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

2004 ◽  
Vol 70 (5) ◽  
pp. 2935-2940 ◽  
Author(s):  
Barbara Morasch ◽  
Hans H. Richnow ◽  
Andrea Vieth ◽  
Bernhard Schink ◽  
Rainer U. Meckenstock
Keyword(s):  
Stable Isotope ◽  
Carbon Isotope ◽  
Isotope Fractionation ◽  
Toluene Degradation ◽  
Fractionation Factor ◽  
Glycyl Radical ◽  
Stable Isotope Fractionation ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme

ABSTRACT 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 (ε) 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 ε (εintrinsic) were calculated. A comparison of εintrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific ε elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of ε found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average ε if no fractionation factor is available for single compounds.

scholarly journals Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway

2015 ◽  
Vol 81 (18) ◽  
pp. 6241-6252 ◽  
Author(s):  
P. M. Martínez-Lavanchy ◽  
Z. Chen ◽  
V. Lünsmann ◽  
V. Marin-Cevada ◽  
R. Vilchez-Vargas ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Fractionation ◽  
Fixed Bed ◽  
Model Systems ◽  
Rrna Gene ◽  
Toluene Degradation ◽  
Content Type ◽  
Stable Isotope Fractionation ◽  
Catabolic Genes ◽  
Fractionation Analysis

ABSTRACTIn the present study, microbial toluene degradation in controlled constructed wetland model systems, planted fixed-bed reactors (PFRs), was queried with DNA-based methods in combination with stable isotope fractionation analysis and characterization of toluene-degrading microbial isolates. Two PFR replicates were operated with toluene as the sole external carbon and electron source for 2 years. The bulk redox conditions in these systems were hypoxic to anoxic. The autochthonous bacterial communities, as analyzed by Illumina sequencing of 16S rRNA gene amplicons, were mainly comprised of the familiesXanthomonadaceae,Comamonadaceae, andBurkholderiaceae, plusRhodospirillaceaein one of the PFR replicates. DNA microarray analyses of the catabolic potentials for aromatic compound degradation suggested the presence of the ring monooxygenation pathway in both systems, as well as the anaerobic toluene pathway in the PFR replicate with a high abundance ofRhodospirillaceae. The presence of catabolic genes encoding the ring monooxygenation pathway was verified by quantitative PCR analysis, utilizing the obtained toluene-degrading isolates as references. Stable isotope fractionation analysis showed low-level of carbon fractionation and only minimal hydrogen fractionation in both PFRs, which matches the fractionation signatures of monooxygenation and dioxygenation. In combination with the results of the DNA-based analyses, this suggests that toluene degradation occurs predominantly via ring monooxygenation in the PFRs.

Evaluation of Toluene Degradation Pathways by Two-Dimensional Stable Isotope Fractionation

2008 ◽  
Vol 42 (21) ◽  
pp. 7793-7800 ◽  
Author(s):  
Carsten Vogt ◽  
Esther Cyrus ◽  
Ilka Herklotz ◽  
Dietmar Schlosser ◽  
Arne Bahr ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Fractionation ◽  
Degradation Pathways ◽  
Two Dimensional ◽  
Toluene Degradation ◽  
Stable Isotope Fractionation

scholarly journals Structure and Function of Benzylsuccinate Synthase and Related Fumarate-Adding Glycyl Radical Enzymes

2016 ◽  
Vol 26 (1-3) ◽  
pp. 29-44 ◽  
Author(s):  
Johann Heider ◽  
Maciej Szaleniec ◽  
Berta M. Martins ◽  
Deniz Seyhan ◽  
Wolfgang Buckel ◽  
...  
Keyword(s):  
Anaerobic Metabolism ◽  
Biochemical Properties ◽  
Initial Structure ◽  
Toluene Degradation ◽  
Methyl Group ◽  
Glycyl Radical ◽  
Benzylsuccinate Synthase ◽  
And Function ◽  
Glycyl Radical Enzyme ◽  
Radical Type

