, nitrogen and phosphorous sources, respectively, and showed a high level of accuracy (82.5 %). We have included reactions for desferrioxamines, ectoine, Chaxamycins, Chaxalactins and for the hybrid polyketides/non-ribosomal peptide synthesized by the halogenase cluster. A detailed Metabolic Flux Balance Analysis was carried out in order to study the metabolic pathways of Chaxalactins, Chaxamycins and the product of the halogenase cluster, by recognizing overexpression targets and useful knock-out sites to increase production of these secondary metabolites. Alternatively we have identified the gene cluster in S. leeuwenhoekii C34 responsible for the biosynthesis of the Chaxamycins and Chaxalactins and have cloned the whole gene cluster in a much more efficient strain of Streptomyces, namely S. coelicolor A3 whose heterologous expression of gene clusters from other Streptomyces strains has been successfully tested. Our recent results concerning these two alternative strategies for identification and overproduction of these important secondary metabolites will be presented and discussed in this presentation

Pitting Corrosion in Austenitic Stainless Steel Water Tanks of Hotel Trains

The water storage tanks of hotel trains suffered pitting corrosion. To identify the cause, the tanks were subjected to a detailed metallographic study and the chemical composition of the austenitic stainless steels used in their construction was determined. Both the tank water and the corrosion products were further examined by physicochemical and microbiological testing. Corrosion was shown to be related to an incompatibility between the chloride content of the water and the base and filler metals of the tanks. These findings formed the basis of recommendations aimed at the prevention and control of corrosion in such tanks. Se han detectado problemas de corrosión por picaduras en los depósitos de agua de trenes hotel. Para identificar las causas se llevó a cabo un detallado estudio metalográfico así como de la composición química de los aceros inoxidables austeníticos utilizados en su construcción. También se realizaron estudios fisicoquímicos y microbiológicos de los productos de corrosión. Se ha encontrado que los problemas de corrosión están relacionados con la incompatibilidad entre el contenido en cloruros del agua y los metales base y de aporte de la soldadura de los tanques. En base a estos hallazgos se proponen una serie de recomendaciones encaminadas a la prevención y control de la corrosión de dichos depósitos

New Insights into Chloramphenicol Biosynthesis in Streptomyces venezuelae ATCC 10712

Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916–sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway

Analysis of the tunicamycin biosynthetic gene cluster of streptomyces chartreusis reveals new insights into tunicamycin production and immunity

The tunicamycin biosynthetic gene cluster of Streptomyces chartreusis consists of 14 genes (tunA to tunN) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters, tunp1 and tunp2. In-frame deletion analysis revealed that just six of these genes (tunABCDEH) are essential for tunicamycin production in the heterologous host Streptomyces coelicolor, while five (tunFGKLN) with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely, tunI and tunJ, which encode a two-component ABC transporter presumably required for export of the antibiotic, and tunM, which encodes a putative S-adenosylmethionine (SAM)-dependent methyltransferase. Expression of tunIJ or tunM in S. coelicolor conferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity

Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2.

The lymphatic system maintains tissue fluid balance, and dysfunction of lymphatic vessels and valves causes human lymphedema syndromes. Yet, our knowledge of the molecular mechanisms underlying lymphatic vessel development is still limited. Here, we show that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of lymphatic vessel development. Endothelial-specific Cdk5 knockdown causes congenital lymphatic dysfunction and lymphedema due to defective lymphatic vessel patterning and valve formation. We identify the transcription factor Foxc2 as a key substrate of Cdk5 in the lymphatic vasculature, mechanistically linking Cdk5 to lymphatic development and valve morphogenesis. Collectively, our findings show that Cdk5-Foxc2 interaction represents a critical regulator of lymphatic vessel development and the transcriptional network underlying lymphatic vascular remodeling