Biology subjects

Buck, M.

Publications and source records attributed to Buck, M..

61 records · Page 4Linked to original sources

bioRxiv2018

Mercury methylating microbial communities of boreal forest soils

The formation of the potent neurotoxic methylmercury (MeHg) is a microbially mediated process that has raised much concern because MeHg poses threats to wildlife and human health. Since boreal forest soils can be a source of MeHg in aquatic networks, it is crucial to understand the biogeochemical processes involved in the formation of this pollutant. High-throughput sequencing of 16S rRNA and the mercury methyltransferase, hgcA, combined with geochemical characterisation of soils, were used to determine the microbial populations contributing to MeHg formation in forest soils across Sweden. The hgcA sequences obtained were distributed among diverse clades, including Proteobacteria, Firmicutes, and Methanomicrobia, with Deltaproteobacteria, particularly Geobacteraceae, dominating the libraries across all soils examined. Our results also suggest that MeHg formation is linked to the composition of also non-mercury methylating bacterial communities, likely providing growth substrate (e.g. acetate) for the hgcA-carrying microorganisms responsible for the actual methylation process. While previous research focused on mercury methylating microbial communities of wetlands, this study provides some first insights into the diversity of mercury methylating microorganisms in boreal forest soils.\n\nImportanceDespite a global state of awareness that mercury, and methylmercury in particular, is a neurotoxin that millions of people continue to be exposed to, there are sizable gaps in our fundamental understanding of the processes and organisms involved in methylmercury formation. In the present study we shed light on the diversity of the microorganisms responsible for methylmercury formation in boreal forest soils. All the microorganisms identified have a relevant role on the processing of organic matter in soils. Moreover, our results show that the formation of methylation formation is not only linked to mercury methylating microorganisms but also to the presence of non-mercury methylating bacterial communities that contribute to methylmercury formation by the appropriate substrate to the microorganisms responsible for the actual methylation process. This study improves current knowledge on the diversity of organisms involved in methylmercury formation in soils.

microbiology↗

bioRxiv2018

Pattern, Pathways and Dynamics of EphA2 SAM and SHIP2 SAM Heterodimer Association Revealed by All-Atom Molecular Dynamics Simulation

Native protein-protein interactions (PPIs) are the cornerstone for understanding the structure, dynamics and mechanisms of function of protein complexes. In this study, we investigate the association of the SAM domains of the EphA2 receptor and SHIP2 enzyme by performing a combined total of 48 s all-atom molecular dynamics (MD) simulations. While the native SAM heterodimer is only predicted at a low rate of 6.7% with the original CHARMM36 force field, the yield is increased to 16.7% and to 18.3% by scaling the vdW solute-solvent interactions (better fitting the solvation free energy of amino acid side chain analogues) and by an increase of vdW radius of guanidinium interactions, and thus a dramatic reduction of electrostatic interaction between Arg and Glu/Asn in CHARMM36m, respectively. These modifications effectively improve the overly sticky association of proteins, such as ubiquitin, using the original potential function. By analyzing the 25 native SAM complexes formed in the simulations, we find that their formation involves a pre-orientation guided by electrostatic interaction, consistent with an electrostatic steering mechanism. The complex could then transform to the native protein interaction surfaces directly from a well pre-orientated position ({Delta}interface-RMSD < 5[A]). In other cases, modest (< 90{degrees}) orientational and/or translational adjustments are needed (5 [A] <{Delta}i-RMSD <10 [A]) to the native complex. Although the tendency for non-native complexes to dissociate has nearly doubled with the modified potential functions, a re-association to the correct complex structure is still rare. Instead a most non-native complexes are undergoing configurational changes/surface searching, which do not lead to native structures on a timescale of 250 ns. These observations provide a rich picture on mechanisms of protein-protein complex formation, and suggest that computational predictions of native complex protein-protein interactions could be improved further.

biophysics↗

bioRxiv2017

A "Tug of War" maintains a dynamic protein-membrane complex: MD simulations of C-Raf RBD-CRD bound to K-Ras4B at an anionic membrane

