Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods.
Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods.
复制标题
使用 BONCAT 和基于 nanoSIMS 的方法研究海洋病毒与宿主的相互作用和元素转移。
DOI：
10.1111/1462-2920.13996
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发表时间：
2018
影响因子：
5.1
通讯作者：
Orphan,VictoriaJ
中科院分区：
文献类型：
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作者：
Pasulka,AlexisL;Thamatrakoln,Kimberlee;Kopf,SebastianH;Guan,Yunbin;Poulos,Bonnie;Moradian,Annie;Sweredoski,MichaelJ;Hess,Sonja;Sullivan,MathewB;Bidle,KayD;Orphan,VictoriaJ
虽然海洋病毒的集体影响在过去十年中变得更加明显，但对病毒宿主动力学和病毒在营养循环中的作用的更深入了解将受益于单病毒水平的直接观察。我们描述了两种新的互补方法-稳定同位素探测与纳米级二次离子质谱（nanoSIMS）和基于荧光的双正交非规范氨基酸标记（BONCAT）-用于研究海洋病毒的活性和生物地球化学影响。这些工具是使用几种生态相关的模型系统（Emiliania huxleyi/EhV 207、Synechococcus sp. WH 8101/Syn 1和Escherichia coli/T7）开发和测试的。通过解析病毒颗粒中的碳和氮富集，我们证明了nanoSIMS示踪实验在获得直接从特定生产途径（通过同位素标记宿主底物）产生的病毒总数的定量估计方面的能力。此外，我们通过实验室实验和试点现场研究表明，BONCAT可用于直接量化病毒生产（通过落射荧光显微镜），只需少量样品操作，不依赖于转换因子。该技术也可用于检测新合成的病毒蛋白。这些工具将有助于填补我们对海洋病毒生物化学影响的理解的关键空白。
While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus‐host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single‐virus level. We describe two new complementary approaches – stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence‐based biorthogonal non‐canonical amino acid tagging (BONCAT) – for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207,Synechococcus sp. WH8101/Syn1 andEscherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labelling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.