Biochemistry of hexose and pentose transformations in soil analyzed by position-specific labeling and 13C-PLFA

© 2014 Elsevier Ltd. Microbial transformations are key processes of soil organic matter (SOM) formation, stabilization and decomposition. Combination of position-specific 13C labeling with compound-specific 13C-PLFA analysis is a novel tool to trace metabolic pathways. This combination was used to analyze short-term transformations (3 and 10 days after tracer application) of two key monosaccharides: glucose and ribose in soil under field conditions. Transformations of sugars were quantified by the incorporation of 13C from individual molecule positions in bulk soil, microbial biomass (by CFE) and in cell membranes of microbial groups classified by 13C-PLFA.The 13C incorporation in the Gram negative bacteria was higher by one order of magnitude compared to all other microbial groups. All of the 13C recovered in soil on day 3 was allocated in microbial biomass. On day 10 however, a part of the 13C was recovered in non-extractable microbial cell components or microbial excretions. As sugars are not absorbed by mineral particles due to a lack of charged functional groups, their quick mineralization from soil solution is generally expected. However, microorganisms transformed sugars to metabolites with a slower turnover. The 13C incorporation from the individual glucose positions into soil and microbial biomass showed that the two main glucose utilizing pathways in organisms - glycolysis and the pentose phosphate pathway - exist in soils in parallel. However, the pattern of 13C incorporation from individual glucose positions into PLFAs showed intensive recycling of the added 13C via gluconeogenesis and a mixing of both glucose utilizing pathways. The pattern of position-specific incorporation of ribose C also shows initial utilization in the pentose phosphate pathway but is overprinted on day 10, again due to intensive recycling and mixing. This shows that glucose and ribose - as ubiquitous substrates - are used in various metabolic pathways and their C is intensively recycled in microbial biomass.Analyzing the fate of individual C atoms by position-specific labeling deeply improves our understanding of the pathways of microbial utilization of sugars (and other compounds) by microbial groups and so, of soil C fluxes

Nitrogen-inputs regulate microbial functional and genetic resistance and resilience to drying–rewetting cycles, with implications for crop yields

Background and aims The increasing input of anthropogenically-derived nitrogen (N) to ecosystems raises a crucial question: how do N inputs modify the soil microbial stability, and thus affect crop productivity? Methods Soils from an 8-year rice-wheat rotation experiment with increasing N-input rates were subjected to drying–rewetting (DW) cycles for investigating the resistance and resilience of soil functions, in terms of abundances of genes (potential functions) and activities of enzymes (quantifiable functions), to this stress, and particularly the contribution of resistance and resilience on crop production was evaluated. Results Although the DW cycles had a stronger effect compared to N fertilization level, the N input was also important in explaining the variation in the resistance and resilience of functional genes and the activities of enzymes involved in C, N and P cycling. Crop yields benefited from both of high resistance and high resilience of soil microbial functions, though the resistance and resilience of soil enzyme activities exhibited a stronger contribution to crop yields compared to the functional genes and the overall contribution strength was conditioned by N input levels. Conclusions In addition to the well-known direct contribution of N fertilization on crop yields, N input plays an indirect role on crop production via conditioning the resistance and resilience of soil functions in response to repeated DW cycles

Decreased rhizodeposition, but increased microbial carbon stabilization with soil depth down to 3.6 m

Despite the importance of subsoil carbon (C) deposition by deep-rooted crops in mitigating climate change and maintaining soil health, the quantification of root C input and its microbial utilization and stabilization below 1 m depth remains unexplored. We studied C input by three perennial deep-rooted plants (lucerne, kernza, and rosinweed) grown in a unique 4-m deep RootTower facility. 13C multiple pulse labeling was applied to trace C flows in roots, rhizodeposition, and soil as well as 13C incorporation into microbial groups by phospholipid fatty acids and the long-term stabilization of microbial residues by amino sugars. The ratio of rhizodeposited 13C in the PLFA and amino sugar pools was used to compare the relative microbial stability of rhizodeposited C across depths and plant species. Belowground C allocation between roots, rhizodeposits, and living and dead microorganisms indicated depth dependent plant investment. Rhizodeposition as a fraction of the total belowground C input declined from the topsoil (0–25 cm) to the deepest layer (360 cm), i.e., from 35%, 45%, and 36%–8.0%, 2.5%, and 2.7% for lucerne, kernza, and rosinweed, respectively, where lucerne had greater C input than the other species between 340 and 360 cm. The relative microbial stabilization of rhizodeposits in the subsoil across all species showed a dominance of recently assimilated C in microbial necromass, thus indicating a higher microbial stabilization of rhizodeposited C with depth. In conclusion, we traced photosynthates down to 3.6 m soil depth and showed that even relatively small C amounts allocated to deep soil layers will become microbially stabilized. Thus, deep-rooted crops, in particular lucerne are important for stabilization and storage of C over long time scales in deep soil

Wie beeinflusst Sorption die Metabolisierung von Alanin durch mikrobielle Gruppen?

