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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 ¹H 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 ¹³C-edited HSQC to monitor changes in concentration of metabolites in selected metabolic pathways.

SUBMITTER: Dass R

PROVIDER: S-EPMC5509914 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The analysis of 1D anti-diagonal spectra from the projections of 2D double-quantum filtered correlation spectroscopy NMR spectra is presented for the determination of the compositions of liquid mixtures of linear and branched alkanes confined within porous media. These projected spectra do not include the effects of line broadening and therefore retain high-resolution information even in the presence of inhomogeneous magnetic fields as are commonly found in porous media. A partial least-square regression analysis is used to characterize the mixture compositions. Two case studies are considered. First, mixtures of 2-methyl alkanes and n-alkanes are investigated. It is shown that estimation of the mol % of branched species present was achieved with a root-mean-square error of prediction (RMSEP) of 1.4 mol %. Second, the quantification of multicomponent mixtures consisting of linear alkanes and 2-, 3-, and 4-monomethyl alkanes was considered. Discrimination of 2-methyl and linear alkanes from other branched isomers in the mixture was achieved, although discrimination between 3- and 4- monomethyl alkanes was not possible. Compositions of the linear alkane, 2-methyl alkane, and the total composition of 3- and 4-methyl alkanes were estimated with a RMSEP <3 mol %. The approach was then used to estimate the composition of the mixtures in terms of submolecular groups of CH3CH2, (CH3)2CH, and CH2CH(CH3)CH2 present in the mixtures; a RMSEP <1 mol % was achieved for all groups. The ability to characterize the mixture compositions in terms of molecular subgroups allows the application of the method to characterize mixtures containing multimethyl alkanes. The motivation for this work is to develop a method for determining the mixture composition inside the catalyst pores during Fischer-Tropsch synthesis. However, the method reported is generic and can be applied to any system in which there is a need to characterize mixture compositions of linear and branched alkanes.

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

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