Fast 2D NMR Spectroscopy for In vivo Monitoring of Bacterial Metabolism in Complex Mixtures

Abstract

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 1 H experiments have been reported in studies of bacterial metabolism monitored in situ. 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 Escherichia coli culture affected by ampicillin and on a sample of Propionibacterium acnes, 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 13 C-edited HSQC to monitor changes in concentration of metabolites in selected metabolic pathways.

Enhancing Compression Level for More Efficient Compressed Sensing and Other Lessons from NMR Spectroscopy

“…The application of NUS and CS in their work allowed to precisely track peak positions and intensities during sample heating. The reactions occurring in complex mixtures were also extensively investigated by NUS/CS NMR spectroscopy [3,84]. Usually, such mixtures require at least 2D NMR experiment to resolve important peaks in a spectrum, which makes monitoring troublesome using conventional sampling.…”

Alternative data processing techniques for serial NMR experiments

“…In case peaks move, separate processing of each of the "frames" using compressed sensing methods is effective. [32][33][34]…”

Fast quantitative 2D NMR for metabolomics and lipidomics: A tutorial

“…Although these methods are associated with sensitivity losses and/or resolution penalties, these limitations are well described and understood. Two of these methods (nonuniform sampling or NUS and ultrafast [UF] NMR) have successfully been used in targeted and untargeted metabolomics and lipidomics workflows …”