我们感谢让-Roch Mouret, 西尔维亚 Dequin 和吉恩-玛丽 Sabalyrolles 为帮助的概念, 机器人辅助发酵系统和马丁 Pradal, 尼古拉布维尔和帕斯卡尔 Brial 为他们的技术支持。这一项目的经费由全国 Ministère 教育、de la 研究等技术提供。

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作者没有什么可透露的。

利用同位素示踪实验, 通过代谢网络定量化化合物, 是了解微生物代谢操作的一种很有前途的方法。这种方法, 虽然成功地应用于一个或两个标记的基板, 目前不能实施研究的代谢各种来源使用多重标记元素同位素 (即, 超过两个基板)。事实上, 可用的分析技术能够准确地确定 proteinogenic 氨基酸和分子的标记模式, 仅当使用单一元素的同位素时, 并且可能时用两个元素进行联合标记。因此, 为了?...

了解微生物代谢如何运作是一个关键问题的设计有效的战略, 以改善发酵过程和调节生产的发酵化合物。在过去的二十年中, 基因组学和功能基因组学的进步, 在很大程度上促进了许多微生物代谢网络拓扑的知识的扩展。对此信息的访问导致了旨在全面概述蜂窝功能1的方法的发展。这些方法通常依赖于基于模型的可测量参数的解释。这些实验数据一方面包括代谢物的吸收率和生产率, 另一方面, 从同位素示踪实验中获得的定量的细胞内信息。这些数据提供了在定义的新陈代谢网络中的不同通路的体内活动的扣除的基本信息2,3,4。目前, 可用的分析技术只能够准确地检测分子在使用单元素同位素时的标记模式, 并可能在两个同位素元素的共同标记下。此外, 在大多数生长条件下, 碳源只由一个或两个化合物组成。因此, 基于碳基板上的13C 同位素示踪剂的方法得到了广泛和成功的应用, 以全面了解碳代谢网络的运行情况5,6,7 ,8。
相比之下, 在许多自然和工业环境中, 支持微生物生长的可用氮资源通常由广泛的分子组成。例如, 在葡萄酒或啤酒发酵过程中, 氮气作为18种氨基酸和铵的混合物提供, 在可变浓度下为9。这一系列的 N 类化合物, 可用于代谢使这些复杂的媒体条件大大不同于那些通常用于生理学研究, 因为后者是通过使用独特的氮源, 典型的铵。
总的来说, 内化的氮化合物可以直接纳入蛋白质或 catabolized。许多微生物的氮代谢网络结构, 包括酵母的酿酒酵母, 是非常复杂的根据基板的多样性。示意性地, 这个系统是基于氮代谢中心核心的组合, 它催化谷氨酰胺、谷氨酸和α-二的转换10,11, 具有转氨酶和 deaminases。通过这个网络, 胺基团从铵或其他氨基酸收集和α-酮酸释放。这些中间体也综合通过中央碳代谢 (CCM)12,13。这种大量的分支反应和中间体, 参与了外源氮源的分解和 proteinogenic 氨基酸的代谢, 满足了细胞的合成代谢要求。通过这些不同的相互连接的路线的活动也导致代谢物的排泄。特别是α-酮酸可以通过艾氏通路重定向产生更高的醇及其乙酸酯衍生物14, 这是产品感官图谱的重要贡献。随后, 氮代谢如何运作在生物质生产和挥发性分子 (香气) 的形成中起着关键作用。
有关氮代谢的反应、酶和基因在文献中有广泛的描述。然而, 在整个代谢网络中分配多种氮源的问题尚未得到解决。解释这种缺乏信息有两个主要原因。首先, 鉴于氮代谢网络的重要复杂性, 需要大量的定量数据才能完全了解它的运作, 直到现在。其次, 许多实验性的约束和分析方法的局限性阻碍了以前用于解释 CCM 函数的方法的实现。
为了克服这些问题, 我们选择了一种系统级的方法, 它是基于对一系列同位素示踪实验数据的核对。该工作流包括: -在相同环境条件下进行的一组发酵, 而不同的营养源 (基质) 每次都被标记。 -结合分析程序 (HPLC、gc-ms), 在发酵的不同阶段, 对被标记的基质的残留浓度和化合物的浓度和同位素富集进行精确测定。标记分子的分解代谢, 包括衍生生物质。 -计算每个被消耗标记的分子的质量和同位素平衡, 并对数据集进行进一步的综合分析, 以获得全球对微生物通过测定通量比值管理多种营养源的概览.
要应用这种方法, 必须注意培养菌株/微生物的可再生行为。此外, 在相同的发酵过程中, 必须采取不同文化的样品。在这篇手稿中所报道的实验工作中, 一个机器人辅助系统用于在线监测发酵, 以此来解释这些限制。
此外, 必须选择一套标签的基板 (化合物, 性质, 和位置的标签), 这是适当的, 以解决科学问题的研究。在这里, 15N 标记铵、谷氨酰胺和精氨酸被选为葡萄汁中发现的三主要氮源。这就允许评估从消耗化合物到 proteinogenic 氨基酸的氮再分配模式。我们还研究了被消耗的氨基酸的碳骨架的命运及其对挥发性分子生产的贡献。为了实现这一目标, 在研究中加入了一致的13C 标记亮氨酸、异亮氨酸、苏氨酸和缬氨酸, 这些氨基酸是从艾氏通路的主要中间体中获得的。
总的来说, 我们定量地探讨了酵母如何管理一个复杂的氮资源, 通过重新分配外源氮源, 以满足其合成代谢的要求, 在整个发酵, 而另外消除过剩的碳前体挥发性分子。本文所报道的实验方法可用于研究其他微生物所使用的多种营养源。这似乎是分析遗传背景或环境条件对微生物代谢行为的影响的适当方法。

