Protein-based stable isotope probing

We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.     

Metagenomic stable isotope probing reveals bacteriophage participation in soil carbon cycling

Soil viruses are important components of the carbon (C) cycle, yet we still know little about viral ecology in soils. We added diverse ¹³C-labelled carbon sources to soil and we used metagenomic-SIP to detect ¹³C assimilation by viruses and their putative bacterial hosts. These data allowed us to link a ¹³C-labelled bacteriophage to its ¹³C-labelled Streptomyces putative host, and we used qPCR to track the dynamics of the putative host and phage in response to C inputs. Following C addition, putative host numbers increased rapidly for 3 days, and then more gradually, reaching maximal abundance on Day 6. Viral abundance and virus:host ratio increased dramatically over 6 days, and remained high thereafter (8.42 ± 2.94). From Days 6 to 30, virus:host ratio remained high, while putative host numbers declined more than 50%. Putative host populations were ¹³C-labelled on Days 3–30, while ¹³C-labelling of phage was detected on Days 14 and 30. This dynamic suggests rapid growth and ¹³C-labelling of the host fueled by new C inputs, followed by extensive host mortality driven by phage lysis. These findings indicate that the viral shunt promotes microbial turnover in soil following new C inputs, thereby altering microbial community dynamics, and facilitating soil organic matter production.     

Monitoring of microbial proteome dynamics using Raman stable isotope probing

Abnormal protein kinetics could be a cause of several diseases associated with essential life processes. An accurate understanding of protein dynamics and turnover is essential for developing diagnostic or therapeutic tools to monitor these changes. Raman spectroscopy in combination with stable isotope probes (SIP) such as carbon-13, and deuterium has been a breakthrough in the qualitative and quantitative study of various metabolites. In this work, we are reporting the utility of Raman-SIP for monitoring dynamic changes in the proteome at the community level. We have used ¹³C-labeled glucose as the only carbon source in the medium and verified its incorporation in the microbial biomass in a time-dependent manner. A visible redshift in the Raman spectral vibrations of major biomolecules such as nucleic acids, phenylalanine, tyrosine, amide I, and amide III were observed. Temporal changes in the intensity of these bands demonstrating the feasibility of protein turnover monitoring were also verified. Kanamycin, a protein synthesis inhibitor was used to assess the feasibility of identifying effects on protein turnover in the cells. Successful application of this work can provide an alternate/adjunct tool for monitoring proteome-level changes in an objective and nondestructive manner.     

Identification of anthracene degraders in leachate-contaminated aquifer using stable isotope probing

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in landfill leachate-contaminated aquifer. It is necessary to identify the microorganisms truly responsible for PAH degradation if bioremediation can be applied as an effective technology. DNA-based stable isotope probing (SIP) in combination with terminal restriction fragment length polymorphism (TRFLP) was used to identify the active anthracene degraders in the contaminated aquifer sediment. One kind of degrader was classified as Variovorax species within class ??-proteobacteria, but another belonged to unclassified bacteria. These findings also suggest novel microorganisms involved in PAH-degrading processes.     

Detecting active methanogenic populations on rice roots using stable isotope probing

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/(13CO2) or N2/(13CO2) with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system.     

Unlocking the 'microbial black box' using RNA-based stable isotope probing technologies

Microbial ecologists have long sought to associate the transformation of compounds in the environment with the microbial clades responsible. The development of stable isotope probing (SIP) has made this possible in many ecological and biotechnological contexts. RNA-based SIP technologies represent a significant leap forward for culture-independent 'functional phylogeny' analyses, where specific consumption of a given compound carrying a (13)C signature can be associated with the small subunit ribosomal RNA molecules of the microbes that consume it. Recent advances have led to the unequivocal identification of microorganisms responsible for contaminant degradation in engineered systems, and to applications enhancing our understanding of carbon flow in terrestrial ecosystems.     

Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing

Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [(13)C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.     

Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing

Arctic lakes are a significant source of the greenhouse gas methane (CH₄), but the role that methane oxidizing bacteria (methanotrophs) play in limiting the overall CH₄ flux is poorly understood. Here, we used stable isotope probing (SIP) techniques to identify the metabolically active aerobic methanotrophs in upper sediments (0–1 cm) from an arctic lake in northern Alaska sampled during ice‐free summer conditions. The highest CH₄ oxidation potential was observed in the upper sediment (0–1 cm depth) with 1.59 µmol g wet weight^?1 day^?1 compared with the deeper sediment samples (1–3 cm, 3–5 cm and 5–10 cm), which exhibited CH₄ oxidation potentials below 0.4 µmol g wet weight^?1 day^?1. Both type I and type II methanotrophs were directly detected in the upper sediment total communities using targeted primer sets based on 16S rRNA genes. Sequencing of 16S rRNA genes and functional genes (pmoA and mxaF) in the ¹³C‐DNA from the upper sediment indicated that type I methanotrophs, mainly Methylobacter, Methylosoma, Methylomonas and Methylovulum miyakonense, dominated the assimilation of CH₄. Methylotrophs, including the genera Methylophilus and/or Methylotenera, were also abundant in the ¹³C‐DNA. Our results show that a diverse microbial consortium acquired carbon from CH₄ in the sediments of this arctic lake.     

Alterations in anaerobic ammonium oxidation of paddy soil following organic carbon treatment estimated using 13C-DNA stable isotope probing

In this study, soil samples from the typical rice-wheat cropping system in Jiangsu Province, China, subjected to different fertilizer application treatments―no carbon (CK), urea (UR), straw (SR), pig manure (PM), starch (ST), and glucose (GL)―were used to determine potential anaerobic ammonium oxidation (anammox) rate and its association with bacterial abundance, diversity, and activity by using DNA stable isotope probing combined with ¹⁵N isotope tracing and molecular techniques. The effects of different organic carbon sources on anammox were significant, in the following order: GL > ST, SR > UR > PM; anammox activity differed significantly across treatments; however, the ¹³C active anammox bacteria were only closely related to Ca. Brocadia. The anammox hydrazine synthase β subunit functional gene sequences were highly associated with the Candidatus genus Brocadia in PM and CK treatments. The different organic carbon sources had different inhibitory effects with anammox rate, which dropped from 3.19 to 1.04 nmol dinitrogen gas g⁻¹ dry soil h⁻¹ among treatments. About 4.2–22.3% of dinitrogen gas emissions were attributed to anammox and indicated that a specific population of anammox bacteria was present and varied with the addition of exogenous organic compounds in paddy soils, although a small part of dinitrogen gas was emitted from the soil via anammox.

     

Functional analysis of an anaerobic m-xylene-degrading enrichment culture using protein-based stable isotope probing

A sulfate-reducing consortium maintained for several years in the laboratory with m-xylene as sole source of carbon and energy was characterized by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of PCR-amplified 16S rRNA genes and stable isotope probing of proteins (Protein-SIP). During growth upon m-xylene or methyl-labeled m-xylene (1,3-dimethyl-(13)C(2)-benzene), a phylotype affiliated to the family Desulfobacteriaceae became most abundant. A second dominant phylotype was affiliated to the phylum Epsilonproteobacteria. In cultures grown with methyl-labeled m-xylene, 331 proteins were identified by LC-MS/MS analysis. These proteins were either not (13)C-labeled (23%) or showed a (13)C-incorporation of 19-22 atom% (13)C (77%), the latter demonstrating that methyl groups of m-xylene were assimilated. (13)C-labeled proteins were involved in anaerobic m-xylene biodegradation, in sulfate reduction, in the Wood-Ljungdahl-pathway, and in general housekeeping functions. Thirty-eight percent of the labeled proteins were affiliated to Deltaproteobacteria. Probably due to a lack of sequence data from Epsilonproteobacteria, only 14 proteins were assigned to this phylum. Our data suggest that m-xylene is assimilated by the Desulfobacteriaceae phylotype, whereas the role of the Epsilonproteobacterium in the consortium remained unclear.     