Mikrobieller Abbau ist ein wesentlicher Transformationsprozess der organischen Bodensubstanz. Sorption von niedermolekularen organischen Substanzen wie Alanin an Mineraloberflächen kann diese vor dem mikrobiellen Abbau und Mineralisierung schützen. Somit wäre Sorption ein entscheidender Stabilisierungsprozess für die organische Bodensubstanz. Dennoch ist es für Mikroorganismen möglich, niedermolekulare organische Substanzen von Mineraloberflächen zu desorbieren. Das exakte Prozessgefüge dieser Desorption und mikrobiellen Verwertung, aber auch die involvierten mikrobiellen Gruppen sind jedoch weitestgehend unbekannt. Wir verwendeten positionsspezifisch C-13-markiertes Alanin, dessen Mineralisation zu CO2 und dessen Einbau in Phospholipid-Fettsäuren (PLFAs) quantifiziert wurde, um die mikrobielle Verwendung von sorbiertem und nicht-sorbiertem Alanin im Boden zu unterscheiden. Um die durch die Sorption bedingten Veränderungen der Verfügbarkeit und Metabolisierung des Alanins zu erfassen, wurden einheitlich- und positionsspezifisch C-13- und C-14-markiertes Alanin in einem Ap-Horizont einer lehmigen Parabraunerde über 10 Tage inkubiert. Das CO2 aus der Respiration wurde in NaOH-Fallen erfasst und dessen C-14-Aktivität bestimmt. Die Verwendung der funktionellen Gruppen des Alanins durch die verschiedenen mikrobiellen Gruppen wurde mittels C-13-PLFA Analyse ermittelt. Keine der mikrobiellen Gruppen bevorzugte das unsorbierte Alanin gegenüber dem sorbierten – der Großteil der Gruppen inkorporierte die gleiche Menge in ihre PLFAs. So zeigten beispielsweise Gram negative Bakterien eine hohe Wettbewerbsfähigkeit hinsichtlich des Alanins, allerdings keine Präferenz bezüglich des Unsorbierten. Nur die Gruppe der Pilze inkorporierten signifikant mehr sorbiertes Alanin: Sie sind prädestiniert dafür, Mineraloberfläche zu umwachsen und daran sorbierte Substanzen mit ihren Hyphen aufzunehmen. Diese Präferenz ist mit einem veränderten Abbauweg, der über Glukoneogenese und den Pentose-Phosphat-Weg führt, verbunden. Sorption kann zu einer kurzfristigen Verzögerung des Abbaus kleiner geladener Moleküle führen, jedoch ist kein langfristiger Stabilisierungseffekt erkennbar. Die meisten mikrobiellen Gruppen nehmen sowohl sorbiertes und nicht-sorbiertes Alanin im gleichen Maße auf und nur einzelne Gruppen, wie Pilze, haben sich auf die präferentielle Aufnahme sorbierter Substanzen spezialisiert

Microbial metabolism in soil at subzero temperatures: Adaptation mechanisms revealed by position-specific ¹³C labeling

© 2017 Bore, Apostel, Halicki, Kuzyakov and Dippold.Although biogeochemical models designed to simulate carbon (C) and nitrogen (N) dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically 13C-labeled glucose at three temperatures: +5 (control), -5, and -20°C. 13C was quantified in CO2, bulk soil, microbial biomass, and dissolved organic carbon (DOC) after 1, 3, and 10 days and also after 30 days for samples at -20°C. Compared to +5°C, CO2 decreased 3- and 10-fold at -5 and -20°C, respectively. High 13C recovery in CO2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5°C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis. A threefold higher 13C recovery in microbial biomass at -5 than +5°C points to synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Less than 0.4% of 13C was recovered in DOC after 1 day, demonstrating complete glucose uptake by microorganisms even at -20°C. Consequently, we attribute the fivefold higher extracellular 13C in soil than in microbial biomass to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. The knowledge of sustained metabolism at subzero temperatures will not only be useful for modeling global C dynamics in ecosystems with periodically or permanently frozen soils, but will also be important in understanding and controlling the adaptive mechanisms of food spoilage organisms

Detection of the DCC gene product in normal and malignant colorectal tissues and its relation to a codon 201 mutation.

