Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community
Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community
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DOI：
10.1128/aem.71.12.7858-7865.2005
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发表时间：
2005-12-01
影响因子：
4.4
通讯作者：
Madsen, EL
中科院分区：
文献类型：
--
作者：
DeRito, CM;Pumphrey, GM;Madsen, EL
这项实地研究的目的是深入了解三种不同的微生物种群参与苯酚的原位代谢。我们的方法测量（CO2）-C-13呼吸[C-13]苯酚和稳定同位素探针（SIP）的土壤DNA在农业现场。传统上，基于SIP的调查一直受到碳交叉喂养所带来的不确定性的影响。通过改变我们的实地为基础的，底物给药的方法，实验的目的是超越初级降解检测营养相关的人口在食物链中。利用气相色谱-质谱（GC/MS），它表明，C-13标记的生物量，来自土壤中的主要苯酚降解菌，是一个合适的生长基质的其他成员的土壤微生物群落。接下来，设计了三种给药方案以原位检查参与苯酚代谢的微生物群落的活性成员：（i）I剂量的[C-13]苯酚，（ii）I日剂量的未标记苯酚，随后I剂量的[C-13]苯酚，和（iii）12日剂量的[C-13]苯酚。GC/MS分析表明，先前暴露于苯酚会使（CO2）-C-13的释放增加10倍。此外，使用二次离子质谱（西姆斯）对C-13处理过的土壤进行成像，证实了单个细菌将C-13掺入其生物量中。来自3种给药方案的C-13标记的土壤DNA的PCR扩增和16 S rRNA基因测序揭示了3个不同的克隆文库：（i）未富集的初级苯酚降解菌是最多样化的，由α-、β-和γ-变形菌和高G + C含量的革兰氏阳性菌组成，（ii）富集的初级苯酚降解菌由库克菌属和葡萄球菌属的成员主导，和（iii）营养相关（碳交叉饲养者）由假单胞菌属的成员占主导地位。这些数据表明，SIP有可能记录基板预暴露引起的人口变化，并遵循陆地微生物食物链的碳流。
The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured (CO2)-C-13 respired from [C-13] phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that C-13-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) I dose of [C-13] phenol, (ii) It daily doses of unlabeled phenol followed by I dose of [C-13] phenol, and (iii) 12 daily doses of [C-13] phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted (CO2)-C-13 evolution by a factor of 10. Furthermore, imaging of C-13-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated C-13 into their biomass. PCR amplification and 16S rRNA gene sequencing of C-13-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of alpha-, beta-, and gamma-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.