稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

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...     

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria

The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of ¹³C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived ¹³C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real‐time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors. Biotechnol. Bioeng. 2009;102: 1527–1536. © 2008 Wiley Periodicals, Inc.     

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.     

Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [¹³C]-α-toluene or [¹³C₇]-toluene as growth substrates. Cells incubated with [¹³C]-α-toluene or [¹³C₇]-toluene incorporated 8–15 at.%¹³C and 51–57 at.%¹³C into total lipid fatty acids, respectively, indicating a lower specific incorporation of ¹³C from [¹³C₇]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [¹³C]-α-toluene and [¹³C₇]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.     

新疆冷泉沉积物葡萄糖利用细菌群落多样性的稳定同位素标记分析

新疆沙湾冷泉低盐且不含硫化物,为了解其沉积物细菌群落的碳源利用,以13C稳定同位素标记的葡萄糖作为底物培养沉积物中的细菌,通过提取和分离带有13C标记的总DNA,结合16S rDNA-PCR克隆文库法以及限制性片段长度多态性方法,对冷泉沉积物中葡萄糖利用细菌群落多样性进行分析.随机挑取417个阳性克隆,HaeⅢ酶切分型,共获得27个OTUs.经测序、序列同源性对比和系统发育学分析,归为细菌域中的9个门,即厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)、硝化螺旋菌门(Nitrospirae)、放线菌门(Actinobacteria)、变形杆菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、浮霉菌门(Planctomycetes)、蓝细菌门(Cyanobacteria)和酸杆菌门(Acidobacteria),其中,厚壁菌门和变形杆菌门为优势类群,分别占克隆文库的28.3％和38.6％.与前期研究比较,以葡萄糖为碳源的细菌OTUs仅占总菌群的31％,表明该环境中可能存在其它碳源利用方式的细菌群落.     

Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.     

Soil microbial community structure and activity in a 100-year-old fertilization and crop rotation experiment

Aims Nitrogen (N) fertilization and lime addition may affect soil microbial and nematode communities and ecosystem functions through changing environmental conditions, such as soil pH and soil organic carbon. The objectives of this experiment were to examine the impact of N input and liming on soil microbial and nematode communities and to identify the key environmental determinant of community composition in a century-old fertilization and crop rotation experiment.Methods The field experiment consisting of a 3-year crop rotation regime was established in 1911 in southeastern USA. Four treatments, (i) no-input control, (ii) NPK with winter legume, (iii) PK with legume and lime and (iv) NPK with legume and lime, were included in this study. Soil samples collected at the 0–5cm depth were used to determine the bacterial growth rate by the 3 H-thymidine incorporation technique. Incorporation of 13 C into neutral lipids, glycolipids and phospholipid fatty acids (PLFAs) was measured after incubation of soil with 13 C-labeled acetate for 24h. Free-living nematodes in fresh soil were extracted using a density sucrose centrifugal flotation method and identified to trophic group level.Important findings Liming resulted in a 10-fold increase in bacterial growth rates compared with the no-input control, whereas N fertilization had no significant effect. Multivariate analysis of PLFA profiles showed that soil microbial community composition was different among the four treatments; the difference was primarily driven by soil pH. PLFAs indicative of Gram-negative bacteria covaried with soil pH, but not those of fungi and actinobacteria. Liming enhanced 13 C incorporation into neutral lipids, glycolipids and phospholipids by 2–15 times. In addition, 13 C incorporation into 16:0, 16:1ω9, 18:1ω9, 18:1ω7 and 18:2ω6 were greater than other PLFAs, suggesting that Gram-negative bacteria and fungi were more active and sensitive to simple C input. Bacterivorous nematodes were the dominant trophic group in the soil, but no significant differences in nematode communities were found among the treatments. Our results suggest that soil pH had a greater impact than N fertilization on soil microbial community composition and activity in a crop rotation system including legumes.     

Insights into the sulfur metabolism of Chlorobaculum tepidum by label-free quantitative proteomics

Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. It can also oxidize sulfide to produce extracellular S⁰ globules, which can be further oxidized to sulfate and used as an electron donor. Here, we performed label-free quantitative proteomics on total cell lysates prepared from different metabolic states, including a sulfur production state (10 h post-incubation [PI]), the beginning of sulfur consumption (20 h PI), and the end of sulfur consumption (40 h PI), respectively. We observed an increased abundance of the sulfide:quinone oxidoreductase (Sqr) proteins in 10 h PI indicating a sulfur production state. The periplasmic thiosulfate-oxidizing Sox enzymes and the dissimilatory sulfite reductase (Dsr) subunits showed an increased abundance in 20 h PI, corresponding to the sulfur-consuming state. In addition, we found that the abundance of the heterodisulfide-reductase and the sulfhydrogenase operons was influenced by electron donor availability and may be associated with sulfur metabolism. Further, we isolated and analyzed the extracellular sulfur globules in the different metabolic states to study their morphology and the sulfur cluster composition, yielding 58 previously uncharacterized proteins in purified globules. Our results show that C. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism in response to the availability of reduced sulfur compounds.     