Protein expression of the putative tumour-suppressor gene DCC on chromosome 18q was evaluated in a panel of 16 matched colorectal cancer and normal colonic tissue samples together with DCC mRNA expression and allelic deletions (loss of heterozygosity, LOH). Determined by a polymerase chain reaction (PCR)-LOH assay, 12 of the 16 (75%) cases were informative with LOH occurring in 2 of the 12 cases. For DCC mRNA, transcripts could be detected in all analysed normal tissues (eight out of eight) by RT-PCR, whereas 6 of the 15 tumours were negative. DCC protein expression, investigated by immunohistochemistry using the monoclonal antibody 15041 A directed against the intracellular domain, was homogeneously positive in all normal tissue samples. In tumour tissues, no DCC protein was seen in 11 out of 16 samples (69%). For the DCC codon 201, we found a loss of a wild-type codon sequence caused by mutation or LOH in at least 8 out of 15 cases (53%) compared with the corresponding normal tissue. DCC protein expression was undetectable in eight of the nine tumours missing both wild-type codons. Only one of the five tumours with retained DCC protein expression had no detectable wild-type codon 201. In addition, 9 out of 15 normal tissue specimens were mutated in codon 201. In two out of three cases with homozygous wild-type codons in peripheral blood lymphocyte (PBL) DNA, mutations were already observed in the tumour adjacent normal colonic mucosa. We conclude that DCC immunostaining should be introduced in the clinicopathological routine because of its strong correlation with the known prognostic markers 18q LOH and mutation of codon 201

Die unterschätzte Rolle des Recyclings intakter Metabolite für die Umsatzraten der organischen Bodensubstanz

Obwohl Sorption an Mineraloberflächen als dominierender Prozess zur Erklärung des langsamen Umsatzes mineralassoziierter organischer Bodensubstanz (OBS) dient, widerspricht diese Idee einer zunehmenden Zahl an Inkubationsstudien, die zeigen, dass für niedermolekulare Substanzen nicht nur die mikrobielle Aufnahme kompetitivier als die Sorption ist, sondern auch sorbierte Substanzen in hohem Maße desorbiert und mikrobiell verwertet werden können. Dabei wurde gezeigt, dass sich die Verstoffwechselung desorbierter Substanzen zugunsten eines erhöhten Recyclings verschiebt. Dies wirft die Frage auf, ob Recycling von intakten Metaboliten, d.h. unter Erhalt des Kohlenstoffgerüstes, generell ein bisher stark unterschätzter Prozess ist, der die relativ hohen 14C Alter der OBS teilweise erklären kann. Nach Applikation hoher Toxindosen konnte nachgewiesen werden, dass die nachfolgende Reetablierung der mikrobiellen Gemeinschaft zu großem Anteil auf Recycling der Nekromasskomponenten der Vorgeneration basiert. Intaktes Metabolitrecycling ist jedoch unter steady-state Bedingungen nur äußerst schwierig von der direkten Stabiliserung zu unterscheiden. Um diesen Prozess im Fließgleichgewicht nachzuweisen muss 1) ein Biomolekül untersucht werden, dessen Biosyntheseweg so aufwändig ist, dass Recycling einen deutlichen Vorteil für die Zelle im Vergleich zur Neusynthese darstellt, 2) dieses Biomolkül in lebenden Zellen in einer anderen Form gebunden sein, als die zu recycelnde Einheit in der Bodenlösung, so dass beide Zustandsformen unterschieden werden können und 3) dieses Biomolekül positionsspezifisch isotopenmarkiert zugegeben werden, so dass über einen identischen Einbau der Positionen die Intaktheit des Kohlenstoffgerüstes nachgewiesen werden kann. Am Beispiel der Alkylketten von Fettsäuren, die in mikrobiellen Zellen primär als Phospholipide in den Membranen gebunden sind, soll dieses Prinzip veranschaulicht werden. Eine erste Abschätzung des intakten Recyclings von Alkylketten durch Mikroorganismen in Böden zeigt, dass von den 0.03% der basierend auf Alkyl-Kohlenstoff neugebildeten PLFA mehr als 75% aus intaktem Recycling dieser Ketten hervor gingen. Obwohl der Beitrag des Recyclings intakter Metabolite zur Umsatzzeit der gesamten OBS aufgrund der geringen Anzahl bisher untersuchter Metabolite noch nicht final quantifiziert werden kann, untermauern die hier vorgestellten Ergebnisse jedoch die hohe Relevanz dieses Prozesses für die Dynamik der OBS

Fluphenazine reduces proteotoxicity in C. elegans and mammalian models of alpha-1-antitrypsin deficiency

The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. Elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo . Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models. © 2014 Li et al

Nitrogen Gain and Loss Along an Ecosystem Sequence: From Semi-desert to Rainforest