The pathway of anaerobic toluene degradation is initiated by a remarkable radical-type enantiospecific addition of the chemically inert methyl group to the double bond of a fumarate cosubstrate to yield <i>(R)</i>-benzylsuccinate as the first intermediate, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. In recent years, it has become clear that benzylsuccinate synthase is the prototype enzyme of a much larger family of fumarate-adding enzymes, which play important roles in the anaerobic metabolism of further aromatic and even aliphatic hydrocarbons. We present an overview on the biochemical properties of benzylsuccinate synthase, as well as its recently solved structure, and present the results of an initial structure-based modeling study on the reaction mechanism. Moreover, we compare the structure of benzylsuccinate synthase with those predicted for different clades of fumarate-adding enzymes, in particular the paralogous enzymes converting <i>p</i>-cresol, 2-methylnaphthalene or <i>n</i>-alkanes.

scholarly journals Improved quantification of microbial CH<sub>4</sub> oxidation efficiency in Arctic wetland soils using carbon isotope fractionation

2012 ◽  
Vol 9 (12) ◽  
pp. 16999-17035 ◽  
Author(s):  
I. Preuss ◽  
C. Knoblauch ◽  
J. Gebert ◽  
E.-M. Pfeiffer
Keyword(s):  
Stable Isotope ◽  
Isotope Fractionation ◽  
Wetland Soils ◽  
Tundra Soils ◽  
Ch4 Oxidation ◽  
Stable Isotope Fractionation ◽  
Oxidation Efficiency ◽  
Water Saturated ◽  
Fractionation Factors

Abstract. Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the Arctic will cause a deeper permafrost thawing followed by increased carbon mineralization and CH4 formation in water saturated tundra soils which might cause a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River Delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and δ13CH4-signatures were measured and the fractionation factors for the processes of oxidation (αox) and diffusion (αdiff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (e.g. landfill cover soils) have assumed a gas transport dominated by advection (αtrans = 1). In tundra soils, however, diffusion is the main gas transport mechanism, aside from ebullition. Hence, diffusive stable isotope fractionation has to be considered. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an αdiff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was αdiff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that αox differs widely between sites and horizons (mean αox, = 1.017 ± 0.009) and needs to be determined individually. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged organic rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost affected ecosystems and their potential strengths in response to global warming.

scholarly journals Silicon uptake and isotope fractionation dynamics by crop species

2020 ◽  
Vol 17 (24) ◽  
pp. 6475-6490
Author(s):  
Daniel A. Frick ◽  
Rainer Remus ◽  
Michael Sommer ◽  
Jürgen Augustin ◽  
Danuta Kaczorek ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Diffusion Process ◽  
Silicic Acid ◽  
Biogenic Silica ◽  
Crop Production ◽  
Isotope Fractionation ◽  
Root Uptake ◽  
Fractionation Factor ◽  
Stable Isotope Fractionation ◽  
Silicon Uptake

