Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors () of −1.5 and −3.9‰, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic (_intrinsic) were calculated. A comparison of _intrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average if no fractionation factor is available for single compounds.     

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.     

Ethylene and methane in the upper water column of the subtropical Atlantic

The vertical distributions of ethylene and methane in the upper water column of the subtropical Atlantic were measured along a transect from Madeira to the Caribbean and compared with temperature, salinity, oxygen, nutrients, chlorophyll-a, and dissolved organic carbon (DOC).Methane concentrations between 41.6 and 60.7 nL L-1 were found in the upper 20 m of the water column giving a calculated average flux of methane into the atmosphere of 0.82 g m-2 h-1. Methane profiles reveal several distinct maxima in the upper 500 m of the water column and short-time variations which are presumably partly related to the vertical migration of zooplankton.Ethylene concentrations in near surface waters varied in the range of 1.8 to 8.2 nL L-1. Calculated flux rates for ethylene into the atmosphere were in the range of 0.41 to 1.35 g m-2 h-1 with a mean of 0.83 g m-2 h-1. Maximum concentrations of up to 39.2 nL L-1 were detected directly below the pycnocline in the western Atlantic. The vertical distributions of ethylene generally showed one maximum at the pycnocline (about 100 m depth) where elevated concentrations of chlorophyll-a, dissolved oxygen, and nutrients were also found; no ethylene was detected below 270 m depth. This suggests that ethylene release is mainly related to one, probably phytoplankton associated, source, while for methane, enhanced net production occurs at various depth horizons. For surface waters, a simple correlation between ethylene and chlorophyll-a or DOC concentrations could not be observed. No considerable diurnal variation was observed for the distribution and concentration of ethylene in the upper water column.     

Changing Feeding Regimes To Demonstrate Flexible Biogas Production: Effects on Process Performance,Microbial Community Structure,and Methanogenesis Pathways

Flexible biogas production that adapts biogas output to energy demand can be regulated by changing feeding regimes. In this study, the effect of changes in feeding intervals on process performance, microbial community structure, and the methanogenesis pathway was investigated. Three different feeding regimes (once daily, every second day, and every 2 h) at the same organic loading rate were studied in continuously stirred tank reactors treating distiller''s dried grains with solubles. A larger amount of biogas was produced after feeding in the reactors fed less frequently (once per day and every second day), whereas the amount remained constant in the reactor fed more frequently (every 2 h), indicating the suitability of the former for the flexible production of biogas. Compared to the conventional more frequent feeding regimes, a methane yield that was up to 14% higher and an improved stability of the process against organic overloading were achieved by employing less frequent feeding regimes. The community structures of bacteria and methanogenic archaea were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA and mcrA genes, respectively. The results showed that the composition of the bacterial community varied under the different feeding regimes, and the observed T-RFLP patterns were best explained by the differences in the total ammonia nitrogen concentrations, H₂ levels, and pH values. However, the methanogenic community remained stable under all feeding regimes, with the dominance of the Methanosarcina genus followed by that of the Methanobacterium genus. Stable isotope analysis showed that the average amount of methane produced during each feeding event by acetoclastic and hydrogenotrophic methanogenesis was not influenced by the three different feeding regimes.     

Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH₂-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Δcys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Δcys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Δcys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.     

Sulfidic acetate mineralization at 45°C by an aquifer microbial community: key players and effects of heat changes on activity and community structure

High-Temperature Aquifer Thermal Energy Storage (HT-ATES) is a sustainable approach for integrating thermal energy from various sources into complex energy systems. Temperatures ≥45°C, which are relevant in impact zones of HT-ATES systems, may dramatically influence the structure and activities of indigenous aquifer microbial communities. Here, we characterized an acetate-mineralizing, sulfate-reducing microbial community derived from an aquifer and adapted to 45°C. Acetate mineralization was strongly inhibited at temperatures ≤25°C and 60°C. Prolonged incubation at 12°C and 25°C resulted in acetate mineralization recovery after 40–80 days whereas acetate was not mineralized at 60°C within 100 days. Cultures pre-grown at 45°C and inhibited for 28 days by incubation at 12°C, 25°C, or 60°C recovered quickly after changing the temperature back to 45°C. Phylotypes affiliated to the order Spirochaetales and to endospore-forming sulfate reducers of the order Clostridiales were highly abundant in microcosms being active at 45°C highlighting their key role. In summary, prolonged incubation at 45°C resulted in active microbial communities mainly consisting of organisms adapted to temperatures between the typical temperature range of mesophiles and thermophiles and being resilient to temporary heat changes.     

