M?ssbauer studies of Escherichia coli sulfite reductase complexes with carbon monoxide and cyanide. Exchange coupling and intrinsic properties of the [4Fe-4S] cluster

M?ssbauer studies of the hemoprotein subunit (SiR) of E. coli sulfite reductase have shown that the siroheme and the [4Fe-4S] cluster are exchange-coupled. Here we report M?ssbauer studies of SiR complexed with either CO or CN- and of SiR in the presence of the chaotropic agent dimethyl sulfoxide (Me2SO). The spectra of one-electron-reduced SiR X CN show that all five iron atoms reside in a diamagnetic environment; the ferroheme X CN complex is low spin and the [4Fe-4S] cluster is in the 2+ oxidation state. Titration with ferricyanide affords a CN- complex of oxidized SiR in which the siroheme iron is low spin ferric, with the cluster remaining in the 2+ state. At low temperatures, paramagnetic hyperfine interactions are observed for the iron sites of the cluster, suggesting that it is exchange-coupled to the heme iron. Reduction of one-electron-reduced SiR X CN and SiR X CO yields complexes with "g = 1.94"-type EPR signals showing that the second electron is accommodated by the iron-sulfur cluster. The fully reduced complexes yield well resolved M?ssbauer spectra which were analyzed in the spin Hamiltonian formalism. The analysis shows that the cluster subsites are equivalent in pairs, one pair having properties reminiscent of ferric sites whereas the other pair has features more typical of ferrous sites. The M?ssbauer spectra of oxidized SiR kept in 60% (v/v) Me2SO are virtually identical with those observed for SiR in standard buffer, implying that the coupling is maintained in the presence of the chaotrope. Fully reduced SiR displays an EPR signal with g values of g = 2.53, 2.29, and 2.07. In 60% Me2SO, this signal vanishes and a g = 1.94 signal develops; this transition is accompanied by a change in the spin state of the heme iron from S = 1 (or 2) to S = O.     

Above-ground biomass references for urban trees from terrestrial laser scanning data

Background and AimsWithin extending urban areas, trees serve a multitude of functions (e.g. carbon storage, suppression of air pollution, mitigation of the ‘heat island’ effect, oxygen, shade and recreation). Many of these services are positively correlated with tree size and structure. The quantification of above-ground biomass (AGB) is of especial importance to assess its carbon storage potential. However, quantification of AGB is difficult and the allometries applied are often based on forest trees, which are subject to very different growing conditions, competition and form. In this article we highlight the potential of terrestrial laser scanning (TLS) techniques to extract highly detailed information on urban tree structure and AGB.MethodsFifty-five urban trees distributed over seven cities in Switzerland were measured using TLS and traditional forest inventory techniques before they were felled and weighed. Tree structure, volume and AGB from the TLS point clouds were extracted using quantitative structure modelling. TLS-derived AGB estimates were compared with AGB estimates based on forest tree allometries dependent on diameter at breast height only. The correlations of various tree metrics as AGB predictors were assessed.Key ResultsEstimates of AGB derived by TLS showed good performance when compared with destructively harvested references, with an R² of 0.954 (RMSE = 556 kg) compared with 0.837 (RMSE = 1159 kg) for allometrically derived AGB estimates. A correlation analysis showed that different TLS-derived wood volume estimates as well as trunk diameters and tree crown metrics show high correlation in describing total wood AGB, outperforming tree height.ConclusionsWood volume estimates based on TLS show high potential to estimate tree AGB independent of tree species, size and form. This allows us to retrieve highly accurate non-destructive AGB estimates that could be used to establish new allometric equations without the need for extensive destructive harvesting.     

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.     

PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability

In the yeast Saccharomyces cerevisiae, PKA and Sch9 exert similar physiological roles in response to nutrient availability. However, their functional redundancy complicates to distinguish properly the target genes for both kinases. In this article, we analysed different phenotypic read-outs. The data unequivocally showed that both kinases act through separate signalling cascades. In addition, genome-wide expression analysis under conditions and with strains in which either PKA and/or Sch9 signalling was specifically affected, demonstrated that both kinases synergistically or oppositely regulate given gene targets. Unlike PKA, which negatively regulates stress-responsive element (STRE)- and post-diauxic shift (PDS)-driven gene expression, Sch9 appears to exert additional positive control on the Rim15-effector Gis1 to regulate PDS-driven gene expression. The data presented are consistent with a cyclic AMP (cAMP)-gating phenomenon recognized in higher eukaryotes consisting of a main gatekeeper, the protein kinase PKA, switching on or off the activities and signals transmitted through primary pathways such as, in case of yeast, the Sch9-controlled signalling route. This mechanism allows fine-tuning various nutritional responses in yeast cells, allowing them to adapt metabolism and growth appropriately.     

