ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking.

COPI-coated vesicles mediate retrograde membrane traffic from the cis-Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER-resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V-dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI•Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.

Ecotoxicity of neutral red (dye) and its environmental applications.

Neutral red (NR) is a synthetic phenazine with promising prospect in environmental biotechnology as an electron shuttle. Recently, NR injections into coal seam associated groundwater in Australia (final dissolved NR concentration: 8 µM ± 0.2) were shown to increase methanogenesis up to ten-fold. However, information about NR toxicity to ecological receptors is sorely lacking. The main aim of this study was to investigate the concentration dependent toxicity of NR in microorganisms and plants. Acute toxicity of NR was determined by the modified Microtox™ assay. Microbial viability was determined using Escherichia coli and Bacillus subtilis. Germination and early growth of plants was studied using Lactuca sativa, Daucus carota, Allium cepa and an Australian native Themeda triandra. Lastly, mutagenicity of the coal seam associated groundwater was assessed using the Ames test. The EC50 of acute NR toxicity was determined to be 0.11 mM. The EC50 of microbial viability was between 1 and 7.1mM NR. Among the concentrations tested, only 0.01, 0.10 and 100mM of NR significantly affected (p<0.001) germination of L. sativa. The EC50 for root elongation in seeds was between 1.2 and 35.5mM NR. Interestingly, root elongation in seeds was significantly stimulated (p<0.001) between 0.25 and 10mM NR, showing a hormetic effect. A significant increase in mutagenicity was only observed in one of the three wells tested. The results suggest that the average dissolved NR concentration (8 µM ± 0.2) deployed in the field trial at Lithgow State Coal Mine, Australia, appears not to negatively impact the ecological receptors tested in this study.

Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines.

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.

Expanding the repertoire of electron acceptors for the anaerobic oxidation of methane in carbonates in the Atlantic and Pacific Ocean.

Authigenic carbonates represent a significant microbial sink for methane, yet little is known about the microbiome responsible for the methane removal. We identify carbonate microbiomes distributed over 21 locations hosted by seven different cold seeps in the Pacific and Atlantic Oceans by carrying out a gene-based survey using 16S rRNA- and mcrA gene sequencing coupled with metagenomic analyses. Based on 16S rRNA gene amplicon analyses, these sites were dominated by bacteria affiliated to the Firmicutes, Alpha- and Gammaproteobacteria. ANME-1 and -2 archaeal clades were abundant in the carbonates yet their typical syntrophic partners, sulfate-reducing bacteria, were not significantly present. Based on mcrA amplicon analyses, the Candidatus Methanoperedens clades were also highly abundant. Our metagenome analysis indicated that methane oxidizers affiliated to the ANME-1 and -2, may be capable of performing complete methane- and potentially short-chain alkane oxidation independently using oxidized sulfur and nitrogen compounds as terminal electron acceptors. Gammaproteobacteria are hypothetically capable of utilizing oxidized nitrogen compounds and may be involved in syntrophy with methane-oxidizing archaea. Carbonate structures represent a window for a more diverse utilization of electron acceptors for anaerobic methane oxidation along the Atlantic and Pacific Margin.

Predictors Associated with Health-Related Heat Risk Perception of Urban Citizens in Germany.

The rising probability of extremely high temperatures and an increasing number of consecutive hot days caused by climate change-combined with the impact of these high temperatures on human health-is widely discussed in the literature. There are calls for the development of heatwave adaptation measures by governmental and scientific institutions. In this research, the predictors of health-related heat risk perception of urban citizens in Augsburg, Germany, were investigated. An online survey was conducted with 468 citizens, asking about their heat risk perception, knowledge about heat risks, and demographic data and health information. Statistical methods (Spearman correlation, unpaired t-test, ANOVA and multiple regression) were used to determine which factors were significant and relevant. The results show that the knowledge of heat risks, heat risk sensitivity and an external locus of control are the most important factors for heat risk perception. The health implication score and chronic disease show significant effects in descriptive statistics. Furthermore, younger people showed the highest heat risk perception of all age groups. Surprisingly, income, education, living alone and gender did not play a role in heat risk perception. The findings imply a need for better and intensified heat risk communication in urban areas-especially among elderly people-and thus are important for creating acceptance towards heat wave risks, which is a prerequisite of willingness to adapt.

Constraining source attribution of methane in an alluvial aquifer with multiple recharge pathways.

