Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases

The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.     

非核糖体肽合成酶(NRPSs)作用机理与应用的研究进展

许多微生物能利用非核糖体肽合成酶(NRPSs)合成结构复杂、种类繁多的的生物活性肽。非核糖体肽因其独特的理化特性和药理学特性已被广泛关注,极具商业开发潜力。NRPSs由多个模块组成,模块的不同空间排列顺序决定其多肽产物的氨基酸序列特异性。NRPSs以多载体巯基化模板机理进行多肽合成,其底物特异性由腺苷酰化结构域和缩合结构域共同实现。目前,人们已经利用天然的NRPSs、某些特定结构域、将已知NRPSs的模块或特定结构域进行组合甚至杂合组合而构建成的新的NRPSs来合成目的多肽。     

Chain initiation in the leinamycin-producing hybrid nonribosomal peptide/polyketide synthetase from Streptomyces atroolivaceus S-140. Discrete, monofunctional adenylation enzyme and peptidyl carrier protein that directly load D-alanine

Nonribosomal peptide natural products are biosynthesized from amino acid precursors by nonribosomal peptide synthetases (NRPSs), which are organized into modules. For a typical NRPS initiation module, an adenylation (A) domain activates an amino acid and installs it onto a peptidyl carrier protein (PCP) domain as a thioester; an elongation module, which has a condensation (C) domain located between every consecutive pair of A and PCP domains, catalyzes the formation of the peptide bond between the upstream aminoacyl/peptidyl-S-PCP and the free amino group of the downstream aminoacyl-S-PCP. D-amino acid constituents in peptide natural products usually arise from the L-enantiomers through the action of integral epimerization (E) domains of an NRPS. The biosynthetic gene cluster for leinamycin, a hybrid nonribosomal peptide/polyketide containing a D-alanine moiety, does not encode a typical NRPS initiation module with the expected A-PCP-E domains; instead, it has only an A protein (LnmQ) and a PCP (LnmP), both of which are encoded by separate genes. Here we show the results of biochemical experiments as follows: (i) we demonstrate that LnmQ directly activates D-alanine as D-alaninyl-AMP and installs it onto LnmP to generate a D-alaninyl-S-PCP intermediate; (ii) we confirm that aminoacylation of LnmP by LnmQ in trans is the result of specific communication between the separate A and PCP proteins; and (iii) we reveal that leinamycin production can be improved by supplementation of exogenous D-alanine in the fermentation broth of Streptomyces atroolivaceous S-140. These findings unveil an unprecedented NRPS initiation module structure that is characterized by a discrete D-alanine-specific A protein and a PCP.     

The linear pentadecapeptide gramicidin is assembled by four multimodular nonribosomal peptide synthetases that comprise 16 modules with 56 catalytic domains

Linear gramicidin is a membrane channel forming pentadecapeptide that is produced via the nonribosomal pathway. It consists of 15 hydrophobic amino acids with alternating l- and d-configuration forming a beta-helix-like structure. It has an N-formylated valine and a C-terminal ethanolamine. Here we report cloning and sequencing of the entire biosynthetic gene cluster as well as initial biochemical analysis of a new reductase domain. The biosynthetic gene cluster was identified on two nonoverlapping fosmids and a 13-kilobase pair (kbp) interbridge fragment covering a region of 74 kbp. Four very large open reading frames, lgrA, lgrB, lgrC, and lgrD with 6.8, 15.5, 23.3, and 15.3 kbp, were identified and shown to encode nonribosomal peptide synthetases with two, four, six, and four modules, respectively. Within the 16 modules identified, seven epimerization domains in alternating positions were detected as well as a putative formylation domain fused to the first module LgrA and a putative reductase domain attached to the C-terminal module of LgrD. Analysis of the substrate specificity by phylogenetic studies using the residues of the substrate-binding pockets of all 16 adenylation domains revealed a good agreement of the substrate amino acids predicted with the sequence of linear gramicidin. Additional biochemical analysis of the three adenylation domains of modules 1, 2, and 3 confirmed the colinearity of this nonribosomal peptide synthetase assembly line. Module 16 was predicted to activate glycine, which would then, being the C-terminal residue of the peptide chain, be reduced by the adjacent reductase domain to give ethanolamine, thereby releasing the final product N-formyl-pentadecapeptide-ethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro.     

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.     

