Effect of Lipid Composition on the Inhibition Mechanism of Amiloride on Alamethicin Ion Channels in Supported Phospholipid Bilayers.

The inhibition effect of amiloride on alamethicin ion channels was studied in a model zwitterionic floating bilayer lipid membrane (fBLM). The EIS studies indicated that amiloride prevents the transport of ions through the alamethicin channels leading to an overall increase in membrane resistance. The PM-IRRAS data demonstrated that amiloride has no influence on the secondary structure of alamethicin but restricts the insertion of the peptides into the bilayer and blocks ion transport through preformed alamethicin channels. The effect of amiloride on ion channel formation in the floating bilayer formed by a zwitterionic lipid was compared to those of previous studies involving negatively charged fBLMs and tethered zwitterionic lipid bilayers. The findings from these studies show that the effects of amiloride on ion channel formation strongly depend on the mobility and charge of the membrane lipids.

Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin.

We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.

Characterisation of UDP-glucuronosyltransferase activity in sea turtle Chelonia mydas.

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest Vmax (1203 pmol·min-1mg·protein-1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.

Discovery, Synthesis, and Optimization of Peptide-Based Antibiotics.

The rise of multidrug resistant bacteria has significantly compromised our supply of antibiotics and poses an alarming medical and economic threat to society. To combat this problem, it is imperative that new antibiotics and treatment modalities be developed, especially those toward which bacteria are less capable of developing resistance. Peptide natural products stand as promising candidates to meet this need as bacterial resistance is typically slow in response to their unique modes of action. They also have additional benefits including favorable modulation of host immune responses and often possess broad-spectrum activity against notoriously treatment resistant bacterial biofilms. Moreover, nature has provided a wealth of peptide-based natural products from a range of sources, including bacteria and fungi, which can be hijacked in order to combat more dangerous clinically relevant infections.This Account highlights recent advances in the total synthesis and development of a range of peptide-based natural product antibiotics and details the medicinal chemistry approaches used to optimize their activity.In the context of antibiotics with potential to treat Gram-positive bacterial infections, this Account covers the synthesis and optimization of the natural products daptomycin, glycocin F, and alamethicin. In particular, the reported synthesis of daptomycin highlights the utility of on-resin ozonolysis for accessing a key kynurenine residue from the canonical amino acid tryptophan. Furthermore, the investigation into glycocin F analogues uncovered a potent lead compound against Lactobacillus plantarum that bears a non-native thioacetal linkage to a N-acetyl-d-glucosamine (GlcNAc) sugar, which is otherwise O-linked in its native form.For mycobacterial infections, this Account covers the synthesis and optimization of teixobactin, callyaerin A, lassomycin, and trichoderin A. The synthesis of callyaerin A, in particular, highlighted the importance of a (Z)-2,3-diaminoacrylamide motif for antimicrobial activity against Mycobacterium tuberculosis, while the synthesis of trichoderin A highlighted the importance of (R)-stereoconfiguration in a key 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD) residue.Lastly, this Account covers lipopeptide antibiotics bearing activity toward Gram-negative bacterial infections, namely, battacin and paenipeptin C. In both cases, optimization of the N-terminal lipid tails led to the identification of analogues with potent activity toward Escherichia coli and Pseudomonas aeruginosa.

Fusaricidin-Type Compounds Create Pores in Mitochondrial and Plasma Membranes of Mammalian Cells.

