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In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31 P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31 P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31 P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.

In Situ Monitoring of Bacteria under Antimicrobial Stress Using 31P Solid-State NMR