Plants and microorganisms, besides the climate, drive nitrogen (N) cycling in ecosystems. Our objective was to investigate N losses and N acquisition strategies along a unique ecosystem-sequence (ecosequence) ranging from arid shrubland through Mediterranean woodland to temperate rainforest. These ecosystems differ in mean annual precipitation, mean annual temperate, and vegetation cover, but developed on similar granitoid soil parent material, were addressed using a combination of molecular biology and soil biogeochemical tools. Soil N and carbon (C) contents, δ15N signatures, activities of N acquiring extracellular enzymes as well as the abundance of soil bacteria and fungi, and diazotrophs in bulk topsoil and rhizosphere were determined. Relative fungal abundance in the rhizosphere was higher under woodland and forest than under shrubland. This indicates toward plants' higher C investment into fungi in the Mediterranean and temperate rainforest sites than in the arid site. Fungi are likely to decompose lignified forest litter for efficient recycling of litter-derived N and further nutrients. Rhizosphere—a hotspot for the N fixation—was enriched in diazotrophs (factor 8 to 16 in comparison to bulk topsoil) emphasizing the general importance of root/microbe association in N cycle. These results show that the temperate rainforest is an N acquiring ecosystem, whereas N in the arid shrubland is strongly recycled. Simultaneously, the strongest 15N enrichment with decreasing N content with depth was detected in the Mediterranean woodland, indicating that N mineralization and loss is highest (and likely the fastest) in the woodland across the continental transect. Higher relative aminopeptidase activities in the woodland than in the forest enabled a fast N mineralization. Relative aminopeptidase activities were highest in the arid shrubland. The highest absolute chitinase activities were observed in the forest. This likely demonstrates that (a) plants and microorganisms in the arid shrubland invest largely into mobilization and reutilization of organically bound N by exoenzymes, and (b) that the ecosystem N nutrition shifts from a peptide-based N in the arid shrubland to a peptide- and chitin-based N nutrition in the temperate rainforest, where the high N demand is complemented by intensive N fixation in the rhizosphere

Einfluss räumlicher Heterogenität von Phosphor auf die mikrobielle P‑Aufnahme und die Zusammensetzung der mikrobiellen Gemeinschaft in Waldböden

Neben Stickstoff ist Phosphor (P) das wichtigste wachstumslimitierende Nährelement in Böden. Dennoch gibt es wenig Information über die räumliche Heterogenität des P-Gehaltes in Waldböden. Darüber hinaus ist der Effekt einer homogenen versus heterogenen P‑Verteilung im Boden auf die mikrobielle P‑Akquirierung und Zusammensetzung der mikrobiellen Gemeinschaft weitgehend unbekannt. Ein Rhizotronexperiment mit P-armem Waldboden wurde durchgeführt um konkurrierende P‑Aufnahmestrategien von Mikroorganismen zu untersuchen. Um den Effekt räumlicher P‑Heterogenität auf pflanzliche und mikrobielle P‑Aufnahme zu eruieren wurden mit F.sylvatica bepflanzte Rhizotrone mit P‑33‑Eisen(III)phosphat, einer relativ immobilen P Quelle, in verschiedenen räumlichen Verteilungen markiert. Die P‑Mobilisierung durch Mikroorganismen wurde mittels einer verbesserten P-33-PLFA-Methode verfolgt, welche die P‑33‑Inkorporierung in Mikroorganismen mit Änderungen in der Zusammensetzung mikrobieller Gemeinschaften in situ verbindet. Die mikrobielle P-Aufnahme war erhöht in Rhizotronen mit hoher P‑Verfügbarkeit, sowie in solchen mit heterogener P‑Verteilung. Charakteristische PLFA weisen auf eine Akkumulation von Ektomykorrhizapilzen, typischerweise assoziiert mit Buchenwurzeln, in P‑reichen Arealen hin. Diese Ektomykorrhizzapilze führen wahrscheinlich zu einer starken Zunahme der P‑Mobilisierung des ausgebrachten P‑33‑Eisen(III)phosphats in stark P-haltigen Habitaten. Im Gegensatz hierzu benötigen Habitate mit niedriger P-Verfügbarkeit eine komplexer zusammengesetzte mikrobielle Gemeinschaft um unzugängliche P-Quellen zu mobilisieren. Entsprechend fördern hohe P‑Vorkommen die Bildung von Pilzhyphen zur P-Mobilisierung – ein Effekt, der mit sinkendem P-Gehalt abnimmt. Des Weiteren zeigen grampositive und ‑negative Bakterien eine massiv erhöhte P‑Aufnahme unter zunehmend heterogenen P-Verteilungen. Sie stellen jedoch einen kleineren Anteil der mikrobiellen Gemeinschaft als in homogen P‑angereicherten Rhizotronen, was auf einen Vorteil filamentöser Organismen bei heterogener P-Verteilung hindeutet. Entsprechend fördert eine heterogene P-Verteilung in Waldböden die P-Aufnahme mikrobieller Gemeinschaften aus mineralischen P-Quellen mit geringer biologischer Verfügbarkeit in Waldböden