Identification of triclosan-degrading bacteria in a triclosan enrichment culture using stable isotope probing

Triclosan, a widely used antimicrobial agent, is an emerging contaminant in the environment. Despite its antimicrobial character, biodegradation of triclosan has been observed in pure cultures, soils and activated sludge. However, little is known about the microorganisms responsible for the degradation in mixed cultures. In this study, active triclosan degraders in a triclosan-degrading enrichment culture were identified using stable isotope probing (SIP) with universally ¹³C-labeled triclosan. Eleven clones contributed from active microorganisms capable of uptake the ¹³C in triclosan were identified. None of these clones were similar to known triclosan-degraders/utilizers. These clones distributed among α-, β-, or γ-Proteobacteria: one belonging to Defluvibacter (α-Proteobacteria), seven belonging to Alicycliphilus (β-Proteobacteria), and three belonging to Stenotrophomonas (γ-Proteobacteria). Successive additions of triclosan caused a significant shift in the microbial community structure of the enrichment culture, with dominant ribotypes belonging to the genera Alicycliphilus and Defluvibacter. Application of SIP has successfully identified diverse uncultivable triclosan-degrading microorganisms in an activated sludge enrichment culture. The results of this study not only contributed to our understanding of the microbial ecology of triclosan biodegradation in wastewater, but also suggested that triclosan degraders are more phylogenetically diverse than previously reported.     

Assimilation of benzene carbon through multiple trophic levels traced by different stable isotope probing methodologies

The flow of benzene carbon along a food chain consisting of bacteria and eukaryotes, including larvae (Diptera: Chironomidae), was evaluated by total lipid fatty acids (TLFAs)-, amino acid- and protein-stable isotope probing (SIP). A coconut-fibre textile, colonized by a benzene-degrading biofilm, was sampled in a system established for the remediation of benzene, toluene, ethylbenzene and xylenes (BTEX)-polluted groundwater and incubated with (12)C- and [(13)C(6)]-benzene (>99 at.%) in a batch-scale experiment for 2-8 days. After 8 days, Chironomus sp. larvae were added to study carbon flow to higher trophic levels. Gas chromatography-combustion-isotope ratio monitoring mass spectrometry of TLFA showed increased isotope ratios in the (13)C-benzene-incubated biofilm. A higher (13)C-enrichment was observed in TLFAs, indicative of Gram-negative bacteria than for Gram-positive. Fatty acid indicators of eukaryotes showed significant (13)C-incorporation, but to a lower extent than bacterial indicators. Fatty acids extracted from larvae feeding on (13)C-biofilm reached an isotopic ratio of 1.55 at.%, illustrating that the larvae feed, to some extent, on labelled biomass. No (13)C-incorporation was detectable in larval proteins after their separation by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis and analysis by nano-liquid-chromatography-mass spectrometry. The flow of benzene-derived carbon could be traced in a food web consisting of bacteria and eukaryotes.     

Water uptake by plants: perspectives from stable isotope composition

Stable isotope studies of hydrogen and oxygen stable isotope ratios of water within plants are providing new information on water sources, competitive interactions and water use patterns under natural conditions. Variation in the utilization of summer rain by aridland species and limited use of stream water by mature riparian trees are two examples of how stable isotope studies have modified our understanding of plant water relations. Analyses of xylem sap and tree rings have the potential of providing both short-term and long-term information on plant water use patterns.     