Fusaricidins and related LI-F compounds are effective bactericides and fungicides. Recently, we have found that they are highly toxic to mammalian cells. Here, we studied the effect of fusaricidin-type compounds (FTCs) on the membranes of mammalian cells. Ethanol extracts from Paenibacillus polymyxa strains, RS10 and I/Sim, were fractionated and analyzed by HPLC and mass spectrometry. The effects of FTCs on mitochondrial functions and integrity were studied by standard methods: measurements of swelling, membrane potential (ΔΨm), respiration rate, cytochrome c release, and pore sizes. Superoxide flashes were registered by 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA). Plasma membrane permeability was assessed by propidium iodide (PI) staining and ATP release. FTCs caused the permeabilization of the inner mitochondria membrane (IMM) to ions and low-molecular-weight (~750 Da) solutes. The permeabilization did not depend on the permeability transition pore (mPTP) but was strongly dependent on ΔΨm. Fusaricidins A plus B, LI-F05a, and LI-F05b-LI-F07b permeabilized IMM with comparable efficiency. They created pores and affected mitochondrial functions and integrity similarly to mPTP opening. They permeabilized the sperm cell plasma membrane to ATP and PI. Thus, the formation of pores in polarized membranes underlays the toxicity of FTCs to mammals. Besides, FTCs appeared to be superior reference compounds for mPTP studies.

Understanding the antimicrobial activity of water soluble γ-cyclodextrin/alamethicin complex.

In our previous investigation (Zhang et. al., J. Functional Foods 40 (2018) 700-706), we have proposed a method of complexation of alamethicin (ALM) with γ-cyclodextrin (γ-CD) to increase the solubility and demonstrated an enhancement in its antimicrobial activity against Listeria monocytogenes. In this study, transmission electron microscopy of L. monocytogenes treated with γ-CD/ALM complex indicated cell lysis due to pore formation. This was further corroborated by fluorescent dye leakage from DMPC/cholesterol liposomes when exposed to γ-CD/ALM complex for molar ratios of 1, 5 and 10. The extent of dye leakage increased with ALM-lipid ratio in the range of 0.00015 to 0.16. Dye leakage of γ-CD/ALM complex was found to be the highest for molar ratio of 5, consistent with our earlier results of antimicrobial activity of the complex against L. monocytogenes. All atom molecular dynamics (MD) simulation showed that γ-CD/ALM complex can effectively bind to the 3:1 POPE/POPG bilayer, a mimic of bacterial cell membrane. In addition, circular dichroism spectrum indicated that ALM in the complex has a helical conformation in solution as well as in the presence of liposome. Transmembrane MD simulation of six γ-CD/ALM complex aggregates in α helical conformation showed water channel with barrel stave like pore structure.

The antibiotic peptaibol alamethicin from Trichoderma permeabilises Arabidopsis root apical meristem and epidermis but is antagonised by cellulase-induced resistance to alamethicin.

BACKGROUND: Trichoderma fungi live in the soil rhizosphere and are beneficial for plant growth and pathogen resistance. Several species and strains are currently used worldwide in co-cultivation with crops as a biocontrol alternative to chemical pesticides even though little is known about the exact mechanisms of the beneficial interaction. We earlier found alamethicin, a peptide antibiotic secreted by Trichoderma, to efficiently permeabilise cultured tobacco cells. However, pre-treatment with Trichoderma cellulase made the cells resistant to subsequent alamethicin, suggesting a potential mechanism for plant tolerance to Trichoderma, needed for mutualistic symbiosis. RESULTS: We here investigated intact sterile-grown Arabidopsis thaliana seedlings germinated in water or growth medium. These could be permeabilised by alamethicin but not if pretreated with cellulase. By following the fluorescence from the membrane-impermeable DNA-binding probe propidium iodide, we found alamethicin to mainly permeabilise root tips, especially the apical meristem and epidermis cells, but not the root cap and basal meristem cells nor cortex cells. Alamethicin permeabilisation and cellulase-induced resistance were confirmed by developing a quantitative in situ assay based on NADP-isocitrate dehydrogenase accessibility. The combined assays also showed that hyperosmotic treatment after the cellulase pretreatment abolished the induced cellulase resistance. CONCLUSION: We here conclude the presence of cell-specific alamethicin permeabilisation, and cellulase-induced resistance to it, in root tip apical meristem and epidermis of the model organism A. thaliana. We suggest that contact between the plasma membrane and the cell wall is needed for the resistance to remain. Our results indicate a potential mode for the plant to avoid negative effects of alamethicin on plant growth and localises the point of potential damage and response. The results also open up for identification of plant genetic components essential for beneficial effects from Trichoderma on plants.

