Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria

Abstract

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.

Determination of bacterial surface charge density via saturation of adsorbed ions

“…The measured time-resolved SHS kinetic traces depicted in Fig. 2 can be fit, using our established model, to determine the molecular transport rates for the various bacterial interfaces (i.e., OM, PM, S-layer, and CM) (20)(21)(22)(23)(24)(25)(26)(27)(28). It is found that MG cation transport across the OM proteins in the OM and the pores in the S-layer is similarly rapid and was deduced as 0.04 and 0.02 s À1 , respectively.…”

“…This characteristic mechanism has previously been employed to monitor molecular adsorption and transport across phospholipid membranes in biomimetic model liposomes (12)(13)(14)(15)(16)(17)(18)(19) and living cells (20)(21)(22)(23)(24)(25)(26)(27)(28). When SHG-active molecules adsorb onto the exterior surface of the membrane, they align with one another because of the similarity of the interaction driving the adsorption process.…”

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

“…Although we observe Ebp puncta at the septal membrane, it is possible that Ebp are polymerized at the septum, possibly within punctate microdomains, and subsequently move within the cell membrane towards the cell hemisphere for anchoring by SrtA. Microdomains in the bacterial cell membrane are dynamic and fluidic in nature (Los and Murata, 2004; Miller et al, 2019). Furthermore, new surface-exposed Ebp were only seen at the cell hemipsheres with or without cell wall inhibition.…”

Determination of Bacterial Surface Charge Density Via Saturation of Adsorbed Ions

“…This characteristic mechanism has previously been employed to monitor molecular adsorption and transport across phospholipid membranes in biomimetic model liposomes (12)(13)(14)(15)(16)(17)(18)(19) and living cells. (20)(21)(22)(23)(24)(25)(26)(27)(28) When SHG-active molecules adsorb onto the exterior surface of the membrane, they align with one another due to the similarity of the interaction driving the adsorption process. This oriented ensemble of SHG-active molecules gives rise to a coherent SHS response, the intensity of which scales as the square of the molecular surface density.…”