Biology subjects

Kopf, S.

Publications and source records attributed to Kopf, S..

7 recordsLinked to original sources

bioRxiv2026

Lipid Hydrogen Stable Isotope Probing Reveals Decadal-Scale Generation Times for Archaea in Hot Spring Sediments

Quantifying the lipid biosynthesis rate of archaea in hot spring sediments is necessary to estimate their generation times and environmental significance, with implications for modern biogeochemical cycling and astrobiology. Here, we performed lipid hydrogen stable isotope probing (LH-SIP) experiments on sediments collected from two high-temperature, suboxic, circumneutral hot springs in Yellowstone National Park (USA) and El Tatio Geyserfield (Chile). We determined the incorporation rate of 2H2O into intact polar lipids (IPLs) during short-duration, ex situ incubations, which provides a taxon- and metabolism-agnostic quantification of biosynthesis under near-natural conditions. We targeted isoprenoid glycerol dialkyl glycerol tetraether lipids (iGDGTs) and recovered structures with 0 to 7 cyclopentyl moieties from both springs. We observed minor 2H-uptake into archaeal IPL-iGDGT-derived biphytanes in spring sediments in Yellowstone, corresponding to decadal-scale apparent generation times (16 +/- 3 years), and no detectable uptake in El Tatio sediments, where lipid turnover was slower than could be resolved under the experimental conditions (maximum resolvable apparent generation times ~ 42 +/- 21 years). We conclude that these slow apparent generation times likely reflect substantial dilution by a large standing IPL pool and/or recycling or re-functionalization of relict lipid components, rather than equivalently slow rates of archaeal cell division. These are the first estimates of archaeal lipid production rates in terrestrial hydrothermal systems using LH-SIP incubations and provide critical constraints for interpreting archaeal lipids in modern systems and ancient hot spring deposits.

microbiology↗

bioRxiv2024

Lipid hydrogen isotope compositions primarily reflect growth water in the model archaeon Sulfolobus acidocaldarius

The stable hydrogen isotope composition ({delta}2H) of lipid biomarkers can track environmental processes and remain stable over geologically relevant time scales, enabling studies of past climate, hydrology, and ecology. Most research has focused on lipids from the domain Eukarya (e.g., plant waxes, long-chain alkanes), and the potential of prokaryotic lipid biomarkers from the domain Archaea to offer unique insights into environments not captured by eukaryotic lipids remains unclear. Here, we investigate the H-isotope composition of biphytanes in Sulfolobus acidocaldarius, a model thermoacidophile and obligate heterotroph. We conducted a series of experiments that varied temperature, pH, shaking rate, electron acceptor availability, or electron donor flux. From these experiments, we quantified the lipid/water H-isotope fractionation (2{varepsilon}L/W) values for core biphytane chains derived from tetraether lipids. The 2{varepsilon}L/W values are consistently negative (-230 to - 180 {per thousand}) and are relatively invariant across all experiments despite a 20-fold change in doubling times and 2-fold change in lipid cyclization. The magnitude and relative invariance of 2{varepsilon}L/W values are consistent with studies on other heterotrophic archaea and suggests archaeal lipids may be faithful recorders of the {delta}2H composition of growth water. Our study highlights the potential of archaeal lipid {delta}2H as a hydrological proxy, offering new insights into environments where traditional proxies, such as plant-derived lipids, are not available, including extreme environments and extraterrestrial settings. ImportanceReconstructing past climates is crucial for understanding Earths environmental history and its responses to changing conditions. This study examines Sulfolobus acidocaldarius, a thermoacidophilic archaeon that thrives in extreme environments like hot springs. These microorganisms incorporate hydrogen water in the growth environment into membrane lipids, creating hydrogen isotope signatures that can reflect hydroclimate conditions. Our findings show that these hydrogen isotope ratios remain consistent even under varying temperature, pH, oxygen levels, and electron donor fluxes, indicating a stable fractionation between lipids and water. This invariance suggests that S. acidocaldarius lipids could serve as a robust proxy for reconstructing ancient water H-isotope values, especially in extreme environments where traditional proxies, such as plant waxes, are absent. This research has broader implications for planetary-scale reconstructions, including potential applications in studying past climates on other planets, such as Mars, where similar microorganisms may have existed in hydrothermal conditions.