The effects of UDP-sugars, UDP and Mg2+on uridine diphosphate glucuronosyltransferase activity in human liver microsomes.

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation. 2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg2+ ions. We also determined whether UDP-sugars other than the co-substrate UDP-glucuronic acid (UDP-GlcA) affected glucuronidation. 3. We show that other UDP-sugars do not significantly influence glucuronidation. We also demonstrate that UDP inhibits HLM UGT activity and that this is reversed by including Mg2+ in the assay. The Mg2+ effect can be offset using EDTA, and is dependent on the concentration of UDP-GlcA in the assay. 4. We propose that formation of a Mg2+-UDP complex prevents UDP from affecting the enzyme. Our results suggest that 5 mM UDP-GlcA and 10 mM Mg2+ be used for UGT assays in fully disrupted HLM.

The Role of Terminal Functionality in the Membrane and Antibacterial Activity of Peptaibol-Mimetic Aib Foldamers.

Peptaibols are peptide antibiotics that typically feature an N-terminal acetyl cap, a C-terminal aminoalcohol, and a high proportion of α-aminoisobutyric acid (Aib) residues. To establish how each feature might affect the membrane-activity of peptaibols, biomimetic Aib foldamers with different lengths and terminal groups were synthesised. Vesicle assays showed that long foldamers (eleven Aib residues) with hydrophobic termini had the highest ionophoric activity. C-terminal acids or primary amides inhibited activity, while replacement of an N-terminal acetyl with an azide group made little difference. Crystallography showed that N3 Aib11 CH2 OTIPS folded into a 310 helix 2.91 nm long, which is close to the bilayer hydrophobic width. Planar bilayer conductance assays showed discrete ion channels only for N-acetylated foldamers. However long foldamers with hydrophobic termini had the highest antibacterial activity, indicating that ionophoric activity in vesicles was a better indicator of antibacterial activity than the observation of discrete ion channels.

Enhancing the Antimicrobial Activity of Alamethicin F50/5 by Incorporating N-terminal Hydrophobic Triazole Substituents.

A simple and efficient strategy is proposed to significantly improve the antibacterial activity of peptaibols and other antimicrobial peptides by N-terminal capping with 1,2,3-triazole bearing various hydrophobic substituents on C-4. Such N-terminal insertions on alamethicin F50/5 could enhance its antimicrobial activity on Gram-positive bacteria without modification of its overall three-dimensional structure. Although the native peptide and its analogues shared comparable helical contents, the crystal structure of one of the most active derivative showed a local slight distortion of the N-terminal extremity, which was also observed in solution using NMR spectroscopy. Importantly, fluorescence studies showed that the N-capped derivatives had increased affinity for liposomes, which may indicate they interacted more strongly with the bacterial membrane than alamethicin F50/5.

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex III.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex II.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site IIf); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site IIf The decrease in matrix [dicarboxylate] allows O2 access to reduced site IIf, thereby making electron donation to O2 possible, explaining the rapid increase in ROS production provided that site IIf is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

Communication: Alamethicin can capture lipid-like molecules in the membrane.

Alamethicin (Alm) is a 19-mer antimicrobial peptide produced by fungus Trichoderma viride. Above a threshold concentration, Alm forms pores across the membrane, providing a mechanism of its antimicrobial action. Here we show that at a small concentration which is below the threshold value, Alm participates in formation of nanoscale lipid-mediated clusters of guest lipid-like molecules in the membrane. These results are obtained by electron spin echo (ESE) technique-a pulsed version of electron paramagnetic resonance-on spin-labeled stearic acid in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer with Alm added at 1/200 peptide-to-lipid ratio. ESE decay measurements are interpreted assuming that stearic acid molecules in the membrane are assembling around the Alm molecule. One may suggest that this Alm capturing effect on the guest lipid-like molecules could be important for the peptide antimicrobial action.