Energy Conversion in Bull Sperm Flagella

With the use of a specially developed incubation chamber the rates of motility, respiration, and fructolysis were measured simultaneously on semen samples. By inhibiting the respiration with antimycin A, and/or the fructolysis with 2-deoxyglucose, the rates of each of the two ATP-producing pathways could be reduced independently. In this way the ratio of the amount of free energy produced by respiration and by fructolysis could be varied at will from 1 to 0. In uninhibited preparations approximately 75% of the free energy derives from respiration, and 25% from fructolysis. By the use of the absolute rates of respiration, fructolysis, and motility, the efficiency of the conversion of free energy into hydrodynamic work was calculated. After correction for the decay of the preparation during the experiment, this conversion efficiency was found to be 30–45% lower for free energy from respiration than for free energy from fructolysis. The difference in distribution of the enzymes for fructolysis and respiration over the flagellum was ruled out as the cause of the efficiency difference. The respiration could be 70% inhibited by oligomycin. It is concluded that approximately one-third of the free energy from respiration is used for maintenance of the mitochondria.     

Synthesis by Ring‐Closing Alkyne Metathesis with Selective Hydrogenation,and Olfactory Comparison of (7E)‐ and (7Z)‐Cyclohexadec‐7‐enone (Aurelione®)

Both C?C‐bond isomers of cyclohexadec‐7‐enone ( 6 , Aurelione^®) were selectively synthesized via cyclohexadec‐7‐ynol ( 16 ) by ring‐closing alkyne metathesis of icosa‐2,18‐diyn‐9‐ol ( 15 ), employing an in situ‐formed catalyst from Mo(CO)₆ and 4‐(trifluoromethyl)phenol. Pyridinium chlorochromate (PCC) oxidation and subsequent Lindlar hydrogenation afforded the (7Z)‐configured isomer (7Z)‐ 6 , while hydrosilylation of the intermediate cyclohexadec‐7‐ynone ( 17 ), followed by desilylation, provided the (7E)‐configured cyclohexadec‐7‐enone ((7E)‐ 6 ). The substrate for the alkyne metathesis was prepared from cycloheptanone ( 7 ) by cycloaddition of chloromethylcarbene to its trimethylsilyl enol ether 8 , and subsequent ring enlargement of the adduct 9 under rearrangement to 2‐methylcyclooct‐2‐enone ( 10 ), which was subjected to Weitz? Scheffer epoxidation and Eschenmoser? Ohloff fragmentation to non‐7‐ynal ( 12 ). Its reaction with the Grignard reagent of 11‐bromoundec‐2‐yne ( 14 ), prepared from the corresponding alcohol 13 by Appel? Lee bromination, furnished the icosa‐2,18‐diyn‐9‐ol ( 15 ). While both isomers of cyclohexadec‐7‐enone ( 6 ) possess warm and powdery musk odors with tobacco‐type ambery accents, (7Z)‐ 6 is more animalic and waxy, whereas (7E)‐ 6 was found to be more floral, sweet, and hay‐like in tonality. Interestingly, however, with odor detection thresholds of 2.0 ng/l air and 2.3 ng/l air, respectively, both (7Z)‐ 6 and (7E)‐ 6 were found to be almost identical in their odor strength, with the (7Z)‐ 6 being only very slightly more powerful.     

Expression of a mutant HSP110 sensitizes colorectal cancer cells to chemotherapy and improves disease prognosis

Heat shock proteins (HSPs) are necessary for cancer cell survival. We identified a mutant of HSP110 (HSP110ΔE9) in colorectal cancer showing microsatellite instability (MSI CRC), generated from an aberrantly spliced mRNA and lacking the HSP110 substrate-binding domain. This mutant was expressed at variable levels in almost all MSI CRC cell lines and primary tumors tested. HSP110ΔE9 impaired both the normal cellular localization of HSP110 and its interaction with other HSPs, thus abrogating the chaperone activity and antiapoptotic function of HSP110 in a dominant-negative manner. HSP110ΔE9 overexpression caused the sensitization of cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of people with CRC. The survival and response to chemotherapy of subjects with MSI CRCs was associated with the tumor expression level of HSP110ΔE9. HSP110 may thus constitute a major determinant for both prognosis and treatment response in CRC.