Association of Raf kinase with activated Ras triggers downstream signaling cascades, towards regulating transcription in the cells nucleus. Dysregulation of Ras: Raf signaling stimulates cancers. We investigate the C-Raf RBD and CRD regions when bound to oncogenic K-Ras4B at the membrane. All-atom molecular dynamics simulations suggest that the membrane plays an integral role in regulating the configurational ensemble of the complex. Remarkably, the complex samples a few states dynamically, reflecting a competition between C-Raf CRD and K-Ras4B- membrane interactions. This competition arises because the interaction between the RBD and K-Ras is strong and the linker between the RBD and CRD is short. This study reveals a mechanism that maintains a modest binding for the overall complex at the membrane to facilitate fast signaling processes. It is likely a common mechanism for other multi-protein, if not multidomain proteins at membranes.

biophysics↗

bioRxiv2017

The RNA-binding site of poliovirus 3C protein doubles as a phosphoinositide-binding domain

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using NMR spectroscopy. The PIP-binding site was located on a highly dynamic -helix that also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses.

biochemistry↗

bioRxiv2017

Vitamin and amino acid auxotrophy in anaerobic consortia operating under methanogenic condition

Syntrophy among Archaea and Bacteria facilitates the anaerobic degradation of organic compounds to CH4 and CO2. Particularly during aliphatic and aromatic hydrocarbon mineralization, as in crude oil reservoirs and petroleum-contaminated sediments, metabolic interactions between obligate mutualistic microbial partners are of central importance1. Using micro-manipulation combined with shotgun metagenomic approaches, we disentangled the genomes of complex consortia inside a short chain alkane-degrading cultures operating under methanogenic conditions. Metabolic reconstruction revealed that only a small fraction of genes in the metagenome-assembled genomes of this study, encode the capacity for fermentation of alkanes facilitated by energy conservation linked to H2 metabolism. Instead, inferred lifestyles based on scavenging anabolic products and intermediate fermentation products derived from detrital biomass was a common feature in the consortia. Additionally, inferred auxotrophy for vitamins and amino acids suggests that the hydrocarbon-degrading microbial assemblages are structured and maintained by multiple interactions beyond the canonical H2-producing and syntrophic alkane degrader-methanogen partnership2. Our study uncovers the complexity of interactomes within microbial consortia mediating hydrocarbon transformation under anaerobic conditions.

microbiology↗

bioRxiv2017

GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation

Ammonium assimilation in E. coli is regulated by two paralogous proteins (GlnB and GlnK), which orchestrate interactions with regulators of gene expression, transport proteins and metabolic pathways. Yet how they conjointly modulate the activity of glutamine synthetase (GS), the key enzyme for nitrogen assimilation, is poorly understood. We combine experiments and theory to study the dynamic roles of GlnB and GlnK during nitrogen starvation and upshift. We measure time-resolved in vivo concentrations of metabolites, total and post-translationally modified proteins, and develop a concise biochemical model of GlnB and GlnK that incorporates competition for active and allosteric sites, as well as functional sequestration of GlnK. The model predicts the responses of GS, GlnB and GlnK under time-varying external ammonium level in the wild type and two genetic knock-outs. Our results show that GlnK is tightly regulated under nitrogen-rich conditions, yet it is expressed during ammonium run-out and starvation. This suggests a role for GlnK as a buffer of nitrogen shock after starvation, and provides a further functional link between nitrogen and carbon metabolisms.

systems biology↗

bioRxiv2017

Model communities hint to promiscuous metabolic linkages between ubiquitous free-living freshwater bacteria

Free-living microorganisms with streamlined genomes are very abundant in the environment. Genome streamlining results in losses in the cells biosynthetic potential generating physiological dependencies between microorganisms. However, there exists no consensus on the specificity of these microbial associations. To verify specificity and extent of these associations, mixed cultures were established from three different freshwater environments. These cultures contained free-living streamlined organisms lacking multiple biosynthetic pathways. Among the co-occurring members of the mixed cultures, there was no clear recurring pattern of metabolic complementarity and dependencies. This, together with weak temporal co-occurrence patterns observed using time-series metagenomics, suggests that free-living freshwater bacteria form loose and unspecific cooperative loops. Comparative genomics suggests that the proportion of accessory genes in populations of streamlined bacteria allows for flexibility in interaction partners. Altogether this renders these free-living bacterial lineages functionally versatile despite their streamlining tendencies.

microbiology↗