Acetate Activation in Methanosaeta thermophila

The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi) and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM=0.27±0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell

Extending the host range of Listeria monocytogenes by rational protein design

SummaryIn causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis

The effect of vertical ocean mixing on the tropical Atlantic in a coupled global climate model

Sea surface temperature (SST) biases in the tropical Atlantic are a long-standing problem among coupled global climate models (CGCMs). They occur in equilibrated state, as well as in initialised seasonal to decadal simulations. The bias is typically characterised by too high SST in upwelling regions and associated errors of wind and precipitation. We examine the SST bias in the state-of-the-art CGCM EC-Earth by means of an upper ocean heat budget analysis. Horizontal advection processes affect the SST bias development only to a small extent, and surface heat fluxes mostly dampen the warm bias. Subgrid-scale upper ocean vertical mixing is too low in EC-Earth when compared to estimates from reanalysis data, potentially giving rise to the warm bias. We perform sensitivity experiments to examine the effect of enhanced vertical mixing on the SST bias in quasi equilibrium present day climate and its impact on projected climate change. Enhanced mixing in historical simulation mode (MixUp pr) reduces the SST bias in the tropical Atlantic compared to the control experiment (Control pr). Associated atmospheric biases of precipitation and surface winds are also reduced in MixUp pr. We further perform climate projections under the RCP8.5 emission scenario (Control fu and MixUp fu). Under increasing greenhouse gas forcing, the tropical Atlantic warms by up to 4.5∘C locally, and maritime precipitation increases in boreal winter and spring. We show that the vertical mixing parameterisation influences future climate. In MixUp fu, SSTs remain 0.5∘C colder in boreal winter and spring, but increase with the same amplitude in summer and fall. The strength and location of the projected intertropical convergence zone also depends on the ocean vertical mixing efficiency. The rain band moves southward in summer, and its strength increases in winter in MixUp fu as compared to Control fu.</p

An EC-Earth coupled atmosphere–ocean single-column model (AOSCM.v1_EC-Earth3) for studying coupled marine and polar processes

Single-column models (SCMs) have been used as tools to help develop numerical weather prediction and global climate models for several decades. SCMs decouple small-scale processes from large-scale forcing, which allows the testing of physical parameterisations in a controlled environment with reduced computational cost. Typically, either the ocean, sea ice or atmosphere is fully modelled and assumptions have to be made regarding the boundary conditions from other subsystems, adding a potential source of error. Here, we present a fully coupled atmosphere–ocean SCM (AOSCM), which is based on the global climate model EC-Earth3. The initial configuration of the AOSCM consists of the Nucleus for European Modelling of the Ocean (NEMO3.6) (ocean), the Louvain-la-Neuve Sea Ice Model (LIM3) (sea ice), the Open Integrated Forecasting System (OpenIFS) cycle 40r1 (atmosphere), and OASIS3-MCT (coupler). Results from the AOSCM are presented at three locations: the tropical Atlantic, the midlatitude Pacific and the Arctic. At all three locations, in situ observations are available for comparison. We find that the coupled AOSCM can capture the observed atmospheric and oceanic evolution based on comparisons with buoy data, soundings and ship-based observations. The model evolution is sensitive to the initial conditions and forcing data imposed on the column. Comparing coupled and uncoupled configurations of the model can help disentangle model feedbacks. We demonstrate that the AOSCM in the current set-up is a valuable tool to advance our understanding in marine and polar boundary layer processes and the interactions between the individual components of the system (atmosphere, sea ice and ocean)

An EC-Earth coupled atmosphere–ocean single-column model (AOSCM.v1_EC-Earth3) for studying coupled marine and polar processes

Single-column models (SCMs) have been used as tools to help develop numerical weather prediction and global climate models for several decades. SCMs decouple small-scale processes from large-scale forcing, which allows the testing of physical parameterisations in a controlled environment with reduced computational cost. Typically, either the ocean, sea ice or atmosphere is fully modelled and assumptions have to be made regarding the boundary conditions from other subsystems, adding a potential source of error. Here, we present a fully coupled atmosphere–ocean SCM (AOSCM), which is based on the global climate model EC-Earth3. The initial configuration of the AOSCM consists of the Nucleus for European Modelling of the Ocean (NEMO3.6) (ocean), the Louvain-la-Neuve Sea Ice Model (LIM3) (sea ice), the Open Integrated Forecasting System (OpenIFS) cycle 40r1 (atmosphere), and OASIS3-MCT (coupler).Results from the AOSCM are presented at three locations: the tropical Atlantic, the midlatitude Pacific and the Arctic. At all three locations, in situ observations are available for comparison. We find that the coupled AOSCM can capture the observed atmospheric and oceanic evolution based on comparisons with buoy data, soundings and ship-based observations. The model evolution is sensitive to the initial conditions and forcing data imposed on the column. Comparing coupled and uncoupled configurations of the model can help disentangle model feedbacks. We demonstrate that the AOSCM in the current set-up is a valuable tool to advance our understanding in marine and polar boundary layer processes and the interactions between the individual components of the system (atmosphere, sea ice and ocean).</p