Identifying the source of methane (CH4) in groundwater is often complicated due to various production, degradation and migration pathways, particularly in settings where there are multiple groundwater recharge pathways. This study demonstrates the ability to constrain the origin of CH4 within an alluvial aquifer that could be sourced from in situ microbiological production or underlying formations at depth. To characterise the hydrochemical and microbiological processes active within the alluvium, previously reported hydrochemical data (major ion chemistry and isotopic tracers (3H, 14C, 36Cl)) were interpreted in the context of CH4 and carbon dioxide (CO2) isotopic chemistry, and the microbial community composition in the groundwater. The rate of observed oxidation of CH4 within the aquifer was then characterised using a Rayleigh fractionation model. The stratification of the hydrochemical facies and microbiological community populations is interpreted to be a result of the gradational mixing of water from river leakage and floodwater recharge with water from basal artesian inflow. Within the aquifer there is a low abundance of methanogenic archaea indicating that there is limited biological potential for microbial CH4 production. Our results show that the resulting interconnection between hydrochemistry and microbial community composition affects the occurrence and oxidation of CH4 within the alluvial aquifer, constraining the source of CH4 in the groundwater to the geological formations beneath the alluvium.

Long-term succession in a coal seam microbiome during in situ biostimulation of coalbed-methane generation.

Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ. Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of 13C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.

Dependency of DNA extraction efficiency on cell concentration confounds molecular quantification of microorganisms in groundwater.

Quantification of microbes in water systems is essential to industrial practices ranging from drinking water and wastewater treatment to groundwater remediation. While quantification using DNA-based molecular methods is precise, the accuracy is dependent on DNA extraction efficiencies. We show that the DNA yield is strongly impacted by the cell concentration in groundwater samples (r = -0.92, P < 0.0001). This has major implications for industrial applications using quantitative polymerase chain reaction (qPCR) to determine cell concentrations in water, including bioremediation. We propose a simple normalization method using a DNA recovery ratio, calculated with the total cell count and DNA yield. Application of this method to enumeration of bacteria and archaea in groundwater samples targeting phylogenetic markers (16S rRNA) demonstrated an increased goodness of fit after normalization (7.04 vs 0.94 difference in Akaike's information criteria). Furthermore, normalization was applied to qPCR quantification of functional genes and combined with DNA sequencing of archaeal and bacterial 16S rRNA genes to monitor changes in abundance of methanogenic archaea and sulphate-reducing bacteria in groundwater. The integration of qPCR and DNA sequencing with appropriate normalization enables high-throughput quantification of microbial groups using increasingly affordable and accessible techniques. This research has implications for microbial ecology and engineering research as well as industrial practice.

Nutrient and acetate amendment leads to acetoclastic methane production and microbial community change in a non-producing Australian coal well.

Coal mining is responsible for 11% of total anthropogenic methane emission thereby contributing considerably to climate change. Attempts to harvest coalbed methane for energy production are challenged by relatively low methane concentrations. In this study, we investigated whether nutrient and acetate amendment of a non-producing sub-bituminous coal well could transform the system to a methane source. We tracked cell counts, methane production, acetate concentration and geochemical parameters for 25 months in one amended and one unamended coal well in Australia. Additionally, the microbial community was analysed with 16S rRNA gene amplicon sequencing at 17 and 25 months after amendment and complemented by metagenome sequencing at 25 months. We found that cell numbers increased rapidly from 3.0 × 104 cells ml-1 to 9.9 × 107 in the first 7 months after amendment. However, acetate depletion with concomitant methane production started only after 12-19 months. The microbial community was dominated by complex organic compound degraders (Anaerolineaceae, Rhodocyclaceae and Geobacter spp.), acetoclastic methanogens (Methanothrix spp.) and fungi (Agaricomycetes). Even though the microbial community had the functional potential to convert coal to methane, we observed no indication that coal was actually converted within the time frame of the study. Our results suggest that even though nutrient and acetate amendment stimulated relevant microbial species, it is not a sustainable way to transform non-producing coal wells into bioenergy factories.

Coat/Tether Interactions-Exception or Rule?

Coat complexes are important for cargo selection and vesicle formation. Recent evidence suggests that they may also be involved in vesicle targeting. Tethering factors, which form an initial bridge between vesicles and the target membrane, may bind to coat complexes. In this review, we ask whether these coat/tether interactions share some common mechanisms, or whether they are special adaptations to the needs of very specific transport steps. We compare recent findings in two multisubunit tethering complexes, the Dsl1 complex and the HOPS complex, and put them into context with the TRAPP I complex as a prominent example for coat/tether interactions. We explore where coat/tether interactions are found, compare their function and structure, and comment on a possible evolution from a common ancestor of coats and tethers.

Corrigendum: Coat/Tether Interactions-Exception or Rule?

[This corrects the article on p. 44 in vol. 4, PMID: 27243008.].

High-rate, High Temperature Acetotrophic Methanogenesis Governed by a Three Population Consortium in Anaerobic Bioreactors.