The inherent flexibility of type I non-ribosomal peptide synthetase multienzymes drives their catalytic activities

Non-ribosomal peptide synthetases (NRPSs) are multienzymes that produce complex natural metabolites with many applications in medicine and agriculture. They are composed of numerous catalytic domains that elongate and chemically modify amino acid substrates or derivatives and of non-catalytic carrier protein domains that can tether and shuttle the growing products to the different catalytic domains. The intrinsic flexibility of NRPSs permits conformational rearrangements that are required to allow interactions between catalytic and carrier protein domains. Their large size coupled to this flexibility renders these multi-domain proteins very challenging for structural characterization. Here, we summarize recent studies that offer structural views of multi-domain NRPSs in various catalytically relevant conformations, thus providing an increased comprehension of their catalytic cycle. A better structural understanding of these multienzymes provides novel perspectives for their re-engineering to synthesize new bioactive metabolites.     

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utility of epimerization domains for the redesign of nonribosomal peptide synthetases

Many pharmacologically important agents are assembled on multimodular nonribosomal peptide synthetases (NRPSs) whose modules comprise a set of core domains with all essential catalytic functions necessary for the incorporation and modification of one building block. Very often, d-amino acids are found in such products which, with few exceptions, are generated by the action of NRPS integrated epimerization (E) domains that alter the stereochemistry of the corresponding peptidyl carrier protein (PCP) bound l-intermediate. In this study we present a quantitative investigation of substrate specificity of four different E domains (two 'peptidyl-' and two 'aminoacyl-'E domains) derived from different NRPSs towards PCP bound peptides. The respective PCP-E bidomain apo-proteins (TycB(3)-, FenD(2)-, TycA- and GrsA-PCP-E) were primed with various peptidyl-CoA precursors by utilizing the promiscuous phosphopantetheinyl transferase Sfp. PCP bound peptidyl-S-Ppant epimerization products were chemically cleaved and analyzed for their l/d-ratios by LCMS. We were able to show that all four E domains tolerate a broad variety of peptidyl-S-Ppant-substrates as evaluated by k(obs) values and final l/d-product equilibria determined for each reaction. The two C-terminal amino acids of the substrate seem to be recognized by 'peptidyl-'E domains. Interestingly, the 'aminoacyl-'E domains GrsA- and TycA-E were also able to convert the elongated intermediates. All four E domains accepted an N-methylated precursor as well and epimerized this substrate with high efficiency. Finally, we could demonstrate that the condensation (C) domain of TycB(1) is also able to process peptidyl substrates transferred by TycA. In conclusion, these findings are of great impact on future engineering attempts.     

Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: A membrane-associated “siderosome”

The peptidic siderophore pyoverdine is the primary iron uptake system of fluorescent pseudomonads, and a virulence factor in the opportunistic pathogen Pseudomonas aeruginosa. Pyoverdine biogenesis is a co-ordinate process requiring several precursor-generating enzymes and large nonribosomal peptide synthetases (NRPSs) in the cytoplasm, followed by extracytoplasmic maturation. By using cell fractionation, protein–protein interaction, and in vivo labeling assays we obtained evidence that, in P. aeruginosa, pyoverdine NRPSs assemble with precursor-generating enzymes into a membrane-bound multi-enzymatic complex, for which we propose the name “siderosome”. The pyoverdine biogenetic complex represents a novel example of subcellular compartmentalization of a secondary metabolic pathway in prokaryotes.     

Serratia marcescens gene required for surfactant serrawettin W1 production encodes putative aminolipid synthetase belonging to nonribosomal peptide synthetase family

Serrawettin W1 produced by Serratia marcescens is a surface active exolipid having various functions supporting behaviors of bacteria on surface environments. Through the genetic analyses of serrawettin-less mutants of S. marcescens 274, the swrW gene encoding putative serrawettin W1 synthetase was identified. Homology analysis of the putative SwrW demonstrated the presence of condensation, adenylation, thiolation, and thioesterase domains which are characteristic for nonribosomal peptide synthetase (NRPS). NRPSs have been known as multi-modular enzymes. Linear alignment of these modules specifying respective amino acids will enable peptide bond formation resulting in a specific amino acid sequence. Putative SwrW was uni-modular NRPS specifying only L-serine. Possible steps in this simple unimodular NRPS for biosynthesis of serrawettin W1 [ cyclo-(D-3-hydroxydecanoyl-L-seryl) (2) ] were predicted by referring to the ingenious enzymatic activity of gramicidin S synthetase (multi-modular NRPS) of Brevibacillus brevis.     