<table><tbody><tr><td>D-Glucose</td><td>PanReac</td><td>141341.0416</td><td></td></tr><tr><td>D-Fructose</td><td>PanReac</td><td>142728.0416</td><td></td></tr><tr><td>DL-Malic acid</td><td>Sigma Aldrich</td><td>M0875</td><td></td></tr><tr><td>Citric acid monohydrate</td><td>Sigma Aldrich</td><td>C7129</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5379</td><td></td></tr><tr><td>Potassium sulfate</td><td>Sigma Aldrich</td><td>P0772</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma Aldrich</td><td>230391</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C7902</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S9625</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A4514</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma Aldrich</td><td>71690</td><td></td></tr><tr><td>Manganese sulfate monohydrate</td><td>Sigma Aldrich</td><td>M7634</td><td></td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Sigma Aldrich</td><td>Z4750</td><td></td></tr><tr><td>Copper (II) sulfate pentahydrate</td><td>Sigma Aldrich</td><td>C7631</td><td></td></tr><tr><td>Potassium iodine</td><td>Sigma Aldrich</td><td>P4286</td><td></td></tr><tr><td>Cobalt (II) chloride hexahydrate</td><td>Sigma Aldrich</td><td>C3169</td><td></td></tr><tr><td>Boric acid</td><td>Sigma Aldrich</td><td>B7660</td><td></td></tr><tr><td>Ammonium heptamolybdate</td><td>Sigma Aldrich</td><td>A7302</td><td></td></tr><tr><td>Myo-inositol</td><td>Sigma Aldrich</td><td>I5125</td><td></td></tr><tr><td>D-Pantothenic acid hemicalcium salt</td><td>Sigma Aldrich</td><td>21210</td><td></td></tr><tr><td>Thiamine, hydrochloride</td><td>Sigma Aldrich</td><td>T4625</td><td></td></tr><tr><td>Nicotinic acid</td><td>Sigma Aldrich</td><td>N4126</td><td></td></tr><tr><td>Pyridoxine</td><td>Sigma Aldrich</td><td>P5669</td><td></td></tr><tr><td>Biotine</td><td>Sigma Aldrich</td><td>B4501</td><td></td></tr><tr><td>Ergost&amp;eacute;rol</td><td>Sigma Aldrich</td><td>E6510</td><td></td></tr><tr><td>Tween 80</td><td>Sigma Aldrich</td><td>P1754</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR Chemicals</td><td>101074F</td><td></td></tr><tr><td>Iron (III) chloride hexahydrate</td><td>Sigma Aldrich</td><td>236489</td><td></td></tr><tr><td>L-Aspartic acid</td><td>Sigma Aldrich</td><td>A9256</td><td></td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td></td></tr><tr><td>L-Alanine</td><td>Sigma Aldrich</td><td>A7627</td><td></td></tr><tr><td>L-Arginine</td><td>Sigma Aldrich</td><td>A5006</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma Aldrich</td><td>C7352</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma Aldrich</td><td>G3126</td><td></td></tr><tr><td>Glycine</td><td>Sigma Aldrich</td><td>G7126</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma Aldrich</td><td>H8000</td><td></td></tr><tr><td>L-Isoleucine</td><td>Sigma Aldrich</td><td>I2752</td><td></td></tr><tr><td>L-Leucine</td><td>Sigma Aldrich</td><td>L8000</td><td></td></tr><tr><td>L-Lysine</td><td>Sigma Aldrich</td><td>L5501</td><td></td></tr><tr><td>L-Methionine</td><td>Sigma Aldrich</td><td>M9625</td><td></td></tr><tr><td>L-Phenylalanine</td><td>Sigma Aldrich</td><td>P2126</td><td></td></tr><tr><td>L-Proline</td><td>Sigma Aldrich</td><td>P0380</td><td></td></tr><tr><td>L-Serine</td><td>Sigma Aldrich</td><td>S4500</td><td></td></tr><tr><td>L-Threonine</td><td>Sigma Aldrich</td><td>T8625</td><td></td></tr><tr><td>L-Tryptophane</td><td>Sigma Aldrich</td><td>T0254</td><td></td></tr><tr><td>L-Tyrosine</td><td>Sigma Aldrich</td><td>T3754</td><td></td></tr><tr><td>L-Valine</td><td>Sigma Aldrich</td><td>V0500</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C5-L-Valine</td><td>Eurisotop</td><td>CLM-2249-H-0.25</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C6-L-Leucine</td><td>Eurisotop</td><td>CLM-2262-H-0.25</td><td></td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;N-Ammonium chloride</td><td>Eurisotop</td><td>NLM-467-1</td><td></td></tr><tr><td>ALPHA-&lt;sup&gt;15&lt;/sup&gt;N-L-Glutamine</td><td>Eurisotop</td><td>NLM-1016-1</td><td></td></tr><tr><td>U-&lt;sup&gt;15&lt;/sup&gt;N4-L-Arginine</td><td>Eurisotop</td><td>NLM-396-PK</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma Aldrich</td><td>270989</td><td></td></tr><tr><td>Ethyl propanoate</td><td>Sigma Aldrich</td><td>112305</td><td></td></tr><tr><td>Ethyl 2-methylpropanoate</td><td>Sigma Aldrich</td><td>246085</td><td></td></tr><tr><td>Ethyl butanoate</td><td>Sigma Aldrich</td><td>E15701</td><td></td></tr><tr><td>Ethyl 2-methylbutanoate</td><td>Sigma Aldrich</td><td>306886</td><td></td></tr><tr><td>Ethyl 3-methylbutanoate</td><td>Sigma Aldrich</td><td>8.08541.0250</td><td></td></tr><tr><td>Ethyl hexanoate</td><td>Sigma Aldrich</td><td>148962</td><td></td></tr><tr><td>Ethyl octanoate</td><td>Sigma Aldrich</td><td>W244910</td><td></td></tr><tr><td>Ethyl decanoate</td><td>Sigma Aldrich</td><td>W243205</td><td></td></tr><tr><td>Ethyl dodecanoate</td><td>Sigma Aldrich</td><td>W244112</td><td></td></tr><tr><td>Ethyl lactate</td><td>Sigma Aldrich</td><td>W244015</td><td></td></tr><tr><td>Diethyl succinate</td><td>Sigma Aldrich</td><td>W237701</td><td></td></tr><tr><td>2-methylpropyl acetate</td><td>Sigma Aldrich</td><td>W217514</td><td></td></tr><tr><td>2-methylbutyl acetate</td><td>Sigma Aldrich</td><td>W364401</td><td></td></tr><tr><td>3-methyl butyl acetate</td><td>Sigma Aldrich</td><td>287725</td><td></td></tr><tr><td>2-phenylethyl acetate</td><td>Sigma Aldrich</td><td>290580</td><td></td></tr><tr><td>2-methylpropanol</td><td>Sigma Aldrich</td><td>294829</td><td></td></tr><tr><td>2-methylbutanol</td><td>Sigma Aldrich</td><td>133051</td><td></td></tr><tr><td>3-methylbutanol</td><td>Sigma Aldrich</td><td>309435</td><td></td></tr><tr><td>Hexanol</td><td>Sigma Aldrich</td><td>128570</td><td></td></tr><tr><td>2-phenylethanol</td><td>Sigma Aldrich</td><td>77861</td><td></td></tr><tr><td>Propanoic acid</td><td>Sigma Aldrich</td><td>94425</td><td></td></tr><tr><td>Butanoic acid</td><td>Sigma Aldrich</td><td>19215</td><td></td></tr><tr><td>2-methylpropanoic acid</td><td>Sigma Aldrich</td><td>58360</td><td></td></tr><tr><td>2-methylbutanoic acid</td><td>Sigma Aldrich</td><td>193070</td><td></td></tr><tr><td>3-methylbutanoic acid</td><td>Sigma Aldrich</td><td>W310212</td><td></td></tr><tr><td>Hexanoic acid</td><td>Sigma Aldrich</td><td>153745</td><td></td></tr><tr><td>Octanoic acid</td><td>Sigma Aldrich</td><td>W279900</td><td></td></tr><tr><td>Decanoic acid</td><td>Sigma Aldrich</td><td>W236403</td><td></td></tr><tr><td>Dodecanoic acid</td><td>Sigma Aldrich</td><td>L556</td><td></td></tr><tr><td>Fermentor 1L</td><td>Legallais</td><td>AT1357</td><td>Fermenter handmade for fermentation</td></tr><tr><td>Disposable vacuum filtration system</td><td>Dominique Deutscher</td><td>029311</td><td></td></tr><tr><td>Fermenters (250 ml)</td><td>Legallais</td><td>AT1352</td><td>Fermenter handmade for fermentation</td></tr><tr><td>Sterile tubes</td><td>Sarstedt</td><td>62.554.502</td><td></td></tr><tr><td>Fermentation locks</td><td>Legallais</td><td>AT1356</td><td>Fermetation locks handmade for fermentation</td></tr><tr><td>BactoYeast Extract</td><td>Becton, Dickinson and Company</td><td>212750</td><td></td></tr><tr><td>BactoPeptone</td><td>Becton, Dickinson and Company</td><td>211677</td><td></td></tr><tr><td>Incubator shaker</td><td>Infors HT</td><td></td><td></td></tr><tr><td>Particle Counter</td><td>Beckman Coulter</td><td>6605697</td><td>Multisizer 3 Coulter Counter</td></tr><tr><td>Centrifuge</td><td>Jouan</td><td></td><td>GR412</td></tr><tr><td>Plate Butler Robotic system</td><td>Lab Services BV</td><td>PF0X-MA</td><td>Automatic instrument</td></tr><tr><td>Plate Butler Software</td><td>Lab Services BV</td><td></td><td>Robot monitor software</td></tr><tr><td>RobView</td><td></td><td></td><td>In-house developed calculation software</td></tr><tr><td>My SQL</td><td></td><td></td><td>International source database</td></tr><tr><td>Cimarec i Telesystem Multipoint Stirrers</td><td>Thermo Fisher Scientific</td><td>50088009</td><td>String Drive 60</td></tr><tr><td>BenchBlotter platform rocker</td><td>Dutscher</td><td>60903</td><td></td></tr><tr><td>Ammonia