纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.     

High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use

Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation—and therefore substrate use—by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % ¹³C and 0.1 atom % ¹⁵N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.     

Application of nanoscale secondary ion mass spectrometry to plant cell research

Imaging resource flow in soil-plant systems remains central to understanding plant development and interactions with the environment. Typically, subcellular resolution is required to fully elucidate the compartmentation, behavior, and mode of action of organic compounds and mineral elements within plants. For many situations this has been limited by the poor spatial resolution of imaging techniques and the inability to undertake studies in situ. Here we demonstrate the potential of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which is capable of the quantitative high-resolution spatial imaging of stable isotopes (e.g., ¹²C, ¹³C, ¹⁴N, ¹⁵N, ¹⁶O, ¹⁸O, ³¹P, ³⁴S) within intact plant-microbial-soil systems. We present examples showing how the approach can be used to investigate competition for ¹⁵N-labelled nitrogen compounds between plant roots and soil microorganisms living in the rhizosphere and the spatial imaging of ³¹P in roots. We conclude that NanoSIMS has great potential to elucidate the flow of isotopically-labelled compounds in complex media (e.g., soil) and opens up countless new opportunities for studying plant responses to abiotic stress (e.g., ¹⁸O₃, elevated ¹³CO₂), signal exchange, nutrient flow and plant-microbial interactions.Key words: mass spectrometry, NanoSIMS, rhizosphere, isotope labelling, soil, nitrogen, carbon, phosphorus, ¹⁵N, ¹³C, ³¹PWe have used the NanoSIMS technique to investigate the flow of nutrients between microbial and plant cells within the rhizosphere. Secondary Ion Mass Spectrometry (SIMS) involves bombarding a sample with a high-energy ion beam, which sputters atoms, molecules and electrons from the sample surface. Ionized species (secondary ions) are extracted to a mass spectrometer, sorted according to their energy and their mass-to-charge ratio, and counted. NanoSIMS, a recent development in SIMS, combines high sensitivity with high spatial resolution (typically 100 nm) to allow elemental and isotopic imaging of secondary ions, such as ¹²C-, ¹⁶O- and ¹²C¹⁴N-, on a range of biological materials at the sub-cellular scale (Fig. 1A and B). An element map is obtained by scanning the primary ion beam over the sample surface and measuring the secondary ion intensities of any given ion species, at each pixel in the image. The intrinsically high mass resolution allows the separation of different ion species at the same nominal atomic mass (e.g., ¹²C¹⁵N- from ¹³C¹⁴N- at mass 27), while the multi-collection capability allows the simultaneous measurement of up to five ion species. This makes it possible to obtain images of different isotopes from the same area simultaneously, from which quantitative isotope ratios from individual components can then be extracted. As such, NanoSIMS offers a means of elucidating processes involved in the transport of ions and molecules into cells and their distribution within cells, at scales and sensitivities not attainable by other methods.^1–5Open in a separate windowFigure 1(A) ¹²C¹⁴N^- and (B) ³¹P^- images of a wheat root cell nucleus from NanoSIMS illustrating the potential to map different elements at the sub-cellular scale; (C) TEM image of two bacteria attached to a cortical cell wall; (D) corresponding ¹⁵N/¹⁴N ratio image from NanoSIMS of the same bacteria. The differential uptake of ¹⁵N is illustrated by the color scale; ranging from natural abundance (blue) to a ¹⁵N/¹⁴N ratio = 1.0 (i.e., 50 at% ¹⁵N) (pink) for the plant cell and bacteria, respectively; (E) Linescan (3.5 µm) illustrating the variation in ¹⁵N/¹⁴N across an enriched bacterium and an un-enriched plant cell wall (line in D). Error bars are based on the Poisson counting statistics for each pixel.We previously demonstrated the use of NanoSIMS to image and map the location of ¹⁵N-labelled bacterial communities artificially introduced into soil microhabitats.^6,7 We extended this approach to a natural ecosystem, by examining the differential partitioning of ¹⁵N-labelled ammonium (¹⁵NH₄⁺) between plant roots and soil microbial communities at the nanometer scale (Fig. 1C and D).⁸ It was shown that introduced ¹⁵N could be detected, and more importantly, mapped, in individual bacterial cells found in the soil matrix, within the rhizosphere, within root hairs, and intra-cellular within the root. The ¹⁵N/¹⁴N ratio data (determined as the ratio between the ¹²C¹⁵N- and the ¹²C¹⁴N- signals) could then be extracted from specific regions of interest—groups of pixels bounding a particular feature, such as a bacterium or a root cell wall, or linescans (Fig. 1E). This unique approach allows the visualization of nutrient flows and metabolic pathways through complex, multi-component ecosystems. Here we consider further the application of the technique to study nutrient availability in plant cell research.     

Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis

A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10⁴Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences.     

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes (¹³C, ¹⁵N, ³⁶S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.     

SIMSISH Technique Does Not Alter the Apparent Isotopic Composition of Bacterial Cells

In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine – iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific ¹³C uptake during labelled methanol anaerobic degradation.     

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction¹, boosting the immune response², pheromone production³, as well as nutrition, including the synthesis of essential amino acids^4, among others.    Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing ¹³C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA⁵. The incorporation of ¹³C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (¹²C) one. In the end, the ¹³C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the ¹²C-unlabeled similar one⁶. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, ¹³C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.     

Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM,TEM/X-EDS,and NanoSIMS

Chemical bioimaging offers an important contribution to the investigation of biochemical functions, biosorption and bioaccumulation processes of trace elements via their localization at the cellular and even at the subcellular level. This paper describes the combined use of high contrast transmission electron microscopy (HC-TEM), energy dispersive X-ray spectroscopy (X-EDS), and nano secondary ion mass spectrometry (NanoSIMS) applied to a model organism, the unicellular green algae Chlamydomonas reinhardtii. HC-TEM providing a lateral resolution of 1 nm was used for imaging the ultrastructure of algae cells which have diameters of 5–10 μm. TEM coupled to X-EDS (TEM/X-EDS) combined textural (morphology and size) analysis with detection of Ca, P, K, Mg, Fe, and Zn in selected subcellular granules using an X-EDS probe size of approx. 1 μm. However, instrumental sensitivity was at the limit for trace element detection. NanoSIMS allowed chemical imaging of macro and trace elements with subcellular resolution (element mapping). Ca, Mg, and P as well as the trace elements Fe, Cu, and Zn present at basal levels were detected in pyrenoids, contractile vacuoles, and granules. Some metals were even localized in small vesicles of about 200 nm size. Sensitive subcellular localization of trace metals was possible by the application of a recently developed RF plasma oxygen primary ion source on NanoSIMS which has shown good improvements in terms of lateral resolution (below 50 nm), sensitivity, and stability. Furthermore correlative single cell imaging was developed combining the advantages of TEM and NanoSIMS. An advanced sample preparation protocol provided adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Thus, the C. reinhardtii cellular ultrastructure could be directly related to the spatial distribution of metals in different cell organelles such as vacuoles and chloroplast.     

Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO₃⁻-N mg of mixed-liquor volatile suspended solids (MLVSS)⁻¹ h⁻¹ to a steady-state value of 0.06 mg of NO₃⁻-N mg of MLVSS⁻¹ h⁻¹ over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [¹³C]methanol to biomark the DNA of the denitrifiers. The extracted [¹³C]DNA and [¹²C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [¹³C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [¹²C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [¹⁴C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

In Vivo Imaging of Tissue-Remodeling Activity Involving Infiltration of Macrophages by a Systemically Administered Protease-Activatable Probe in Colon Cancer Tissues

This study evaluated the detection of tumors using in vivo imaging with a commercially available and systemically administered protease-activatable fluorescent probe, ProSense. To this end, we analyzed the delivery and uptake of ProSense as well as the target protease and its cellular source in a mouse xenograft tumor model. In vivo and ex vivo multi wavelength imaging revealed that ProSense signals accumulated within tumors, with preferential distribution in the vascular leakage area that correlates with vasculature development at the tumor periphery. Immunohistochemically, cathepsin B, which is targeted by ProSense, was specifically localized in macrophages. The codistribution of tenascin C immunoreactivity and gelatinase activity provided evidence of tissue-remodeling at the tumor periphery. Furthermore, in situ zymography revealed extracellular ProSense cleavage in such areas. Colocalization of cathepsin B expression and ProSense signals showing reduction by addition of cathepsin B inhibitor was confirmed in cultured macrophage-derived RAW264.7 cells. These results suggest that increased tissue-remodeling activity involving infiltration of macrophages is a mechanism that may be responsible for the tumor accumulation of ProSense signals in our xenograft model. We further confirmed ProSense signals at the tumor margin showing cathepsin B⁺ macrophage infiltration in a rat colon carcinogenesis model. Together, these data demonstrate that systemically administered protease-activatable probes can effectively detect cancer invasive fronts, where tissue-remodeling activity is high to facilitate neoplastic cell invasion.     

Bacterial genome replication at subzero temperatures in permafrost

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with ¹³C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to −20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize ¹³C-labeled DNA when supplemented with ¹³C-acetate at temperatures of 0 to −20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of ¹³C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.     