microbiology↗

bioRxiv2023

Quantitative measurement of microbial growth rate with Raman microspectroscopy

Rates of microbial activity and growth are fundamental to understanding environmental geochemistry and ecology. However, measuring the heterogeneity of microbial activity at the single-cell level, especially within complex populations and environmental matrices, remains a forefront challenge. Stable Isotope Probing (SIP) is a standard method for assessing microbial activity and involves measuring the incorporation of an isotopically labeled compound into microbial biomass. Here, we assess the utility of Raman microspectroscopy as a SIP technique, specifically focusing on the measurement of deuterium (2H), a tracer of microbial biomass production. We generate calibrations of microbial biomass 2H values and find that Raman microspectroscopy reliably quantifies 2H incorporation ranging between 0 and 40 at. %. Applying the results of this calibration to a SIP model, we explicitly parameterize the factors controlling microbial growth quantification, demonstrating how Raman-SIP can measure the growth of microorganisms with doubling times ranging from hours to years. Furthermore, we correlatively compare our Raman-derived measurements with those of nanoscale secondary ion mass spectrometry (nanoSIMS) to compare the relative strengths of nanoSIMS- and Raman-based SIP approaches. We find that Raman microspectroscopy is a robust, accessible methodology that can readily differentiate and quantify the growth of individual microbial cells in complex samples. ImportanceGrowth rate, the rate at which organisms grow and reproduce, is a key metric with which to evaluate microbial physiology and contributions to system-level processes. The heterogeneity of microbial growth across space, time, and populations is often difficult to capture with bulk-scale techniques. Single-cell methods hold promise for measuring the heterogeneity of microbial growth rates and responses to changing conditions in situ, without the need for cultivation of microbial isolates. In this study, we evaluated the ability of Raman microspectroscopy, a non-destructive and rapid technique, to measure the assimilation of isotopically labeled water into individual microbial cells and thereby calculate their rates of growth. We explicitly parameterize the factors controlling the quantification of microbial growth rate and compare this technique to standard methods. The framework we report allows researchers to couple single-cell and aggregate rate measurements to functional or system-level properties, a forefront challenge in microbiology.

microbiology↗

bioRxiv2023

The hydrogen isotope signatures of autotrophy versus heterotrophy recorded in archaeal tetraether lipids

The stable hydrogen isotope composition of archaeal lipids is emerging as a potential paleoenvironmental proxy, adding to the well-established application of plant leaf wax-derived n-alkanes in paleohydrological reconstruction. A handful of studies reported relatively invariant and depleted hydrogen isotope compositions for archaeal lipids despite the range of different organisms and growth conditions explored. However, how modes of metabolism and physiological state (growth phase) affect the hydrogen isotope signatures of archaeal lipids remains poorly understood, limiting our ability to interpret archaeal lipid biomarker records from the environment. Here we conducted water isotope label experiments with a metabolically flexible and well-studied model archaeon Archaeoglobus fulgidus and quantified the hydrogen isotope fractionation between lipids and water in response to different carbon substrates and electron donor-acceptor pairs. The 2H/1H fractionation between lipids and water ({varepsilon}L/W) was overall negative, ranging from -280 to -226 {per thousand}, and overlapped with the ranges observed for other archaea in previous studies. Isotope flux-balance model results suggest that [≥]80 % and [≥]50 % of lipid-bound H in A. fulgidus cultures directly reflect water isotope compositions (i.e., not via organic substrate or H2) during autotrophy and heterotrophy, respectively. The model results also suggest the final saturation during isoprenoid lipid biosynthesis catalyzed by a flavoenzyme geranylgeranyl reductase likely contributes to the large 2H/1H fractionation observed in this study, consistent with previous isotope flux-balance model results for a different archaeon. Finally, we synthesized available data to compare {varepsilon}L/W patterns across all three domains of life: Bacteria, Archaea and Eukarya. Emerging patterns between archaeal and eukaryotic lipids are consistent with the notion of highly fractionating geranylgeranyl reductase, and the patterns between archaeal and bacterial lipids suggest that the general state of energy limitation may also contribute to large, negative values of {varepsilon}L/W observed in prokaryotic lipids. Altogether, these findings lend further support for the potential of archaeal lipid {varepsilon}L/W as a paleohydrological proxy and provide a broader insight into the 2H/1H fractionation mechanisms potentially shared among prokaryotic and eukaryotic lipid biomarkers.

microbiology↗

bioRxiv2022

Hydrogen stable isotope probing of lipids demonstrates slow rates of microbial growth in soil