Abstract. That silicon is an important element in global biogeochemical cycles is widely recognised. Recently, its relevance for global crop production has gained increasing attention in light of possible deficits in plant-available Si in soil. Silicon is beneficial for plant growth and is taken up in considerable amounts by crops like rice or wheat. However, plants differ in the way they take up silicic acid from soil solution, with some species rejecting silicic acid while others actively incorporate it. Yet because the processes governing Si uptake and regulation are not fully understood, these classifications are subject to intense debate. To gain a new perspective on the processes involved, we investigated the dependence of silicon stable isotope fractionation on silicon uptake strategy, transpiration, water use, and Si transfer efficiency. Crop plants with rejective (tomato, Solanum lycopersicum, and mustard, Sinapis alba) and active (spring wheat, Triticum aestivum) Si uptake were hydroponically grown for 6 weeks. Using inductively coupled plasma mass spectrometry, the silicon concentration and isotopic composition of the nutrient solution, the roots, and the shoots were determined. We found that measured Si uptake does not correlate with the amount of transpired water and is thus distinct from Si incorporation expected for unspecific passive uptake. We interpret this lack of correlation to indicate a highly selective Si uptake mechanism. All three species preferentially incorporated light 28Si, with a fractionation factor 1000×ln (α) of −0.33 ‰ (tomato), −0.55 ‰ (mustard), and −0.43 ‰ (wheat) between growth medium and bulk plant. Thus, even though the rates of active and passive Si root uptake differ, the physico-chemical processes governing Si uptake and stable isotope fractionation do not. We suggest that isotope fractionation during root uptake is governed by a diffusion process. In contrast, the transport of silicic acid from the roots to the shoots depends on the amount of silicon previously precipitated in the roots and the presence of active transporters in the root endodermis, facilitating Si transport into the shoots. Plants with significant biogenic silica precipitation in roots (mustard and wheat) preferentially transport silicon depleted in 28Si into their shoots. If biogenic silica is not precipitated in the roots, Si transport is dominated by a diffusion process, and hence light silicon 28Si is preferentially transported into the tomato shoots. This stable Si isotope fingerprinting of the processes that transfer biogenic silica between the roots and shoots has the potential to track Si availability and recycling in soils and to provide a monitor for efficient use of plant-available Si in agricultural production.

scholarly journals Improved quantification of microbial CH<sub>4</sub> oxidation efficiency in arctic wetland soils using carbon isotope fractionation

2013 ◽  
Vol 10 (4) ◽  
pp. 2539-2552 ◽  
Author(s):  
I. Preuss ◽  
C. Knoblauch ◽  
J. Gebert ◽  
E.-M. Pfeiffer
Keyword(s):  
Stable Isotope ◽  
Isotope Fractionation ◽  
Wetland Soils ◽  
Tundra Soils ◽  
Ch4 Oxidation ◽  
Stable Isotope Fractionation ◽  
Oxidation Efficiency ◽  
Water Saturated ◽  
Fractionation Factors

Abstract. Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the arctic will cause deeper permafrost thawing, followed by increased carbon mineralization and CH4 formation in water-saturated tundra soils, thus creating a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and δ13CH4 signatures were measured and the fractionation factors for the processes of oxidation (αox) and diffusion (αdiff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (such as landfill cover soils) have assumed a gas transport dominated by advection (αtrans = 1). In tundra soils, however, diffusion is the main gas transport mechanism and diffusive stable isotope fractionation should be considered alongside oxidative fractionation. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an αdiff = 1.001 &amp;pm; 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was αdiff = 1.013 &amp;pm; 0.003 (n = 18). Furthermore, it was found that αox differs widely between sites and horizons (mean αox = 1.017 ± 0.009) and needs to be determined on a case by case basis. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged, organic-rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic–aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost-affected ecosystems and their potential strengths in response to global warming.

scholarly journals Benzylsuccinate Synthase is Post-Transcriptionally Regulated in the Toluene-Degrading Denitrifier Magnetospirillum sp. Strain 15-1

2020 ◽  
Vol 8 (5) ◽  
pp. 681 ◽  
Author(s):  
Ingrid Meyer-Cifuentes ◽  
Sylvie Gruhl ◽  
Sven-Bastiaan Haange ◽  
Vanessa Lünsmann ◽  
Nico Jehmlich ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Toluene Degradation ◽  
Regulation Of Expression ◽  
Glycyl Radical ◽  
Copy Numbers ◽  
Catalytically Active ◽  
Oxygen Sensitive ◽  
Benzylsuccinate Synthase ◽  
Glycyl Radical Enzyme ◽  
In Cells