High resolution microarray assay for rapid taxonomic assessment of Pseudo-nitzschia spp. (Bacillariophyceae) in the field

Deployable methods facilitating rapid microbial identification provide opportunities to respond quickly when pathogenic or toxin-producing organisms threaten water quality. We developed a microarray assay to streamline identification of microorganisms in the field, focusing on the harmful algal bloom diatom Pseudo-nitzschia. The assay employed electrochemical signal detection and a simplified protocol, allowing identification of specific taxa onboard research vessels within 7 h of water collection. Microarrays targeted the internal transcribed spacer (ITS1) region between the 18S and 28S ribosomal RNA genes using 307 oligonucleotide probes. The probes, ranging from broadly specific to unique, represented 118 Pseudo-nitzschia ribotypes from 15 species available at the time of assay development. Hybridization signals from multiple probes for each target strain were integrated using a novel algorithm for data analysis. Designated the ‘integrated sumscore data analysis’ (ISDA), the algorithm used probe specificity metrics for signal integration, with uniqueness corresponding to higher probe weight values. The integrated signals provided a ‘sumscore’ for each ribotype represented on the array, indicating its presence or absence in the sample. The algorithm was ‘trained’ by comparison with data from scanning electron microscopy, and cloning and sequence analysis of Pseudo-nitzschia ITS1 ribotypes from 7 laboratory cultures and a complex environmental sample. The ISDA provided correct identification and target sequences for all tested strains. Through design of custom probes (up to 12,000 on a microarray chip), this approach may be used to identify additional microbial taxa of interest and provides rapid, reliable shipboard assays for basic research or water quality monitoring.     

Unraveling uncultivable pesticide degraders via stable isotope probing (SIP)

Uncultivable microorganisms account for over 99% of all species on earth, playing essential roles in ecological processes such as carbon/nitrogen cycle and chemical mineralization. Their functions remain unclear in ecosystems and natural habitats, requiring cutting-edge biotechnologies for a deeper understanding. Stable isotope probing (SIP) incorporates isotope-labeled elements, e.g. ^13?C, ^18?O or ^15?N, into the cellular components of active microorganisms, serving as a powerful tool to link phylogenetic identities to their ecological functions in situ. Pesticides raise increasing attention for their persistence in the environment, leading to severe damage and risks to the ecosystem and human health. Cultivation and metagenomics help to identify either cultivable pesticide degraders or potential pesticide metabolisms within microbial communities, from various environmental media including the soil, groundwater, activated sludge, plant rhizosphere, etc. However, the application of SIP in characterizing pesticide degraders is limited, leaving considerable space in understanding the natural pesticide mineralization process. In this review, we try to comprehensively summarize the fundamental principles, successful cases and technical protocols of SIP in unraveling functional-yet-uncultivable pesticide degraders, by raising its shining lights and shadows. Particularly, this study provides deeper insights into various feasible isotope-labeled substrates in SIP studies, including pesticides, pesticide metabolites, and similar compounds. Coupled with other techniques, such as next-generation sequencing, nanoscale secondary ion mass spectrometry (NanoSIMS), single cell genomics, magnetic-nanoparticle-mediated isolation (MMI) and compound-specific isotope analysis (CSIA), SIP will significantly broaden our understanding of pesticide biodegradation process in situ.     

Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ± 3.7% [mean ± standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r² = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.     

Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA

We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine ¹³C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [¹³C]glucose, [¹³C]propionate, or [¹³C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that ¹³C labeling can be much lower, to the extent that δ¹³C ratios can be studied even at their natural abundances.Linking microbial phylogeny to community function provides us with insights into the roles that microorganisms play in global elemental cycling. In recent years, stable isotope-tracing approaches combined with biomarkers have been widely applied to environmental studies (8, 27, 40). Tracking stable- or radioisotope-labeled atoms from particular substrates into components of microbial cells (biomarkers) can reveal which organisms are involved in the consumption of the substrate and also yield information on rates of specific biogeochemical transformation (8).Dissimilatory sulfate reduction is a major pathway for organic carbon mineralization in coastal marine sediments, accounting for, on average, 50% of the total carbon mineralization (18, 36). Sulfate-reducing prokaryotes are a diverse and ubiquitous component of the bacterial community. The diversity of sulfate-reducing bacteria (SRB) in marine sediments has been investigated by using clone libraries of 16S rRNA (38) and dissimilatory sulfite reductase genes (11) and by fluorescence in situ hybridization-related techniques (33, 41). Desulfobacteraceae, a group of complete-oxidizing SRB belonging to the Deltaproteobacteria, have generally been found to be a major group of SRB in marine sediments.Phospholipid-derived fatty acids (PLFA) were the first type of biomarkers to be used in combination with stable isotope probing (SIP) (8). PLFA-SIP provides high sensitivity in terms of the amount of ¹³C label needed, but the phylogenetic resolution offered is low and requires reference signatures of closely related culturable relatives (8). The main advantage of DNA- and RNA-SIP is that they offer improved phylogenetic resolution (27, 40). These two methods are based on the separation of the “heavier” ¹³C-labeled nucleic acid from unlabeled nucleic acid by density centrifugation. Subsequently, organisms incorporating the greatest proportion of label into their DNA or RNA are identified by various molecular-fingerprinting techniques or by constructing clone libraries. RNA has a higher turnover rate than DNA, resulting in faster labeling, and incubation times can therefore be substantially shortened (27, 42). RNA is also more likely to reflect the phylogenetic composition of the metabolically active community, since it is highly susceptible to chemical and enzymatic degradation, and its cellular levels are often tightly regulated (19, 32), although some prokaryotes maintain high numbers of ribosomes during starvation (13).MacGregor et al. (25, 26) developed a related approach, SIP combined with magnetic-bead capture hybridization (here called Mag-SIP), which is based on the isolation of small subunit rRNA from particular phylogenetic groups and the detection of ¹³C-labeling levels by isotope ratio mass spectrometry (IRMS). rRNA is captured by hybridization with specific biotin-labeled oligonucleotide probes, followed by retrieval of hybridized target rRNA using streptavidin-coated magnetic beads. The main advantage of Mag-SIP over other nucleic acid-based SIP methods is that in principle much lower labeling levels can be applied (about 0.001% versus >10% ¹³C, respectively), as label detection is based on IRMS methods. For instance, it has been shown that the method can be used to study the effects of oil pollution on natural δ¹³C ratios of bacterial communities in sediments (37). Moreover, Mag-SIP is not based on PCR, as the isotope ratio of the target rRNA is directly measured without amplification of nucleic acid, avoiding possible PCR artifacts. However, the large amounts (1 to 10 μg) of RNA needed for an accurate isotope ratio analysis by traditional elemental-analyzer (EA)-IRMS has limited the use of Mag-SIP to general domain-specific probes (25, 26). Recently, several methods, such as liquid chromatography (LC) combined with IRMS and spooling-wire microcombustion combined with IRMS, have been introduced that allow isotopic analysis of much smaller samples than with the traditional EA-IRMS systems (20, 43).In this study, we used the wet-oxidation interface of LC-IRMS as a micro-EA (μEA)-IRMS (20). The use of μEA-IRMS substantially lowers the detection limit of isotope ratio measurements in terms of the amount of rRNA needed for an analysis but also calls for modifications of the Mag-SIP protocol in order to decrease protocol blanks (carbon from materials and reagents used in the protocol). We tested a nested set of three biotin-labeled oligonucleotide probes to capture 16S rRNA derived from Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae. The target specificities and stringencies of these probes were tested against pure cultures of both target and nontarget organisms. Moreover, phylogenetic analysis of captured 16S rRNA from environmental samples was done to check specificity and, where necessary, to adjust probe stringency. Finally, we demonstrated Mag-SIP with a study of in situ substrate use by sulfate-reducing Deltaproteobacteria in intertidal anoxic marine sediment. A generalized scheme for Mag-SIP is shown in Fig. ​Fig.11.Open in a separate windowFIG. 1.A generalized scheme for Mag-SIP. The control was sediment incubated without substrate.     

A critical review of NanoSIMS in analysis of microbial metabolic activities at single-cell level

Over 3.8 billion years of evolution has enabled many microbial species a versatile metabolism. However, limited by experimental methods, some unique metabolism remains unknown or unclear. A major obstacle is to attribute the incorporation of certain nutrients into a noncultivable species out of a complex microbial community. Such difficulty could be solved if we are able to directly observe substrate uptake at the single-cell level. Nanoscale secondary ion mass spectrometry (NanoSIMS) is a powerful tool for revealing element distribution in nanometer-scale resolution in the fields such as material sciences, geosciences and astronomy. In this review, we focus on another applicability of NanoSIMS in microbiology. In such fields, physiological properties and metabolic activities of microorganisms can be revealed with a single-cell scale resolution by NanoSIMS solely or in combination with other techniques. This review will highlight the features of NanoSIMS in analyzing the metabolic activities of carbon, nitrogen, metal irons by mixed-culture assemblies. Some values of NanoSIMS in environmental microbiology are expected to be discussed via this review.     

In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.