The rate at which microorganisms grow and reproduce is fundamental to our understanding of microbial physiology and ecology. While soil microbiologists routinely quantify soil microbial biomass levels and the growth rates of individual taxa in culture, there is a limited understanding of how quickly microbes actually grow in soil. For this work, we posed the simple question: what are the growth rates of soil microorganisms? In this study, we measure these rates in three distinct soil environments using hydrogen stable isotope probing of lipids with 2H-enriched water. This technique provides a taxa-agnostic quantification of in situ microbial growth from the degree of 2H enrichment of intact polar lipid compounds ascribed to bacteria and fungi. We find that average apparent generation times in soil are quite slow (20 to 64 days) but also highly variable at the compound-specific level (6 to 1137 days), suggesting differential growth rates between community subsets. We observe that low-biomass communities can exhibit more rapid growth rates than high-biomass communities, highlighting that biomass quantity alone does not predict microbial productivity in soil. Furthermore, within a given soil, the rates at which specific lipids are being synthesized do not relate to their quantity, suggesting a general decoupling of microbial abundance and growth in soil microbiomes. More generally, we demonstrate the utility of lipid stable isotope probing for measuring microbial growth rates in soil and highlight the importance of measuring growth rates to complement more standard analyses of soil microbial communities. SignificanceGeneration times, how quickly organisms grow and reproduce, are a key feature of biology. However, there are few measurements of microbial generation times in soil, despite the crucial importance of soil microbes to terrestrial ecosystems. By measuring the rate at which isotopically labeled water is incorporated into microbial membranes, we find that the generation times of soil microorganisms are far longer than those typically observed in culture. Surprisingly, we observe that lower-biomass soils exhibited faster growth rates than high-biomass soils. More abundant microorganisms are not necessarily the fastest growing and most soil microorganisms are slow growers. Our results underscore the importance of considering slow and variable growth rates when studying microbial communities and their contributions to ecosystem processes.

microbiology↗

bioRxiv2021

Reversal of the renal hyperglycemic memory by targeting sustained tubular p21 expression

A major therapeutic obstacle in diabetes mellitus is the metabolic or hyperglycemic memory: the persistence of impaired organ function despite improvement of blood glucose. Therapies reversing the hyperglycemic memory and thus improving already established organ-dysfunction are lacking, but urgently needed considering the increasing prevalence of diabetes mellitus worldwide. Here we show that glucose-mediated changes in gene expression largely persist in diabetic kidney disease (DKD) despite reversing hyperglycemia. The senescence-associated cyclin-dependent kinase inhibitor p21 (Cdkn1a) was the top hit among genes persistently induced by hyperglycemia and was associated with sustained induction of the p53-p21 pathway. Persistent p21 induction was confirmed in various animal models, in several independent human samples and in in vitro models. Tubular p21 expression and urinary p21-levels were associated with DKD severity and remained elevated despite improved blood glucose levels in humans, suggesting that p21 may be a biomarker indicating persistent ("memorized") kidney damage. Glucose-mediated p21 induction and tubular senescence were enhanced in mice with reduced levels of the disease resolving protease activated protein C (aPC). Mechanistically, glucose-induced and sustained tubular p21 expression is linked with demethylation of its promoter and reduced DNMT1 expression. aPC reverses already established p21 expression independent of its anticoagulant function through receptor signaling. Accordingly, new pharmacological approaches specifically mimicking aPC signaling (3K3A-aPC, parmodulin-2) enabled the reversal of glucose-mediated sustained tubular p21 expression, tubular senescence, and DKD. Thus, p21-dependent tubular senescence contributes to the hyperglycemic memory but can be therapeutically targeted. Single sentence summaryaPC signaling targets persistent p21 expression and tubular senescence and reverses the hyperglycemic memory in diabetic kidney disease.

molecular biology↗

bioRxiv2018

Refining the application of microbial lipids as tracers of Staphylococcus aureus growth rates in cystic fibrosis sputum

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effect of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. Microbial community composition was measured by NanoString DNA analysis and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two different anteiso fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae and hence their isotopic enrichment was interpreted as a growth proxy for S. aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients, thus deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small non-microbial background source detected in some patient samples, a-C15:0 fatty acid still appear to be a relatively robust proxy for CF pathogens, revealing a median generation time of ~1.5 days, similar to prior observations. Contrary to our expectations, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two best practices for application of stable isotope probing in CF sputum: (1) parallel determination of microbial community composition in CF sputum using culture-independent tools, and (2) analysis of samples with a minimum a-C15:0 concentration of 0.1 weight percent of saturated fatty acids.\n\nIMPORTANCEIn chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop an new workflow for the application of stable isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

microbiology↗