Stable Hydrogen and Carbon Isotope Fractionation during Microbial Toluene Degradation: Mechanistic and Environmental Aspects

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d₈ and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d₈ and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = −1.219), Desulfobacterium cetonicum (b = −1.196), Thauera aromatica (b = −0.816), and Geobacter metallireducens (b = −1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = −2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the ¹³C/¹²C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d₈ and nonlabeled toluene for sulfate-reducing strain TRM1 (b = −0.728) and D. cetonicum (b = −0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37°C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20°C (b = −4.086) and minimum fractionation at 35°C (b = −2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d₅ in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)⁻¹. The D/H isotope fractionation (b = −1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.     

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.     

Sulfur and Oxygen Isotope Fractionation During Bacterial Sulfur Disproportionation Under Anaerobic Haloalkaline Conditions

Sulfur and oxygen isotope fractionation of elemental sulfur disproportionation at anaerobic haloalkaline conditions was evaluated for the first time. Isotope enrichment factors of the strains Desulfurivibrio alkaliphilus and Dethiobacter alkaliphilus growing at pH 9 or 10 were ?0.9‰ to ?1‰ for sulfide (³⁴?), +3.6‰ to +4.7‰ for sulfate (³⁴?), and +3.5‰ to +7.7‰ for oxygen in sulfate (¹⁸?). These values are significantly smaller compared to previously published values of sulfur disproportionators at neutral pH. We propose that this discrepancy is caused by masking effects due to preferential formation of polysulfides at high pH leading to accelerated internal sulfur turnover rates, but cannot rule out distinct isotope effects due to specific enzymatic disproportionation reactions under haloalkaline conditions. The results imply that the microbial sulfur cycle in haloalkaline environments is characterized by specific stable sulfur and oxygen isotope patterns.     

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

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Expression of the Dominant-Negative Tail of Myosin Va Enhances Exocytosis of Large Dense Core Vesicles in Neurons

Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of myosin Va in this process, neurons were transfected with the dominant-negative tail domain of myosin Va (myosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This value was doubled to 1.80% of the vesicles when myosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and myosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by myosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium–calmodulin-dependent kinase II. We suggest that myosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Carbon Isotope Fractionation during Anaerobic Degradation of Methyl tert-Butyl Ether under Sulfate-Reducing and Methanogenic Conditions

Methyl tert-butyl ether (MTBE), an octane enhancer and a fuel oxygenate in reformulated gasoline, has received increasing public attention after it was detected as a major contaminant of water resources. Although several techniques have been developed to remediate MTBE-contaminated sites, the fate of MTBE is mainly dependent upon natural degradation processes. Compound-specific stable isotope analysis has been proposed as a tool to distinguish the loss of MTBE due to biodegradation from other physical processes. Although MTBE is highly recalcitrant, anaerobic degradation has been demonstrated under different anoxic conditions and may be an important process. To accurately assess in situ MTBE degradation through carbon isotope analysis, carbon isotope fractionation during MTBE degradation by different cultures under different electron-accepting conditions needs to be investigated. In this study, carbon isotope fractionation during MTBE degradation under sulfate-reducing and methanogenic conditions was studied in anaerobic cultures enriched from two different sediments. Significant enrichment of ¹³C in residual MTBE during anaerobic biotransformation was observed under both sulfate-reducing and methanogenic conditions. The isotopic enrichment factors () estimated for each enrichment were almost identical (−13.4 to −14.6; r² = 0.89 to 0.99). A value of −14.4 ± 0.7 was obtained from regression analysis (r² = 0.97, n = 55, 95% confidence interval), when all data from our MTBE-transforming anaerobic cultures were combined. The similar magnitude of carbon isotope fractionation in all enrichments regardless of culture or electron-accepting condition suggests that the terminal electron-accepting process may not significantly affect carbon isotope fractionation during anaerobic MTBE degradation.     

Novel clades of soil biphenyl degraders revealed by integrating isotope probing,multi-omics,and single-cell analyses

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used ¹³C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of ¹³C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade {"type":"entrez-protein","attrs":{"text":"UBA11222","term_id":"2096922005"}}UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.Subject terms: Microbial ecology, Biogeochemistry, Biogeochemistry, Soil microbiology, Biogeochemistry     

Stable Isotope Fractionation of Tetrachloroethene during Reductive Dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. Strain PCE-S and Abiotic Reactions with Cyanocobalamin

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B₁₂) was studied. Fractionation was largest during the reaction with cyanocobalamin with αC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (αC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (αC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, αC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.     

Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B12) was studied. Fractionation was largest during the reaction with cyanocobalamin with alphaC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (alphaC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (alphaC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, alphaC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.