A combination of acetate oxidation and acetoclastic methanogenesis has been previously identified to enable high-rate methanogenesis at high temperatures (55 to 65°C), but this capability had not been linked to any key organisms. This study combined RNA-stable isotope probing on 13C-labelled acetate and 16S amplicon sequencing to identify the active micro-organisms involved in high-rate methanogenesis. Active biomass was harvested from three bench-scale thermophilic bioreactors treating waste activated sludge at 55, 60 and 65°C, and fed with 13-C labelled and 12C-unlabelled acetate. Acetate uptake and cumulative methane production were determined and kinetic parameters were estimated using model-based analysis. Pyrosequencing performed on 13C- enriched samples indicated that organisms accumulating labelled carbon were Coprothermobacter (all temperatures between 55 and 65°C), acetoclastic Methanosarcina (55 to 60°C) and hydrogenotrophic Methanothermobacter (60 to 65°C). The increased relative abundance of Coprothermobacter with increased temperature corresponding with a shift to syntrophic acetate oxidation identified this as a potentially key oxidiser. Methanosarcina likely acts as both a hydrogen utilising and acetoclastic methanogen at 55°C, and is replaced by Methanothermobacter as a hydrogen utiliser at higher temperatures.

N-Acetylglucosamine Inhibits LuxR, LasR and CviR Based Quorum Sensing Regulated Gene Expression Levels.

N-acetyl glucosamine, the monomer of chitin, is an abundant source of carbon and nitrogen in nature as it is the main component and breakdown product of many structural polymers. Some bacteria use N-acyl-L-homoserine lactone (AHL) mediated quorum sensing (QS) to regulate chitinase production in order to catalyze the cleavage of chitin polymers into water soluble N-acetyl-D-glucosamine (NAG) monomers. In this study, the impact of NAG on QS activities of LuxR, LasR, and CviR regulated gene expression was investigated by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50-80% reduction at low millimolar concentrations (0.25-5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this study shows that NAG down-regulates CviR induced violacein production while simultaneously up-regulating CviR dependent extracellular enzymes, suggesting that an unknown NAG dependent regulatory component influences phenotype expression. The quorum sensing inhibiting activity of NAG also adds to the list of compounds with known quorum sensing inhibiting activities.

Acetoclastic methane formation from Eucalyptus detritus in pristine hydrocarbon-rich river sediments by Methanosarcinales.

Pristine hydrocarbon-rich river sediments in the Greater Blue Mountains World Heritage Area (Australia) release substantial amounts of methane. The present study aimed to unravel for the first time the active methanogens mediating methane formation and exploiting the bacterial diversity potentially involved in the trophic network. Quantitative PCR of 16S rRNA gene and functional genes as well as 454 pyrosequencing were used to address the unknown microbial diversity and abundance. Methane-releasing sediment cores derived from three different river sites of the Tootie River. Highest methane production rates of 10.8 ± 0.5 µg g(-1)(wet weight) day(-1) were detected in 40 cm sediment depth being in congruence with the detection of the highest abundances of the archaeal 16S rRNA gene and the methyl-coenzyme M reductase (mcrA) genes. Stable carbon and hydrogen isotopic signatures of the produced methane indicated an acetoclastic origin. Long-term enrichment cultures amended with either acetate or H2/CO2 revealed acetoclastic methanogenesis as key methane-formation process mediated by members of the order Methanosarcinales. Conditions prevailing in the river sediments might be suitable for hydrocarbon-degrading bacteria observed in the river sediments that were previously unclassified or closely related to the Bacteroidetes/Chlorobi group, the Firmicutes and the Chloroflexi group fuelling acetoclastic methanogensis in pristine river sediments.

Acetate-utilizing bacteria at an oxic-anoxic interface in the Baltic Sea.

Pelagic redoxclines represent chemical gradients of elevated microbial activities. While chemolithoautotrophic microorganisms in these systems are well known as catalysts of major biogeochemical cycles, comparable knowledge on heterotrophic organisms is scarce. Thus, in this study, identity and biogeochemical involvement of active heterotrophs were investigated in stimulation experiments and activity measurements based on samples collected from pelagic redoxclines of the central Baltic Sea in 2005 and 2009. In the 2009 samples, (13)C-acetate 16S rRNA stable isotope probing (16S rRNA-SIP) identified gammaproteobacteria affiliated with Colwellia sp. and Neptunomonas sp. in addition to epsilonproteobacteria related to Arcobacter spp. as active heterotrophs at the oxic-anoxic interface layer. Incubations from sulfidic waters were dominated by two phylogenetic subgroups of Arcobacter. In the 2005 samples, organics, manganese(IV), and iron(III) were added to the sulfidic waters, followed by the determination of metal reduction and identification of the stimulated organisms. Here, the same Arcobacter and Colwellia subgroups were stimulated as in 2009, with Arcobacter predominating in samples, in which manganese(IV) reduction was highest. Our results offer new insights into the heterotrophic bacterial assemblage of Baltic Sea pelagic redoxclines and suggest Arcobacter spp. as a heterotroph with presumed relevance also for manganese cycling.

Impact of different in vitro electron donor/acceptor conditions on potential chemolithoautotrophic communities from marine pelagic redoxclines.

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO2 dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO2 dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO2 dark fixation rates, ranging from 0.7 to 1.4 micromol liter(-1) day(-1), were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO2 dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 micromol liter(-1) CO2 day(-1). Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 micromol liter(-1) NO3(-) day(-1). Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.