Folding and membrane insertion of the pore-forming peptide gramicidin occur as a concerted process

Many antibiotic peptides function by binding and inserting into membranes. Understanding this process provides an insight into the fundamentals of both membrane protein folding and antibiotic peptide function. For the first time, in this work, flow-aligned linear dichroism (LD) is used to study the folding of the antibiotic peptide gramicidin. LD provides insight into the combined processes of peptide folding and insertion and has the advantage over other similar techniques of being insensitive to off-membrane aggregation events. By combining LD data with conventional measurements of protein fluorescence and circular dichroism, the mechanism of gramicidin insertion is elucidated. The mechanism consists of five separately assignable steps that include formation of a water-insoluble gramicidin aggregate, dissociation from the aggregate, partitioning of peptide to the membrane surface, oligomerisation on the surface and concerted insertion and folding of the peptide to the double-helical form of gramicidin. Measurement of the rates of each step shows that although changes in the fluorescence signal cease 10 s after the initiation of the process, the insertion of the peptide into the membrane is actually not complete for a further 60 min. This last membrane insertion phase is only apparent by measurement of LD and circular dichroism signal changes. In summary, this study demonstrates the importance of multi-technique approaches, including LD, in studies of membrane protein folding.     

Gramicidin structure and disposition in highly curved membranes

With a view to deciphering aspects of the mechanism of membrane protein crystallization in lipidic mesophases (in meso crystallization), an examination of the structure and disposition of the pore-forming peptide, gramicidin, in the lipidic cubic phase was undertaken. At its simplest, the cubic phase consists of lipid and water in the form of a molecular 'sponge.' The lipid exists as a continuous, highly curved bilayer that divides the aqueous component into two interpenetrating but non-contacting channels. In this study, we show that gramicidin reconstitutes into the lipid bilayer of the cubic phase and that it adopts the channel, or helical dimer, conformation therein. Fluorescence quenching with brominated lipid was used to establish the bilayer location of the peptide. Electronic absorption and emission spectroscopies corroborated this finding. Peptide conformation in the cubic phase membrane was determined by circular dichroism. The identity and microstructure of the mesophases, and their capacity to accommodate gramicidin and other system components (sodium dodecyl sulfate, trifluoroethanol), was established by small-angle X-ray diffraction. Beyond a limiting concentration, gramicidin destabilized the cubic phase in favor of the inverted hexagonal phase. While gramicidin remained bilayer bound as membrane thickness changed, its conformation responded to the degree of bilayer mismatch with the hydrophobic surface of the peptide. These findings support the hypothesis that reconstitution into the lipid bilayer is an integral part of the in meso crystallization process as applied to membrane proteins. They also suggest ways for improving the process of membrane protein crystallogenesis.     

Aldehyde modification of peptide immunogen enhances protein-reactive antibody response to toxic shock syndrome toxin-1.

Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled peptide without the use of strong adjuvants. Synthetic peptides representing the N-terminal (residues 1-16) and internal (residues 53-65) epitopes of toxic shock syndrome toxin-1 (TSST-1) were coupled to carrier protein, and carbonyl tags were introduced by Amadori reaction with glycolaldehyde. Modified and unmodified antigens in alum were used to immunize rabbits and the reactivities of antisera were compared. Aldehyde modification augmented the response detected by ELISA, which included enhanced binding to peptides and to native TSST-1. In western blot, TSST-1 was detected by antiserum elicited to the N-terminal peptide, but not that generated to the peptide representing the internal sequence. The same antiserum also neutralized TSST-1 activity in a lymphocyte proliferation assay. The circular dichroism spectrum of the N-terminal peptide indicated a propensity for helical conformation, similar to the structure at the corresponding sequence of the native protein. These data suggest that aldehyde modification can boost immunogenicity of peptide-based vaccines, generating epitope-specific immune responses against the cognate protein antigens without using potent adjuvants.     

Analyzing heat capacity profiles of peptide-containing membranes: cluster formation of gramicidin A

The analysis of peptide and protein partitioning in lipid membranes is of high relevance for the understanding of biomembrane function. We used statistical thermodynamics analysis to demonstrate the effect of peptide mixing behavior on heat capacity profiles of lipid membranes with the aim to predict peptide aggregation from c(P)-profiles. This analysis was applied to interpret calorimetric data on the interaction of the antibiotic peptide gramicidin A with lipid membranes. The shape of the heat capacity profiles was found to be consistent with peptide clustering in both gel and fluid phase. Applying atomic force microscopy, we found gramicidin A aggregates and established a close link between thermodynamics data and microscopic imaging. On the basis of these findings we described the effect of proteins on local fluctuations. It is shown that the elastic properties of the membrane are influenced in the peptide environment.     

Generation of formic acid and ethanolamine from serine in biosynthesis of linear gramicidin by a cell-free preparation of Bacillusbrevis (ATCC 8185)

A growing organism that produces antibiotic peptide was incubated with L-(U-¹⁴C)serine for labeling linear gramicidin. Linear gramicidin was isolated by a simple chromatographic method from tyrothricin (mixture of linear gramicidin and tyrocidine) applied to a column of basic aluminum oxide. The hydrolysate of labeled linear gramicidin on thin layer chromatography showed that L-(U-¹⁴C)serine was one of a precursor of ethanolamine moiety by autoradiography. L-(3-¹⁴C)serine generated formic acid in the presence of tetrahydrofolic acid by an enzyme fraction prepared with ammonium sulfate, and further formed ethanolamine binding to the protein. Formylvaline was biosynthesized by it with tetrahydrofolic acid and ATP, and subsequently released from the protein.     

