Isolation and characterization of the naphthocyclinone gene cluster from Streptomyces arenae DSM 40737 and heterologous expression of the polyketide synthase genes

Streptomyces arenae produces the aromatic polyketide naphthocyclinone, which exhibits activity against Gram-positive bacteria. A cosmid clone containing the putative naphthocyclinone gene cluster was isolated from a genomic library of S. arenae by hybridization with a conserved region from the actinorhodin PKS of S. coelicolor. Sequence analysis of a 5.5-kb DNA fragment, which hybridizes with the actI probe, revealed three open reading frames coding for the minimal polyketide synthase. A strong sequence similarity was found to several previously described ketosynthases, chain length factors and acyl carrier proteins from other polyketide gene clusters. An additional open reading frame downstream of the PKS genes of S. arenae showed 53% identity to act VII probably encoding an aromatase. Another open reading frame was identified in a region of 1.436 bp upstream of the PKS genes, which, however, had no similarity to known genes in the database. Approximately 8 kb upstream of the PKS genes, a DNA fragment was identified that hybridizes to an actVII--actIV specific probe coding for a cyclase and a putative regulatory protein, respectively. Disruption of the proposed naphthocyclinone gene cluster by insertion of a thiostrepton resistance gene completely abolished production of naphthocyclinones in the mutant strain, showing that indeed the naphthocyclinone gene cluster had been isolated. Heterologous expression of the minimal PKS genes in S. coelicolor CH999 in the presence of the act ketoreductase led to the production of mutactin and dehydromutactin, indicating that the S. arenae polyketide synthase forms a C-16 backbone that is subsequently dimerized to build naphthocyclinone. The functions of the proposed cyclase and aromatase were examined by coexpression with genes from different polyketide core producers.     

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.     

Polycistronic gene expression in yeast versus cryptic promoter elements

Saccharomyces cerevisiae is a much preferred host for biotechnological applications. However, the expression of entire heterologous pathways, required for some potential products, is technically challenging in yeast. A possible tool would be polycistronic gene expression. Recent studies demonstrated that short 5' untranslated regions (5'UTRs) found upstream of certain genes support cap-independent translation in vitro. In this study 5'UTRs were used as linkers between genes in polycistronic constructs. Expression levels of genes located in the first, second and third position after a promoter were studied by replacing the respective gene by a promoterless green fluorescence protein (GFP) gene. S. cerevisiae transformed with these constructs was grown on different carbon sources and GFP expression was assayed. Our results demonstrate that (i) ribosomal read-through does not suffice for polycistronic gene expression in vivo, (ii) 5'TFIID and 5'HAP4 but not 5'L-A significantly improve the expression of a reporter gene located second in a bicistron, (iii) 5'TFIID, 5'HAP4 and 5'YAP1 but not 5'L-A can drive expression of a promoterless reporter gene, and (iv) expression driven from 5'TFIID, 5'HAP4 and 5'YAP1 is induced in the presence of raffinose or galactose but not in the presence of glucose. This implies that these elements unlike typical internal ribosome entry site-like structures contain small, potentially useful promoters which support carbon source-regulated expression.     

Oxygene dissous et saturation dans un milieu de forte production organique (Etang de Berre). Role d'une halocline sur leur distribution et sur les rapports oxygene-phosphate. Notion de bilan

Résumé L'évolution des structures de distribution de l'oxygène (concentration et saturation) a été suivie, dans les eaux de l'Etang de Berre, durant plusieurs années. A l'existence d'une pycnocline (halocline) à gradient très exprimé, correspond celle d'une oxycline aboutissant à l'anoxie dans certain cas. L'analyse des relations entre oxygène et phosphore minéral, en conditions de pénurie d'oxygène, montre que l'existence d'anomalies dans ces relations tient au fonctionnement du système consommation-production-régénération. Un concept de bilan théorique d'oxygène dans un bassin saumâtre est proposé.

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The evolution of oxygen distribution parameters (concentration and saturation) has been monitored in brackish waters of lake Etang de Berre during several years. In relation with the occurrence of a well-defined pycnocline (halocline) an oxycline is shown to be present, leading to anoxic conditions in some cases. The relationships between oxygen and nutrients under conditions of oxygen depletion are examined and it is shown that anomalies are due to the dynamic aspects of the consumption-production-regeneration system. A concept of theoretical oxygen budget in a brackish water basin is proposed.

     

A Further Investigation on the Primary Productivity in the Divergence Zone Near the French Mediterranean Coast

The present investigation re-examines hydrological features, the inorganic phosphate distribution and primary productivity near the “Hyères” Islands in the French Mediterranean in the early summer divergence zone as previously studied by MINAS and BLANC (1970). In addition, concentrations of particulate nitrogen, Chl-a and nitrate have been determined. Uptake rates of ¹⁵N-labelled nitrate (“new production”) and the oxygen consumption (ETS-activity) by the phytoplankton population have been estimated.     

