Dass Rupashree, Grudzia Ż Katarzyna, Ishikawa Takao, Nowakowski Michał, Dȩbowska Renata, Kazimierczuk Krzysztof
Centre of New Technologies, University of WarsawWarsaw, Poland.
Faculty of Chemistry, Biological and Chemical Research Centre, University of WarsawWarsaw, Poland.
Front Microbiol. 2017 Jul 14;8:1306. doi: 10.3389/fmicb.2017.01306. eCollection 2017.
The biological toolbox is full of techniques developed originally for analytical chemistry. Among them, spectroscopic experiments are very important source of atomic-level structural information. Nuclear magnetic resonance (NMR) spectroscopy, although very advanced in chemical and biophysical applications, has been used in microbiology only in a limited manner. So far, mostly one-dimensional H experiments have been reported in studies of bacterial metabolism monitored . However, low spectral resolution and limited information on molecular topology limits the usability of these methods. These problems are particularly evident in the case of complex mixtures, where spectral peaks originating from many compounds overlap and make the interpretation of changes in a spectrum difficult or even impossible. Often a suite of two-dimensional (2D) NMR experiments is used to improve resolution and extract structural information from internuclear correlations. However, for dynamically changing sample, like bacterial culture, the time-consuming sampling of so-called indirect time dimensions in 2D experiments is inefficient. Here, we propose the technique known from analytical chemistry and structural biology of proteins, i.e., time-resolved non-uniform sampling. The method allows application of 2D (and multi-D) experiments in the case of quickly varying samples. The indirect dimension here is sparsely sampled resulting in significant reduction of experimental time. Compared to conventional approach based on a series of 1D measurements, this method provides extraordinary resolution and is a real-time approach to process monitoring. In this study, we demonstrate the usability of the method on a sample of culture affected by ampicillin and on a sample of , an acne causing bacterium, mixed with a dose of face tonic, which is a complicated, multi-component mixture providing complex NMR spectrum. Through our experiments we determine the exact concentration and time at which the anti-bacterial agents affect the bacterial metabolism. We show, that it is worth to extend the NMR toolbox for microbiology by including techniques of 2D z-TOCSY, for total "fingerprinting" of a sample and 2D C-edited HSQC to monitor changes in concentration of metabolites in selected metabolic pathways.
生物工具箱中充满了最初为分析化学开发的技术。其中，光谱实验是原子级结构信息的非常重要的来源。核磁共振（NMR）光谱学虽然在化学和生物物理应用中非常先进，但在微生物学中的应用却很有限。到目前为止，在监测细菌代谢的研究中大多只报道了一维氢实验。然而，低光谱分辨率和关于分子拓扑结构的有限信息限制了这些方法的实用性。这些问题在复杂混合物的情况下尤为明显，其中来自许多化合物的光谱峰相互重叠，使得对光谱变化的解释变得困难甚至不可能。通常会使用一套二维（2D）NMR实验来提高分辨率并从核间相关性中提取结构信息。然而，对于动态变化的样品，如细菌培养物，二维实验中所谓间接时间维度的耗时采样效率很低。在此，我们提出了蛋白质分析化学和结构生物学中已知的技术，即时间分辨非均匀采样。该方法允许在快速变化的样品情况下应用二维（及多维）实验。这里的间接维度进行稀疏采样，从而显著减少实验时间。与基于一系列一维测量的传统方法相比，该方法提供了非凡的分辨率，并且是一种用于过程监测的实时方法。在本研究中，我们证明了该方法在受氨苄青霉素影响的培养物样品以及与一定剂量爽肤水混合的痤疮丙酸杆菌样品上的可用性，后者是一种复杂的多组分混合物，提供复杂的NMR光谱。通过我们的实验，我们确定了抗菌剂影响细菌代谢的确切浓度和时间。我们表明，通过纳入二维z-TOCSY技术进行样品的全面“指纹识别”以及二维C编辑HSQC来监测选定代谢途径中代谢物浓度的变化，值得为微生物学扩展NMR工具箱。