Specific enrichment of nonribosomal peptide synthetase module by an affinity probe for adenylation domains

We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5′-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated l-Phe-AMS (l-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates l-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes.     

Interkingdom gene transfer of a hybrid NPS/PKS from bacteria to filamentous Ascomycota

Nonribosomal peptides (NRPs) and polyketides (PKs) are ecologically important secondary metabolites produced by bacteria and fungi using multidomain enzymes called nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively. Previous phylogenetic analyses of fungal NRPSs and PKSs have suggested that a few of these genes were acquired by fungi via horizontal gene transfer (HGT) from bacteria, including a hybrid NPS/PKS found in Cochliobolus heterostrophus (Dothideomycetes, Ascomycota). Here, we identify this hybrid gene in fungi representing two additional classes of Ascomycota (Aspergillus spp., Microsporum canis, Arthroderma spp., and Trichophyton spp., Eurotiomycetes; Chaetomium spp. and Metarhizium spp., Sordariomycetes) and use phylogenetic analyses of the most highly conserved domains from NRPSs (adenylation (A) domain) and PKSs (ketoacyl synthase (KS) domain) to examine the hypothesis that the hybrid NPS7/PKS24 was acquired by fungi from bacteria via HGT relatively early in the evolution of the Pezizomycotina. Our results reveal a unique ancestry of the A domain and KS domain in the hybrid gene relative to known fungal NRPSs and PKSs, provide strong evidence for HGT of the hybrid gene from a putative bacterial donor in the Burkholderiales, and suggest the HGT event occurred early in the evolution of the filamentous Ascomycota.     

Roles of type II thioesterases and their application for secondary metabolite yield improvement

A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an “assembly line” and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such “unnatural” natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)—discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.     

Synthesis and channel properties of [Tau 16]gramicidin A

Des(ethanolamine)-taurine16-gramicidin A ([Tau 16]gramicidin A) was synthesized by the solid phase method and its channel-forming behavior in planar lipid bilayers was examined. The purified monovalent anionic peptide formed channels when applied to the aqueous compartments on both sides of the bilayer, but not when applied to one side only. The single-channel conductance was measured for KCl concentrations between 0.1 and 1.0 M and was found to be higher than that of gramicidin A in each case. Single-channel lifetimes were similar to those of gramicidin A suggesting that the channels have the beta 6.3 helix structure.     

Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S

The cyclic beta-sheet structure possessed by the 10-residue antibiotic peptide gramicidin S was taken as the structural framework for the de novo design of biologically active peptides with membrane-active properties. We have shown from previous studies that gramicidin S is a broad-spectrum antibiotic effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, but is toxic to human red blood cells. We tested the effect of ring size on antimicrobial activity and hemolytic activity on peptides varying from 4 to 16 residues. Interestingly, we were able to dissociate hemolytic activity and antimicrobial activity by increasing the ring size of the peptide to 14 residues (peptide GS14). Furthermore, we increased specificity for microbial membranes while decreasing toxicity to red blood cells by substituting enantiomers (D-amino acids for L-amino acids and vice versa) into the GS14 sequence. The enantiomeric substitutions all disrupted beta-sheet structure in benign medium and decreased peptide amphipathicity. The least amphipathic peptide, produced by substituting a D-Lys at position 4 of GS14 (peptide GS14K4), also had the highest therapeutic index, i.e., highest degree of specificity for microbial cells over human cells. Solution structures of GS14 analogs solved by NMR spectroscopy showed that the D-amino acid side chain was located on the nonpolar face of GS14K4. Another analog, a beta-sheet peptide with reduced amphipathicity (peptide GS14 K3L4), also had a lysine (lysine 3) on the nonpolar face as determined by the NMR structure. Both GS14K4 and GS14 K3L4 had reduced amphipathicity relative to GS14 and much higher therapeutic indices. Finally, the alteration of the nonpolar face hydrophobicity of GS14K4 analogs provided a range of activities and specificities, where the peptides with the intermediate hydrophobicities among the series had the highest therapeutic indices. The optimal peptide hydrophobicities varied depending on the microorganism being tested, with higher hydrophobicity requirements against Gram-positive bacteria and yeast compared with Gram-negative microorganisms. The net result of these studies suggests that it is possible to rationally design a cyclic membrane-active antimicrobial peptide with high specificity towards prokaryotic (bacterial and fungal) membranes and minimal toxicity to eukaryotic (e.g., mammalian) membranes.