miR-184 Regulates Pancreatic β-Cell Function According to Glucose Metabolism

In response to fasting or hyperglycemia, the pancreatic β-cell alters its output of secreted insulin; however, the pathways governing this adaptive response are not entirely established. Although the precise role of microRNAs (miRNAs) is also unclear, a recurring theme emphasizes their function in cellular stress responses. We recently showed that miR-184, an abundant miRNA in the β-cell, regulates compensatory proliferation and secretion during insulin resistance. Consistent with previous studies showing miR-184 suppresses insulin release, expression of this miRNA was increased in islets after fasting, demonstrating an active role in the β-cell as glucose levels lower and the insulin demand ceases. Additionally, miR-184 was negatively regulated upon the administration of a sucrose-rich diet in Drosophila, demonstrating strong conservation of this pathway through evolution. Furthermore, miR-184 and its target Argonaute2 remained inversely correlated as concentrations of extracellular glucose increased, underlining a functional relationship between this miRNA and its targets. Lastly, restoration of Argonaute2 in the presence of miR-184 rescued suppression of miR-375-targeted genes, suggesting these genes act in a coordinated manner during changes in the metabolic context. Together, these results highlight the adaptive role of miR-184 according to glucose metabolism and suggest the regulatory role of this miRNA in energy homeostasis is highly conserved.     

Differential sensitivity of total and active soil microbial communities to drought and forest management

Climate change will affect semiarid ecosystems through severe droughts that increase the competition for resources in plant and microbial communities. In these habitats, adaptations to climate change may consist of thinning—that reduces competition for resources through a decrease in tree density and the promotion of plant survival. We deciphered the functional and phylogenetic responses of the microbial community to 60 years of drought induced by rainfall exclusion and how forest management affects its resistance to drought, in a semiarid forest ecosystem dominated by Pinus halepensis Mill. A multiOMIC approach was applied to reveal novel, community‐based strategies in the face of climate change. The diversity and the composition of the total and active soil microbiome were evaluated by 16S rRNA gene (bacteria) and ITS (fungal) sequencing, and by metaproteomics. The microbial biomass was analyzed by phospholipid fatty acids (PLFAs), and the microbially mediated ecosystem multifunctionality was studied by the integration of soil enzyme activities related to the cycles of C, N, and P. The microbial biomass and ecosystem multifunctionality decreased in drought‐plots, as a consequence of the lower soil moisture and poorer plant development, but this decrease was more notable in unthinned plots. The structure and diversity of the total bacterial community was unaffected by drought at phylum and order level, but did so at genus level, and was influenced by seasonality. However, the total fungal community and the active microbial community were more sensitive to drought and were related to ecosystem multifunctionality. Thinning in plots without drought increased the active diversity while the total diversity was not affected. Thinning promoted the resistance of ecosystem multifunctionality to drought through changes in the active microbial community. The integration of total and active microbiome analyses avoids misinterpretations of the links between the soil microbial community and climate change.     

A patchwork pathway for oxygenase‐independent degradation of side chain containing steroids

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen‐independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo‐, phyto‐ and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin‐containing steroid dehydrogenases are involved in C25‐hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26‐oic acids. Side chain degradation to androsta‐1,4‐diene‐3,17‐dione (ADD) via aldolytic C–C bond cleavages involves acyl‐CoA synthetases/dehydrogenases specific for the respective 26‐, 24‐ and 22‐oic acids/‐oyl‐CoAs and promiscuous MaoC‐like enoyl‐CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA‐ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase‐dependent reactions.