enzymatic kit</td><td>R-Biopharm AG</td><td>5390</td><td></td></tr><tr><td>Spectrophotometer cuvettes</td><td>VWR</td><td>634-0678</td><td></td></tr><tr><td>Spectrophotometer UviLine 9400</td><td>Secomam</td><td></td><td></td></tr><tr><td>Amino acids standards physiological - acidics and neutrals</td><td>Sigma Aldrich</td><td>A6407</td><td></td></tr><tr><td>Amino acids standards physiological - basics</td><td>Sigma Aldrich</td><td>A6282</td><td></td></tr><tr><td>Citrate lithium buffers - Ultra ninhydrin reagent</td><td>Biochrom</td><td>BC80-6000-06</td><td></td></tr><tr><td>Sulfosalycilic acid</td><td>Sigma Aldrich</td><td>S2130</td><td></td></tr><tr><td>Norleucine</td><td>Sigma Aldrich</td><td>N1398</td><td></td></tr><tr><td>Biochrom 30 AAA</td><td>Biochrom</td><td></td><td></td></tr><tr><td>EZChrom Elite</td><td>Biochrom</td><td></td><td>Instrument control and Data analysis software</td></tr><tr><td>Ultropac 8 resin Lithium</td><td>Biochrom</td><td>BC80-6002-47</td><td>Lithium High Resolution Physiological Column</td></tr><tr><td>Filter Millex GV</td><td>Merck Millipore</td><td>SLGVX13NL</td><td>Millex GV 13mm (pore size 0.22 &amp;micro;m)</td></tr><tr><td>Membrane filter PALL</td><td>VWR</td><td>514-4157</td><td>Supor-450 47mm 0.45&amp;micro;m</td></tr><tr><td>Vacuum pump Millivac Mini</td><td>Millipore</td><td>XF5423050</td><td></td></tr><tr><td>Aluminium smooth weigh dish 70mm</td><td>VWR</td><td>611-1380</td><td></td></tr><tr><td>Precision balance</td><td>Mettler</td><td></td><td>Specifications AE163</td></tr><tr><td>Dimethyl sulfoxid dried</td><td>Merck</td><td>1029310161</td><td>(max. 0.025% H&lt;sub&gt;2&lt;/sub&gt;O) SeccoSolv</td></tr><tr><td>Combustion oven</td><td>Legallais</td><td></td><td></td></tr><tr><td>Pierce BCA protein assay kit</td><td>Interchim</td><td>UP40840A</td><td></td></tr><tr><td>Formic acid</td><td>Fluka</td><td>94318</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich</td><td>H1009</td><td></td></tr><tr><td>Hydrochloric Acid Fuming 37% Emsure</td><td>Merck</td><td>1003171000</td><td>Grade ACS,ISO,Reag. Ph Eur</td></tr><tr><td>Lithium acetate buffer</td><td>Biochrom</td><td>80-2038-10</td><td></td></tr><tr><td>Commercial solution of hydrolyzed amino acids</td><td>Sigma Aldrich</td><td>AAS18</td><td></td></tr><tr><td>L-Methionine sulfone</td><td>Sigma Aldrich</td><td>M0876</td><td></td></tr><tr><td>L-Cysteic acid monohydrate</td><td>Sigma Aldrich</td><td>30170</td><td></td></tr><tr><td>Pyrex glass culture tubes</td><td>Sigma Aldrich</td><td>Z653586</td><td></td></tr><tr><td>Pyridine</td><td>Acros Organics</td><td>131780500</td><td>99% Extrapure</td></tr><tr><td>Ethyl chloroformate</td><td>Sigma Aldrich</td><td>23131</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>32222</td><td></td></tr><tr><td>Vials</td><td>Sigma Aldrich</td><td>854165</td><td></td></tr><tr><td>Microinserts for 1.5ml vials</td><td>Sigma Aldrich</td><td>SU860066</td><td></td></tr><tr><td>GC/MS</td><td>Agilent Technologies</td><td>5890 GC/5973 MS</td><td></td></tr><tr><td>Chemstation</td><td>Agilent Technologies</td><td></td><td>Instrument control and data analysis software</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>34860</td><td>Chromasolv, for HPLC</td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>34998</td><td>ChromasolvPlus, for HPLC</td></tr><tr><td>N,N-Dimethylformamide dimethyl acetal</td><td>Sigma Aldrich</td><td>394963</td><td></td></tr><tr><td>BSTFA</td><td>Sigma Aldrich</td><td>33024</td><td></td></tr><tr><td>DB-17MS column</td><td>Agilent Technologies</td><td>122-4731</td><td>30m*0.25mm*0.15&amp;micro;m</td></tr><tr><td>Sodium sulfate, anhydrous</td><td>Sigma Aldrich</td><td>238597</td><td></td></tr><tr><td>Technical nitrogen</td><td>Air products</td><td>14629</td><td></td></tr><tr><td>Zebron ZB-WAX column</td><td>Phenomenex</td><td>7HG-G007-11</td><td>30m*0.25mm*0.25&amp;micro;m</td></tr><tr><td>Helium BIP</td><td>Air products</td><td>26699</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>VWR</td><td>612-1702</td><td></td></tr></tbody></table>