新一代高通量测序与稳定性同位素示踪DNA/RNA技术研究稻田红壤甲烷氧化的微生物过程

[目的]利用新一代高通量测序技术分析复杂土壤环境中整体微生物群落结构的变化规律,研究特定功能微生物生理过程的分子机制;利用稳定性同位素示踪微生物核酸DNA/RNA,研究复杂土壤中关键元素转化的微生物调控机制.[方法]针对我国第四纪红色粘土母质发育的3种稻田红壤,围绕13C-甲烷好氧氧化的微生物过程,在DNA和RNA水平高通量测序土壤微生物群落16S rRNA基因和16S rRNA,通过超高速密度梯度离心土壤微生物总核酸获得13C-标记的DNA/RNA,进一步采用克隆文库技术研究稻田红壤甲烷好氧氧化的微生物作用者.[结果]新一代高通量测序结果表明,3种稻田红壤甲烷的好氧氧化过程中,甲烷好氧氧化菌占土壤整体微生物群落的丰度显著增加,RNA水平的增幅显著高于DNA水平,能够更为灵敏地反映土壤甲烷好氧氧化的微生物过程.3种稻田红壤甲烷的好氧氧化过程中,类型Ⅰ和类型Ⅱ甲烷好氧氧化菌在湖南古市土壤中显著增加,湖南桃源土壤中类型Ⅱ甲烷好氧氧化菌增加明显,而类型Ⅰ甲烷好氧氧化菌在广东雷州土壤中增幅最大.进一步利用13C-DNA和13C-RNA分别构建pmoA基因和16S rRNA克隆文库,发现类型Ⅰ甲烷好氧氧化菌主导了湖南古市和广东雷州稻田红壤甲烷的好氧氧化过程,类型Ⅱ甲烷好氧氧化菌主导了湖南桃源稻田红壤甲烷的好氧氧化过程.[结论]新一代高通量测序技术能够在整体微生物群落水平,清楚反映复杂土壤中特定功能微生物的生理生态过程,而RNA较DNA水平的分析更为灵敏;稳定性同位素示踪微生物核酸DNA/RNA技术能够准确地揭示复杂土壤重要过程的微生物作用者.     

Identifying low pH active and lactate-utilizing taxa within oral microbiome communities from healthy children using stable isotope probing techniques

Background

Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized in vitro, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for in vitro temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species.

Methodology/Principal Findings

Supragingival plaque samples from caries-free children incubated with ¹³C-substrates under imposed healthy (buffered, pH 7) and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic acid from glucose metabolism. Rapid lactate utilization upon glucose depletion was observed under pH 7 conditions. SIP analyses revealed a number of genera containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5. The diversity of active species decreased significantly at pH 4.5 and was dominated by Lactobacillus and Propionibacterium species, both of which have been previously found within carious lesions from children.

Conclusions/Significance

Our approach allowed for identification of species that metabolize carbohydrates under different pH conditions and supports the importance of Lactobacilli and Propionibacterium in the development of childhood caries. Identification of species within healthy subjects that are active at low pH can lead to a better understanding of oral caries onset and generate appropriate targets for preventative measures in the early stages.     

定量稳定性同位素探针技术及其在微生物生态学研究中的应用

定量稳定性同位素探针技术(qSIP)是将生态系统中微生物分类性状与代谢功能联系起来的有效工具,能够定量测定特定环境中单个微生物类群暴露于同位素示踪剂后微生物代谢活动或生长速率.qSIP技术采用定量PCR与高通量测序技术并结合稳定同位素探针技术(SIP),通过向环境样品添加标记底物进行培养,提取微生物生物标记物,利用超高...     

DNA buoyant density shifts during 15N-DNA stable isotope probing

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from ¹⁵N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of ¹⁵N into DNA increased BD by 0.015±0.002 g mL⁻¹ for E. coli and 0.013±0.002 g mL⁻¹ for M. luteus. The DNA BD shift was greatly increased (0.045 g mL⁻¹) when dual isotope (¹³C plus ¹⁵N) labeling was employed. Despite the limited DNA BD shift following ¹⁵N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.     

稳定同位素探针技术在有机污染物生物降解中的应用

稳定同位素探针技术(Stable isotope probing,SIP)是稳定同位素标记技术和各种分子生物学手段相结合的一系列技术总称。将其应用于探查污染物降解的功能微生物,实现了不经过分离培养直接把微生物的代谢功能、微生物间相互作用与微生物种群结合起来,从而克服了传统分离培养的缺陷,扩大了微生物资源的利用空间,具有广阔的发展前景。本文介绍了稳定同位素探针技术的基本原理和技术路线,对常规PLFA-SIP、DNA-SIP、RNA-SIP的特点进行了阐述和对比;综述了SIP在有机污染物——苯系物、多环芳烃、多氯联苯生物降解方面的研究进展,提出SIP应用于根际研究是今后该技术在生物降解研究中的一个发展方向。