Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response.

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, ∆mscL, ∆mscS, a double knockout ∆mscS ∆mscK, and a triple knockout ∆mscL ∆mscS ∆mscK, in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains, MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the ∆mscL strain, suggesting either a crosstalk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants.

Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response.

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, Δ mscL , Δ mscS , a double knockout Δ mscS Δ mscK , and a triple knockout Δ mscL Δ mscS Δ mscK in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the Δ mscL strain, suggesting either a cross-talk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants. Summary for the table of contents: The kinetics of hypotonic osmolyte release from E. coli is analyzed in conjunction with bacterial survival. It is shown that MscL, the high-threshold 'emergency release valve', rescues bacteria from down-shocks only in the presence of MscS, MscK or other low-threshold channels that are necessary to pacify MscL at the end of the release phase.

Partitioning of Seven Different Classes of Antibiotics into LPS Monolayers Supports Three Different Permeation Mechanisms through the Outer Bacterial Membrane.

The outer membrane (OM) of Gram-negative (G-) bacteria presents a barrier for many classes of antibacterial agents. Lipopolysaccharide (LPS), present in the outer leaflet of the OM, is stabilized by divalent cations and is considered to be the major impediment for antibacterial agent permeation. However, the actual affinities of major antibiotic classes toward LPS have not yet been determined. In the present work, we use Langmuir monolayers formed from E. coli Re and Rd types of LPS to record pressure-area isotherms in the presence of antimicrobial agents. Our observations suggest three general types of interactions. First, some antimicrobials demonstrated no measurable interactions with LPS. This lack of interaction in the case of cefsulodin, a third-generation cephalosporin antibiotic, correlates with its low efficacy against G- bacteria. Ampicillin and ciprofloxacin also show no interactions with LPS, but in contrast to cefsulodin, both exhibit good efficacy against G- bacteria, indicating permeation through common porins. Second, we observe substantial intercalation of the more hydrophobic antibiotics, novobiocin, rifampicin, azithromycin, and telithromycin, into relaxed LPS monolayers. These largely repartition back to the subphase with monolayer compression. We find that the hydrophobic area, charge, and dipole all show correlations with both the mole fraction of antibiotic retained in the monolayer at the monolayer-bilayer equivalence pressure and the efficacies of these antibiotics against G- bacteria. Third, amine-rich gentamicin and the cationic antimicrobial peptides polymyxin B and colistin show no hydrophobic insertion but are instead strongly driven into the polar LPS layer by electrostatic interactions in a pressure-independent manner. Their intercalation stably increases the area per molecule (by up to 20%), which indicates massive formation of defects in the LPS layer. These defects support a self-promoted permeation mechanism of these antibiotics through the OM, which explains the high efficacy and specificity of these antimicrobials against G- bacteria.

Differential Interactions of Piscidins with Phospholipids and Lipopolysaccharides at Membrane Interfaces.

Piscidins 1 and 3 (P1 and P3) are potent antimicrobial peptides isolated from striped bass. Their mechanism of action involves formation of amphipathic α-helices on contact with phospholipids and destabilization of the microbial cytoplasmic membrane. The peptides are active against both Gram-positive and Gram-negative bacteria, suggesting easy passage across the outer membrane. Here, we performed a comparative study of these two piscidins at the air-water interface on lipopolysaccharide (LPS) monolayers modeling the outer bacterial surface of Gram-negative organisms and on phospholipid monolayers, which mimic the inner membrane. The results show that P1 and P3 are highly surface active (log KAW ∼ 6.8) and have similar affinities to phospholipid monolayers (log Klip ≈ 7.7). P1, which is more potent against Gram negatives, exhibits a much stronger partitioning into LPS monolayers (log KLPS = 8.3). Pressure-area isotherms indicate that under increasing lateral pressures, inserted P1 repartitions from phospholipid monolayers back to the subphase or to a more shallow position with in-plane areas of ∼170 Å2 per peptide, corresponding to fully folded amphipathic α-helices. In contrast, peptide expulsion from LPS occurs with areas of ∼35 Å2, suggesting that the peptides may not form the similarly oriented, rigid secondary structures when they avidly intercalate between LPS molecules. Patch-clamp experiments on Escherichia coli spheroplasts show that when P1 and P3 reach the outer surface of the bacterial cytoplasmic membrane, they produce fluctuating conductive structures at voltages above 80 mV. The data suggests that the strong activity of these piscidins against Gram-negative bacteria begins with the preferential accumulation of peptides in the outer LPS layer followed by penetration into the periplasm, where they form stable amphipathic α-helices upon contact with phospholipids and attack the energized inner membrane.