Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells

Abstract

Solid-state NMR is a powerful and non-perturbative method to measure and define chemical composition and architecture in bacterial cell walls, even in the context of whole cells. Most NMR studies on whole cells have used selectively labeled samples. Here, we introduce an NMR sequence relay using frequency-selective REDOR (fsREDOR) and spin diffusion elements to probe a unique amine contribution in uniformly 13 C-and 15 N-labeled Staphylococcus aureus whole cells that we attribute to the D-alanine of teichoic acid. In addition to the primary peptidoglycan structural scaffold, cell walls can contain significant amounts of teichoic acid that contribute to cell-wall function. When incorporated into teichoic acid, D-alanine is present as an ester, connected via its carbonyl to a ribitol carbon, and thus has a free amine. Teichoic acid D-Ala is removed during cell-wall isolations and can only be detected in the context of whole cells. The sequence presented here begins with fsREDOR and a chemical shift evolution period for 2D data acquisition, followed by DARR spin diffusion and then an additional fsREDOR period. fsREDOR elements were used for 13 C observation to avoid complications from 13 C-13 C couplings due to uniform labeling and for 15 N dephasing to achieve selectivity in the nitrogens serving as dephasers. The results show that the selected amine nitrogen of interest is near to teichoic acid ribitol carbons and also the methyl group carbon associated with alanine. In addition, its carbonyl is not significantly dephased by amide nitrogens, consistent with the expected microenvironment around teichoic acid.

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance

“…The peak at 42 ppm has been ascribed as having contributions from the D-Ala of teichoic acids. 43 We confirmed this assignment and demonstrated here that the 42-ppm peak is entirely due to teichoic acid D-Ala by comparison with the whole-cell NMR spectrum of a dltA mutant, which lacks the dltA gene responsible for the D-alanylation of both wall and lipoteichoic acids. 53 In the dltA mutant, the 42-ppm signal was abolished (Fig 4).…”

“…Samples are either examined at natural abundance 13 C or through previously-reported uniform labeling in which all carbons are 13 C-labeled or all nitrogens are 15 N-labeled in the cell wall and whole cell samples. 42,43 We present an extensive comparative sample analysis, including ten whole-cell NMR samples and cells treated with a teichoic acid inhibitor (tunicamycin) to emphasize the sensitivity of the whole-cell NMR approach.…”

“…We present here a new strategy, demonstrating that interpretable and quantifiable spectral changes in cell-wall and whole-cell samples can report on changes to PG and WTA without any selective labeling. Samples are examined either at natural abundance 13 C or through previously reported uniform labeling in which all carbons are labeled with 13 C or all nitrogens are labeled with 15 N in the cell-wall and whole-cell samples. , We present an extensive comparative sample analysis, including 10 whole-cell NMR samples and cells treated with a teichoic acid inhibitor (tunicamycin), to emphasize the sensitivity of the whole-cell NMR approach.…”

Magnetic Resonance Spectroscopy as a Tool for Assessing Macromolecular Structure and Function in Living Cells

“…By using the high-resolution magic-angle spinning (HR-MAS) technique, Jachymek et al (93) demonstrated that the immunodominant O-polysaccharide on bacterial surfaces had the same structure as that in lipopolysaccharides and in isolated PSs. HR-MAS was also used to study capsular PSs, particularly sugar and capsular glycan modifications on Campylobacter cells (94), as well as the chemical composition and architecture of the bacterial and plant cell wall (95)(96)(97)(98)(99) and whole microalgae (100). In addition, evolution of the O-acetylation of the O-antigen on the outer bacteria membrane has been traced by HR-MAS (30).…”

Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells

“…Atomic-level detail for DNA packaging in bacteriophage T4 capsids employed N­{P} REDOR to describe the electrostatic interactions and mechanisms for DNA charge balance in capsid packaging . Furthermore, we have been developing approaches to identify and compare spectral signatures of specific biomolecules in the context of intact whole cells. − For example, we were able to distinguish whether bacteria were treated with a cell-wall-targeting antibiotic (fosfomycin) or a protein synthesis inhibitor targeting the ribosome (chloramphenicol) by inspecting the natural abundance 13 C NMR spectra of antibiotic-treated whole cells …”