Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. To capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two-photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG not only can monitor the movement of small molecules across membrane leaflets but also is sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.

Advancements in antibiotic drug design are often hindered by missing information on how these small molecules interact with living cells. The antibiotic, daptomycin, has found clinical success and an emerging resistance, but a comprehensive picture of its mechanism of action has remained elusive. Using a surface-specific spectroscopy technique, second harmonic generation, we are able to quantitatively assess the binding of daptomycin to living cell membranes without the addition of exogenous labels. Our results reveal similar binding affinities for both Gram-positive and Gram-negative bacteria studied, including Escherichia coli. More importantly, we show that the presence of phosphate ions influences the binding of daptomycin to the Gram-positive bacterium Enterococcus faecalis. The role of environmental phosphate has not previously been considered in any proposed mechanism, and its implications are expected to be important in vivo.

Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. In order to capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membranespecific probe molecules. Our results show that SHG can not only monitor the movement of small molecules across membrane leaflets, but is also sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics. STATEMENT OF SIGNIFICANCEBacterial membranes are highly adept at discerning and modifying their interactions with different small molecules in their environment. Here we show how second harmonic generation (SHG) spectroscopy can track the dynamics of structurally similar membrane probes in two gram-positive bacterial species. Our results reveal behavior that is dependent on both the probe molecule and the membrane composition. Specifically, we observe flip-flop between leaflets for one molecule, while the other molecule produces a signal indicative of larger scale ordering in the membrane. These phenomena can all be explained by considering potential differences in the membrane fluidity and surface charge between the two bacterial species. Overall, our work highlights the dynamic differences between bacterial membranes and SHG's sensitivity to probing these systems.

Advancements in antibiotic drug design are often hindered by missing information in how these small molecules interact with living cells. The antibiotic, daptomycin, has found clinical success, and emerging resistance, but a comprehensive picture of its mechanism of action has remained elusive. Using the surface-specific spectroscopy, second harmonic generation, we are able to quantitatively assess the binding of daptomycin to living cell membranes without the addition of exogenous labels. Our results reveal similar binding affinities for both gram-positive and gram-negative bacteria studied, including <i>E. coli</i>. More importantly, we show that phosphate ions influence the binding of daptomycin to the gram-positive bacterium <i>E. faecalis</i>. The role of environmental phosphate has not previously been considered in any proposed mechanism and its implications are expected to be important <i>in vivo</i>.

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