环境微生物群落功能研究的新方法和新策略

微生物群落在驱动生物地球化学循环中扮演着重要角色,传统的研究方法可对微生物群落进行遗传结构的解析,但不能有效地与功能研究耦联.概述了近年发展起来的基于核酸和蛋白质水平的分子生物学新方法--环境mRNA 和 rRNA同时荧光原位杂交(FISH)、寡核苷酸微阵列技术(Oligonucleotide Microarray)、 稳定性同位素联合宏基因组学(SIP-enabled Metagenomics)和环境蛋白质组学(Metaproteomics)在环境微生物群落功能研究中的应用,并且对其发展趋势进行了分析和展望.     

磷脂脂肪酸谱图分析方法及其在微生物生态学领域的应用

应用磷脂脂肪酸谱图分析技术对微生物群落进行定量分布,克服了传统的微生物培养方法和显微技术的局限性。介绍了磷脂脂肪酸谱图分析方法及其在微生物生态学领域中的应用,包括对微生物群落的生物量、群落结构、营养状况和新陈代谢活动等方面的研究。     

Differential sensitivity of total and active soil microbial communities to drought and forest management

Climate change will affect semiarid ecosystems through severe droughts that increase the competition for resources in plant and microbial communities. In these habitats, adaptations to climate change may consist of thinning—that reduces competition for resources through a decrease in tree density and the promotion of plant survival. We deciphered the functional and phylogenetic responses of the microbial community to 60 years of drought induced by rainfall exclusion and how forest management affects its resistance to drought, in a semiarid forest ecosystem dominated by Pinus halepensis Mill. A multiOMIC approach was applied to reveal novel, community‐based strategies in the face of climate change. The diversity and the composition of the total and active soil microbiome were evaluated by 16S rRNA gene (bacteria) and ITS (fungal) sequencing, and by metaproteomics. The microbial biomass was analyzed by phospholipid fatty acids (PLFAs), and the microbially mediated ecosystem multifunctionality was studied by the integration of soil enzyme activities related to the cycles of C, N, and P. The microbial biomass and ecosystem multifunctionality decreased in drought‐plots, as a consequence of the lower soil moisture and poorer plant development, but this decrease was more notable in unthinned plots. The structure and diversity of the total bacterial community was unaffected by drought at phylum and order level, but did so at genus level, and was influenced by seasonality. However, the total fungal community and the active microbial community were more sensitive to drought and were related to ecosystem multifunctionality. Thinning in plots without drought increased the active diversity while the total diversity was not affected. Thinning promoted the resistance of ecosystem multifunctionality to drought through changes in the active microbial community. The integration of total and active microbiome analyses avoids misinterpretations of the links between the soil microbial community and climate change.     

Changes in soil microbial community composition induced by cometabolism of toluene and trichloroethylene

The effects of trichloroethylene (TCE) on microbial community composition were analyzed by reverse sample genome probing. Soil enrichments were incubated in dessicators containing an organic phase of either 1 or 10% (w/w) toluene in vacuum pump oil, delivering constant equilibrium aqueous concentrations of 16 and 143mg/l, respectively. Increasing the equilibrium aqueous concentration of TCE from 0 to 10mg/l led to shifts in community composition at 16, but not at 143mg/l of toluene. In closed system co-degradation studies, TCE at an aqueous concentration of 1mg/1 was effectively degraded by toluene-degrading soil enrichments once the aqueous toluene concentration dropped below 25mg/l. Little TCE degradation was observed at higher toluene concentrations (50–250mg/l). The results indicate that TCE changes microbial community composition under conditions where it is being actively metabolized.     

基于胃含物分析和稳定同位素技术研究鳀的摄食生态

鳀是重要的渔业资源捕捞对象,同时也是生态系统营养动力学研究的关键种.基于2020年和2008-2009年东海区采集的鳀样品,结合胃含物分析和肌肉碳、氮稳定同位素技术,分析了鳀的食物组成、食性昼夜差异、不同发育阶段的食性转变及其营养级,研究鳀的摄食生态.胃含物分析显示,鳀主要摄食浮游甲壳类和小型鱼类,优势饵料依次为太平洋...