Hypothiocyanite and hypothiocyanous acid (OSCN − /HOSCN) are pseudohypohalous acids released by the innate immune system which are capable of rapidly oxidizing sulfur-containing amino acids, causing significant protein aggregation and damage to invading bacteria. HOSCN is abundant in saliva and airway secretions and has long been considered a highly specific antimicrobial that is nearly harmless to mammalian cells. However, certain bacteria, commensal and pathogenic, are able to escape damage by HOSCN and other harmful antimicrobials during inflammation, which allows them to continue to grow and, in some cases, cause severe disease. The exact genes or mechanisms by which bacteria respond to HOSCN have not yet been elucidated. We have found, in Escherichia coli , that the flavoprotein RclA, previously implicated in reactive chlorine resistance, reduces HOSCN to thiocyanate with near-perfect catalytic efficiency and strongly protects E. coli against HOSCN toxicity. This is notable in E. coli because this species thrives in the chronically inflamed environment found in patients with inflammatory bowel disease and is able to compete with and outgrow other important commensal organisms, suggesting that HOSCN may be a relevant antimicrobial in the gut, which has not previously been explored. RclA is conserved in a variety of epithelium-colonizing bacteria, implicating its HOSCN reductase activity in a variety of host–microbe interactions. We show that an rclA mutant of the probiotic Limosilactobacillus reuteri is sensitive to HOSCN and that RclA homologs from Staphylococcus aureus , Streptococcus pneumoniae , and Bacteroides thetaiotaomicron all have potent protective activity against HOSCN when expressed in E. coli .

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.

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.