The facultative denitrifying alphaproteobacterium Magnetospirillum sp. strain 15-1 had been isolated from the hypoxic rhizosphere of a constructed wetland model fed with toluene. This bacterium can catabolize toluene anaerobically but not aerobically. Here, we used strain 15-1 to investigate regulation of expression of the highly oxygen-sensitive glycyl radical enzyme benzylsuccinate synthase, which catalyzes the first step in anaerobic toluene degradation. In cells growing aerobically with benzoate, the addition of toluene resulted in a ~20-fold increased transcription of bssA, encoding for the catalytically active subunit of the enzyme. Under anoxic conditions, bssA mRNA copy numbers were up to 129-fold higher in cells growing with toluene as compared to cells growing with benzoate. Proteomics showed that abundance of benzylsuccinate synthase increased in cells growing anaerobically with toluene. In contrast, peptides of this enzyme were never detected in oxic conditions. These findings show that synthesis of benzylsuccinate synthase was under stringent post-transcriptional control in the presence of oxygen, which is a novel level of regulation for glycyl radical enzymes.

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

2001 ◽  
Vol 67 (10) ◽  
pp. 4842-4849 ◽  
Author(s):  
Barbara Morasch ◽  
Hans H. Richnow ◽  
Bernhard Schink ◽  
Rainer U. Meckenstock
Keyword(s):  
Carbon Isotope ◽  
Hydrogen Isotope ◽  
Isotope Fractionation ◽  
Aerobic Bacterium ◽  
Toluene Degradation ◽  
Geobacter Metallireducens ◽  
Carbon Isotope Fractionation ◽  
Sulfate Reducing ◽  
Cell Extracts ◽  
Benzylsuccinate Synthase

ABSTRACT 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-d 8 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-d 8 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 putidamt-2 (b = −2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the13C/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-d 8 and nonlabeled toluene for sulfate-reducing strain TRM1 (b = −0.728) andD. 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-d 5 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.

scholarly journals Silicon isotope fractionation and uptake dynamics of three crop plants: laboratory studies with transient silicon concentrations

2020 ◽  
Author(s):  
Daniel A. Frick ◽  
Rainer Remus ◽  
Michael Sommer ◽  
Jürgen Augustin ◽  
Friedhelm von Blanckenburg
Keyword(s):  
Stable Isotope ◽  
Plant Growth ◽  
Diffusion Process ◽  
Silicic Acid ◽  
Isotope Fractionation ◽  
Crop Plants ◽  
Transfer Efficiency ◽  
Silicon Isotope ◽  
Fractionation Factor ◽  
Stable Isotope Fractionation

Abstract. Silicon has been recognized an important element in global biogeochemical cycles for a long time. Recently, its relevance for global crop production gains increasing attention. Silicon is beneficial for plant growth and is taken up in considerable amounts by crops, likewise rice or wheat. The incorporation of silicic acid from the soil solution into the plants is accomplished by a variety of strategies (rejective, passive and active) that are subject to an intense debate. To forge a new perspective on the underlying processes, we investigated how the silicon stable isotope fractionation during plant growth depends on uptake strategy, transpiration, water use, and Si transfer efficiency. Crop plants with a rejective (tomato, Solanum lycopersicum and mustard, Sinapis alba) and active (spring wheat, Triticum aestivum) uptake were hydroponically grown for 6 weeks. Using inductively coupled plasma mass spectrometry, the silicon amounts and the isotopic composition of the nutrient solution, the roots, and the shoots were determined. Wheat revealed the highest Si transfer efficiency from root to shoot followed by tomato and mustard. All three species preferentially incorporated light 28Si, with a fractionation factor 1000∙ln(α) of −0.33 ‰ (tomato), −0.55 ‰ (mustard) and −0.43 ‰ (wheat). Even though the rates of active and passive Si root uptake differ, the physico-chemical processes governing Si uptake and stable isotope fractionation do not, they are governed by a diffusion process. In contrast, the transport of silicic acid from the roots to the shoots depends on the preceding precipitation of silicic acid in the roots and the presence of active transporters at the root endodermis. Plants with a significant biogenic silica precipitation in roots (mustard, and wheat), preferentially transport silicon enriched in 30Si into their shoots, whereas the transport in tomato is governed by a diffusion process and hence preferentially transports light silicon 28Si into the shoots.

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