Alamethicin for using in bioavailability studies? - Re-evaluation of its effect.

A major pathway for the elimination of drugs is the biliary and renal excretion following the formation of more hydrophilic secondary metabolites such as glucuronides. For in vitro investigations of the phase II metabolism, hepatic microsomes are commonly used in the combination with the pore-forming peptide alamethicin, also to give estimates for the in vivo situation. Thus, alamethicin may represent a neglected parameter in the characterization of microsomal in vitro assays. In the present study, the influence of varying alamethicin concentrations on glucuronide formation of selected phenolic compounds was investigated systematically. A correlation between the alamethicin impact and the lipophilicity of the investigated substrates was analyzed as well. Lipophilicity was determined by the logarithm of the octanol-water partition coefficient. For every substrate, a distinct alamethicin concentration could be detected leading to a maximal glucuronidation activity. Further increase of the alamethicin application led to negative effects. The differences between the maximum depletion rates with and without alamethicin addition varied between 2.7% and 18.2% depending on the substrate. A dependence on the lipophilicity could not be confirmed. Calculation of the apparent intrinsic clearance led to a more than 2-fold increase using the most effective alamethicin concentration compared to the alamethicin free control.

Common Mechanism of Cross-Resistance Development in Pathogenic Bacteria Bacillus cereus Against Alamethicin and Pediocin Involves Alteration in Lipid Composition.

To understand the mechanism of development of cross-resistance in food pathogen Bacillus cereus against an antimicrobial peptide pediocin and antibiotic alamethicin, the present study was designed. Pediococcus pentosaceus was taken as a source of pediocin, and it was purified by ammonium sulphate precipitation followed by cation exchange chromatography with 14.01-fold purity and 14.4 % recovery. B. cereus strains alamethicin-resistant strains (IC50 3.23 µg/ml) were selected from sensitive population with IC50 2.37 µg/ml. The development of resistance in B. cereus against alamethicin was associated with decrease in alamethicin-membrane interaction observed by in vitro assay. Resistant strain of B. cereus was found to harbour one additional general lipid as compared to sensitive strain, one amino group lacking phospholipid and one amino group containing phospholipid (ACP). In addition, ACP content was increased in resistant mutant (29.7 %) as compared to sensitive strain (14.56 %). The alamethicin-resistant mutant B. cereus also showed increased IC50 (58.8 AU/ml) for pediocin as compared to sensitive strain (IC50 47.8 AU/ml). Cross-resistance to pediocin and alamethicin in resistant mutant of B. cereus suggested a common mechanism of resistance. Therefore, this understanding could result in the development of peptide which will be effective against the resistant strains that share same mechanism of resistance.

Incorporation of ß-Silicon-ß3-Amino Acids in the Antimicrobial Peptide Alamethicin Provides a 20-Fold Increase in Membrane Permeabilization.

Incorporation of silicon-containing amino acids in peptides is known to endow the peptide with desirable properties such as improved proteolytic stability and increased lipophilicity. In the presented study, we demonstrate that incorporation of ß-silicon-ß3-amino acids into the antimicrobial peptide alamethicin provides the peptide with improved membrane permeabilizing properties. A robust synthetic procedure for the construction of ß-silicon-ß3-amino acids was developed and the amino acid analogues were incorporated into alamethicin at different positions of the hydrophobic face of the amphipathic helix by using SPPS. The incorporation was shown to provide up to 20-fold increase in calcein release as compared with wild-type alamethicin.

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide.

1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency. 2. Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. 3. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. 4. These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.

Cellular and molecular insight into the inhibition of primary root growth of Arabidopsis induced by peptaibols, a class of linear peptide antibiotics mainly produced by Trichoderma spp.

