Solid-State NMR on Bacterial Cells: Selective Cell Wall Signal Enhancement and Resolution Improvement using Dynamic Nuclear Polarization

Abstract

Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool.

Hydrolysis of peptidoglycan is modulated by amidation of meso‐diaminopimelic acid and Mg2+ in Bacillus subtilis

“…Nevertheless, the effect of Mg 2+ on inhibition of lysis has now been shown both in Gram‐positive (present study) and in phylogenetically distant Gram‐negative organisms (Gschwender and Hofschneider, 1969; Rayman and MacLeod, 1975; Leduc et al ., 1982), possibly suggesting the existence of a common inhibitory mechanism. The reported affinity of wild‐type cell wall for Mg 2+ is on the order of approximately 0.6 mM (Takahashi et al ., 2013; Kern et al ., 2010). At milimolar concentrations the vast majority of binding sites on the cell wall are occupied by Mg 2+ .…”

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

“…In this way, DNP can also enable the selective observation of compositional contributions from the outside of the cell versus those from inside the cell, for example, if the polarizing agent stays on the outside and cannot penetrate the cell [62].…”

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

“…Furthermore, we can selectively isolate for investigation those cellular constituents that are closely associated with the pigment and thereby protected from both environmental effects and chemical degradation. Finally, high resolution solid-state nuclear magnetic resonance (NMR) approaches can yield direct insights into the atomic level structure, dynamics, and action mechanisms of noncrystalline bioassemblies, including plant and microbial complexes that have polysaccharide or lipid constituents (11)(12)(13)(14)(15)(16). Thus, it is feasible to circumvent the difficulties of solubilization or crystallization to access macromolecular structure for pigments derived from known small molecule isotopically enriched precursors by using the C. neoformans fungal melanin and solid-state NMR.…”