Oil pollution in the North Sea—a microbiological point of view

In this study we determined oil degradation rates in the North Sea under most natural conditions. We used the heavy fuel oil, Bunker C, the major oil pollutant of the North Sea, as the model oil. Experiments were conducted in closed systems with water sampled during winter and repeated under identical conditions with water collected during summer. No nitrogen or phosphorous was added and conditions were chosen such that neither oxygen nor nutrients, present in the water, would become limiting during the experiments. We detected a fourfold increased degradation rate for water samples taken in summer (18°C water temperature) as compared to water sampled in winter (4°C water temperature). Under the assumption that biodegradation of oil can be regarded as a Michaelis-Menten type kinetic reaction, the kinectic constants V_max and K_M were determined for oil biodegradation at 4°C and 18°C. At both temperatures K_M was about 40 ppm, whereas V_max was 3–4 times higher at 18°C. From both V_max and the results of fermentation studies, we determined the maximum rates of Bunker C oil degradation in the North Sea as ∼20 g m⁻³a⁻¹ at 4°C in winter and 60–80 g m⁻³a⁻¹ at 18°C in summer. Furthermore, while over 25% of the oil was degraded within 6 weeks in summer, only 6.6% of the oil was degraded in winter. A higher incubation temperature in winter (18°C) increased both the rate and the percentage of oil degraded, but degradation did not reach the level obtained during the summer. While these data reflect the oxidation only of the hydrocarbons, we conducted experiments directly in the open sea to determine the contribution of abiotic factors to oil removal. Approximately 42% of the oil was lost within 6 weeks under these conditions in summer and 65% in winter. However, GC-MS analysis of the recovered oil showed no significant change in the alkane pattern that would indicate enhanced degradation. Thus, mainly abiotic factors such as erosion and dispersion rather than degradation were responsible for enhanced oil removal. Especially the high loss during winter can be attributed to frequent storms resulting in greater dispersion. In conclusion, the higher oil degrading potential of the microbial population in the North Sea was represented by a four times faster oil degradation during the summer. In-situ experiments showed that abiotic factors can have an equal (summer) or even higher (winter) impact on oil removal.     

Exhaled breath condensate pH as a biomarker of COPD severity in ex-smokers

Endogenous airway acidification, as assessed by exhaled breath condensate (EBC) pH, is present in patients with stable COPD. The aim of this study was to measure EBC pH levels in a large cohort of COPD patients and to evaluate associations with functional parameters according to their smoking status.EBC was collected from 161 patients with stable COPD and 112 controls (current and ex-smokers). EBC pH was measured after Argon deaeration and all subjects underwent pulmonary function testing.EBC pH was lower in COPD patients compared to controls [7.21 (7.02, 7.44) vs. 7.50 (7.40, 7.66); p < 0.001] and ex-smokers with COPD had lower EBC pH compared to current smokers [7.16 (6.89, 7.36) vs 7.24 (7.09, 7.54), p = 0.03]. In ex-smokers with COPD, EBC pH was lower in patients with GOLD stage III and IV compared to patients with stage I disease (p = 0.026 and 0.004 respectively). No differences were observed among current smokers with different disease severity. EBC pH levels in ex-smokers were associated with static hyperinflation (as expressed by IC/TLC ratio), air trapping (as expressed by RV/TLC ratio) and diffusing capacity for carbon monoxide, whereas no associations were observed in current smokers.Endogenous airway acidification is related to disease severity and to parameters expressing hyperinflation and air trapping in ex-smokers with COPD. The possible role of EBC pH in COPD needs to be further evaluated in longitudinal studies.     

Distance-based differential analysis of gene curves

MOTIVATION: Time course gene expression experiments are performed to study time-varying changes in mRNA levels of thousands of genes. Statistical methods from functional data analysis (FDA) have recently gained popularity for modelling and exploring such time courses. Each temporal profile is treated as the realization of a smooth function of time, or curve, and the inferred curve becomes the basic unit of statistical analysis. The task of identifying genes with differential temporal profiles then consists of detecting statistically significant differences between curves, where such differences are commonly quantified by computing the area between the curves or the l? distance. RESULTS: We propose a general test statistic for detecting differences between gene curves, which only depends on a suitably chosen distance measure between them. The test makes use of a distance-based variance decomposition and generalizes traditional MANOVA tests commonly used for vectorial observations. We also introduce the visual l? distance, which is shown to capture shape-related differences in gene curves and is robust against time shifts, which would otherwise inflate the traditional l? distance. Other shape-related distances, such as the curvature, may carry biological significance. We have assessed the comparative performance of the test on realistically simulated datasets and applied it to human immune cell responses to bacterial infection over time.