workflow based on the combination of isotopic tracer experiments to investigate microbial metabolism of multiple nutrient sources

微生物学领域的研究依赖于各种方法的实施。特别是, 适当的方法的发展大大有助于提供广泛的知识代谢的微生物生长在化学定义的媒体含有独特的氮和碳源。相比之下, 通过多种营养来源的新陈代谢的管理, 尽管它们在自然或工业环境中的广泛存在, 仍然几乎未被探索。这种情况主要是由于缺乏适当的方法, 妨碍了调查工作。
我们报告一个实验策略, 以定量和全面地探讨新陈代谢是如何运作的, 当一个营养素作为不同的分子,即, 一个复杂的资源的混合物提供。在这里, 我们描述了它的应用, 通过酵母代谢网络评估多氮源的划分。该工作流结合了稳定同位素示踪实验中所获得的信息, 使用选定的13C 或15N 标记基板。它首先包括平行和可再生的发酵在同一个媒介, 包括 N 包含分子的混合物;但是, 每次选定的氮源都被标记。结合分析程序 (HPLC, gc-ms), 以评估目标化合物的标记模式, 并量化的消耗和回收的基质在其他代谢物。完整的数据集的综合分析提供了在细胞内消耗的基质的命运的概述。这种方法需要一个精确的协议, 收集 samples–facilitated 的机器人辅助系统的在线监测 fermentations–and 实现了大量的耗时分析。尽管有这些限制, 它还是第一次允许对酵母代谢网络中的多个氮源进行划分。我们阐明了从更丰富的来源向其他 n-化合物的氮的再分配, 并确定了挥发性分子和 proteinogenic 氨基酸的代谢来源。