Role of wind stress in driving SST biases in the tropical Atlantic

Coupled climate models used for long-term future climate projections and seasonal or decadal predictions share a systematic and persistent warm sea surface temperature (SST) bias in the tropical Atlantic. This study attempts to better understand the physical mechanisms responsible for the development of systematic biases in the tropical Atlantic using the so-called Transpose-CMIP protocol in a multi-model context. Six global climate models have been used to perform seasonal forecasts starting both in May and February over the period 2000-2009. In all models, the growth of SST biases is rapid. Significant biases are seen in the first month of forecast and, by six months, the root-mean-square SST bias is 80% of the climatological bias. These control experiments show that the equatorial warm SST bias is not driven by surface heat flux biases in all models, whereas in the south-eastern Atlantic the solar heat flux could explain the setup of an initial warm bias in the first few days. A set of sensitivity experiments with prescribed wind stress confirm the leading role of wind stress biases in driving the equatorial SST bias, even if the amplitude of the SST bias is model dependent. A reduced SST bias leads to a reduced precipitation bias locally, but there is no robust remote effect on West African Monsoon rainfall. Over the south-eastern part of the basin, local wind biases tend to have an impact on the local SST bias (except in the high resolution model). However, there is also a non-local effect of equatorial wind correction in two models. This can be explained by sub-surface advection of water from the equator, which is colder when the bias in equatorial wind stress is corrected. In terms of variability, it is also shown that improving the mean state in the equatorial Atlantic leads to a beneficial intensification of the Bjerknes feedback loop. In conclusion, we show a robust effect of wind stress biases on tropical mean climate and variability in multiple climate models

The Genome Characteristics and Predicted Function of Methyl-Group Oxidation Pathway in the Obligate Aceticlastic Methanogens, Methanosaeta spp

In this work, we report the complete genome sequence of an obligate aceticlastic methanogen, Methanosaeta harundinacea 6Ac. Genome comparison indicated that the three cultured Methanosaeta spp., M. thermophila, M. concilii and M. harundinacea 6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from Methanosaeta strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of fwd and mch, genes involved in methyl-group oxidation. Moreover, 13C-labeling experiments suggested that the Methanosaeta strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the mch mutants of Methanosarcina barkeri or M. acetivorans failed to grow on acetate, we suggest that Methanosaeta may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An fpo operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the Methanosaeta strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F420H2 was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr), hdrABC, and hdrED, respectively, were found in the three Methanosaeta genomes. Quantitative PCR determined that the expression of hdrED was about ten times higher than hdrABC, suggesting that hdrED plays a major role in aceticlastic methanogenesis

5-Keto-D-Fructose, a Natural Diketone and Potential Sugar Substitute, Significantly Reduces the Viability of Prokaryotic and Eukaryotic Cells

5-Keto-D-fructose (5-KF) is a natural diketone occurring in micromolar concentrations in honey, white wine, and vinegar. The oxidation of D-fructose to 5-KF is catalyzed by the membrane-bound fructose dehydrogenase complex found in several acetic acid bacteria. Since 5-KF has a sweetening power comparable to fructose and is presumably calorie-free, there is great interest in making the diketone commercially available as a new sugar substitute. Based on a genetically modified variant of the acetic acid bacterium Gluconobacter oxydans 621H, an efficient process for the microbial production of 5-KF was recently developed. However, data on the toxicology of the compound are completely lacking to date. Therefore, this study aimed to investigate the effect of 5-KF on the viability of prokaryotic and eukaryotic cells. It was found that the compound significantly inhibited the growth of the gram-positive and gram-negative model organisms Bacillus subtilis and Escherichia coli in a concentration-dependent manner. Furthermore, cell viability assays confirmed severe cytotoxicity of 5-KF toward the colon cancer cell line HT-29. Since these effects already occurred at concentrations of 5 mM, the use of 5-KF in the food sector should be avoided. The studies performed revealed that in the presence of amines, 5-KF promoted a strong Maillard reaction. The inherent reactivity of 5-KF as well as the Maillard products formed could be the trigger for the observed inhibition of prokaryotic and eukaryotic cells

Role of nickel in high rate methanol degradation in anaerobic granular sludge bioreactors

The effect of nickel deprivation from the influent of a mesophilic (30°C) methanol fed upflow anaerobic sludge bed (UASB) reactor was investigated by coupling the reactor performance to the evolution of the Methanosarcina population of the bioreactor sludge. The reactor was operated at pH 7.0 and an organic loading rate (OLR) of 5–15 g COD l−1 day−1 for 191 days. A clear limitation of the specific methanogenic activity (SMA) on methanol due to the absence of nickel was observed after 129 days of bioreactor operation: the SMA of the sludge in medium with the complete trace metal solution except nickel amounted to 1.164 (±0.167) g CH4-COD g VSS−1 day−1 compared to 2.027 (±0.111) g CH4-COD g VSS−1 day−1 in a medium with the complete (including nickel) trace metal solution. The methanol removal efficiency during these 129 days was 99%, no volatile fatty acid (VFA) accumulation was observed and the size of the Methanosarcina population increased compared to the seed sludge. Continuation of the UASB reactor operation with the nickel limited sludge lead to incomplete methanol removal, and thus methanol accumulation in the reactor effluent from day 142 onwards. This methanol accumulation subsequently induced an increase of the acetogenic activity in the UASB reactor on day 160. On day 165, 77% of the methanol fed to the system was converted to acetate and the Methanosarcina population size had substantially decreased. Inclusion of 0.5 μM Ni (dosed as NiCl2) to the influent from day 165 onwards lead to the recovery of the methanol removal efficiency to 99% without VFA accumulation within 2 days of bioreactor operation