Trichoderma spp. are well known biocontrol agents that produce a variety of antibiotics. Peptaibols are a class of linear peptide antibiotics mainly produced by Trichoderma Alamethicin, the most studied peptaibol, is reported as toxic to plants at certain concentrations, while the mechanisms involved are unclear. We illustrated the toxic mechanisms of peptaibols by studying the growth-inhibitory effect of Trichokonin VI (TK VI), a peptaibol from Trichoderma longibrachiatum SMF2, on Arabidopsis primary roots. TK VI inhibited root growth by suppressing cell division and cell elongation, and disrupting root stem cell niche maintenance. TK VI increased auxin content and disrupted auxin response gradients in root tips. Further, we screened the Arabidopsis TK VI-resistant mutant tkr1. tkr1 harbors a point mutation in GORK, which encodes gated outwardly rectifying K(+)channel proteins. This mutation alleviated TK VI-induced suppression of K(+)efflux in roots, thereby stabilizing the auxin gradient. The tkr1 mutant also resisted the phytotoxicity of alamethicin. Our results indicate that GORK channels play a key role in peptaibol-plant interaction and that there is an inter-relationship between GORK channels and maintenance of auxin homeostasis. The cellular and molecular insight into the peptaibol-induced inhibition of plant root growth advances our understanding of Trichoderma-plant interactions.

Mechanism of voltage-gated channel formation in lipid membranes.

Although several molecular models for voltage-gated ion channels in lipid membranes have been proposed, a detailed mechanism accounting for the salient features of experimental data is lacking. A general treatment accounting for peptide dipole orientation in the electric field and their nucleation and growth kinetics with ion channel formation is provided. This is the first treatment that explains all the main features of the experimental current-voltage curves of peptides forming voltage-gated channels available in the literature. It predicts a regime of weakly voltage-dependent conductance, followed by one of strong voltage-dependent conductance at higher voltages. It also predicts values of the parameters expressing the exponential dependence of conductance upon voltage and peptide bulk concentration for both regimes, in good agreement with those reported in the literature. Most importantly, the only two adjustable parameters involved in the kinetics of nucleation and growth of ion channels can be varied over broad ranges without affecting the above predictions to a significant extent. Thus, the fitting of experimental current-voltage curves stems naturally from the treatment and depends only slightly upon the choice of the kinetic parameters.

Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis.

Alamethicin is a well-studied antimicrobial peptide (AMP) that kills Gram-positive bacteria. It forms narrow, barrel-stave pores in planar lipid bilayers. We present a detailed, time-resolved microscopy study of the sequence of events during the attack of alamethicin on individual, live Bacillus subtilis cells expressing GFP in the cytoplasm. At the minimum inhibitory concentration (MIC), the first observed symptom is the halting of growth, as judged by the plateau in measured cell length vs time. The data strongly suggest that this growth-halting event occurs prior to membrane permeabilization. Gradual degradation of the proton-motive force, inferred from a decrease in pH-dependent GFP fluorescence intensity, evidently begins minutes later and continues over about 5 min. There follows an abrupt permeabilization of the cytoplasmic membrane to the DNA stain Sytox Orange and concomitant loss of small osmolytes, causing observable cell shrinkage, presumably due to decreased turgor pressure. This permeabilization of the cytoplasmic membrane occurs uniformly across the entire membrane, not locally, on a timescale of 5s or less. GFP gradually leaks out of the cell envelope, evidently impeded by the shrunken peptidoglycan layer. Disruption of the cell envelope by alamethicin occurs in stages, with larger and larger species permeating the envelope as time evolves over tens of minutes. Some of the observed symptoms are consistent with the formation of barrel-stave pores, but the data do not rule out "chaotic pore" or "carpet" mechanisms. We contrast the effects of alamethicin and the human cathelicidin LL-37 on B. subtilis.