图 3提供了工作流的示意图, 该流程图是为了调查在葡萄酒发酵过程中发现的多种氮源的酵母的管理情况而实施的。 对于不同的取样点, 生物 parameters–growth 特性、氮耗形态和 proteinogenic 氨基 acids–show 的剖面在发酵中具有很高的重现性 (图 4)。这种一致性根据在一组14独立发酵中生成的数据的组合来验证方法的相关性。
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Watch this Scientific Journal Video about Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources at JoVE.com

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

本议定书描述了一个实验过程, 定量和全面地研究多种营养来源的代谢。这一工作流程, 基于同位素示踪实验和分析程序的结合, 使被消耗的营养素的命运和由微生物综合的分子的新陈代谢的起源被确定。

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

workflow-based-on-combination-isotopic-tracer-experiments-to

Research

JoVE Journal

Bioengineering

9.2K 次观看.  Université de Montpellier. 本议定书描述了一个实验过程, 定量和全面地研究多种营养来源的代谢。这一工作流程, 基于同位素示踪实验和分析程序的结合, 使被消耗的营养素的命运和由微生物综合的分子的新陈代谢的起源被确定。

视频: Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

科研

环境科学

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

生物化学

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

生物学

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

<meta charset="utf8"></head><body>使用 OPTIR-FISH 进行细胞鉴定和代谢分析的综合方案

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol&#39;s standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

<meta charset="utf8"></head><body>作者聚焦:用于复杂环境中单细胞代谢和身份分析的集成 OPTIR-FISH

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

光学光热红外荧光原位杂交 （OPTIR-FISH）

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

摘要

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植物与放射性示踪剂测量矿质营养和毒物的通量

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

对于代谢物分析基于法 - 串联液相色谱 - 质谱法

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

代谢途径的确认和发现通过¹³ C - Proteinogenic氨基酸的标签

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

摘要

探索更多视频

此视频中的章节

植物与放射性示踪剂测量矿质营养和毒物的通量

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

对于代谢物分析基于法 - 串联液相色谱 - 质谱法

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

光学光热红外荧光原位杂交 （OPTIR-FISH）

光学光热红外荧光原位杂交 （OPTIR-FISH）

光学光热红外荧光原位杂交 （OPTIR-FISH）

全细胞Bioreporter方法评估交通有机污染物的生物利用度和水饱和系统

代谢途径的确认和发现通过¹³ C - Proteinogenic氨基酸的标签

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）

光学光热红外荧光原位杂交（OPTIR-FISH）