RESUMO
The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A 19F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state 19F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes.
Assuntos
Bicamadas Lipídicas , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodosRESUMO
The acid lability of aliphatic ketals, which often serve as protection groups for 1,2-diols, is influenced by their local structural environment. The acetonide of the protected amino acid cis-dihydroxyproline (Dyp) is a typical protecting group cleavable by traces of TFA. The tricyclic acetonide of the dipeptide d-HotâTap is resistant to TFA and thus can serve as a bioorthogonal modification of bioactive peptides. With the aim of improving antimicrobial activity and hemolytic properties, we use these reactivity differences to scale the membrane affinity of the decapeptide Gramicidin S cyclo(d-Phe-Pro-Val-Orn-Leu-)2 (GS). The cis-dihydroxylated amino acids are used to increase the polarity of GS or obversely decrease the polarity by stereoselective ketal formation with an aliphatic ketone. While Dyp (GS mimetic 15) has only minimal influence on the biological properties of GS, d-HotâTap at the position of d-Phe1-Pro2 eradicates the biological activity (GS mimetic 16). The acid-stable ketals 17-19 are bioorthogonal modifications which reconstitute the biological activity of GS. We describe an improved synthesis of orthogonally protected Fmoc-Dyp-acetonide (9) and of several Fmoc-d-HotâTap-ketals for solid-phase peptide synthesis.
Assuntos
Anti-Infecciosos/química , Gramicidina/química , Cetonas/química , Acetais/química , Interações Hidrofóbicas e Hidrofílicas , Hidroxilação , Estrutura MolecularRESUMO
Conformationally constrained non-racemizing trifluoromethyl-substituted lysine isosteres [(E)- and (Z)-TCBLys] with charged side chains are presented as a new type of 19 F-NMR labels for peptide studies. Design of the labels, their synthesis, incorporation into peptides and experimental demonstration of their application for solid state NMR studies of membrane-active peptides are described. A series of fluorine-labeled analogues of the helical amphipathic antimicrobial peptide PGLa(Nle) was obtained, in which different lysine residues in the original peptide sequence were replaced, one at a time, by either (E)- or (Z)-TCBLys. Antimicrobial activities of the synthesized analogues were practically the same as those of the parent peptide. The structural and orientational parameters of the helical PGLa(Nle) peptide in model bilayers, as determined using the novel labels confirmed and refined the previously known structure. (E)- and (Z)-TCBLys, as a set of cationic 19 F-NMR labels, were shown to deliver structural information about the charged face of amphipathic peptides by solid state 19 F-NMR, previously inaccessible by this method.
RESUMO
A conformationally restricted monofluorinated α-amino acid, (3-fluorobicyclo[1.1.1]pentyl)glycine (F-Bpg), was designed as a label for the structural analysis of membrane-bound peptides by solid-state 19 F NMR spectroscopy. The compound was synthesized and validated as a 19 F label for replacing natural aliphatic α-amino acids. Calculations suggested that F-Bpg is similar to Leu/Ile in terms of size and lipophilicity. The 19 F NMR label was incorporated into the membrane-active antimicrobial peptide PGLa and provided information on the structure of the peptide in a lipid bilayer.
RESUMO
Multidrug-resistant Pseudomonas aeruginosa is a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against different P. aeruginosa isolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of ≤0.5. Remarkably, several clinical P. aeruginosa isolates that grew as small-colony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and ≤0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistant P. aeruginosa and biofilms of P. aeruginosa strain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Gramicidina/farmacologia , Polimixina B/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade MicrobianaRESUMO
Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly (13)C/(15)N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.
Assuntos
Bacillales/metabolismo , Isótopos de Carbono/metabolismo , Gramicidina/metabolismo , Marcação por Isótopo/métodos , Isótopos de Nitrogênio/metabolismo , Análise Custo-Benefício , Meios de Cultura/química , Fermentação , Espectroscopia de Ressonância MagnéticaRESUMO
Magainin 2 (MAG2) and PGLa are two related antimicrobial peptides found in the skin of the African frog Xenopus laevis with a pronounced synergistic activity, which act by permeabilizing bacterial membranes. To probe the influence of hydrophobic peptide-lipid and peptide-peptide interactions on the antimicrobial activity and on synergy, the sequence of MAG2 was modified by replacing single amino acids either with a small alanine or with the stiff and bulky hydrophobic 3-(trifluoromethyl)-L-bicyclopent-[1.1.1]-1-ylglycine side chain. The minimum inhibitory concentration of 14 MAG2 analogs was strongly influenced by these single substitutions: the antimicrobial activity was consistently improved when the hydrophobicity was increased on the hydrophobic face of the amphiphilic helix, while the activity decreased when the hydrophobicity was reduced. The synergy with PGLa, on the other hand, was rather insensitive to mutations of hydrophobic residues. It thus seems that the antimicrobial effect of MAG2 on its own depends strongly on the hydrophobicity of the peptide, while the synergy with PGLa does not depend on the overall hydrophobicity of MAG2.
Assuntos
Substituição de Aminoácidos , Bactérias/efeitos dos fármacos , Magaininas/química , Magaininas/farmacologia , Proteínas de Xenopus/química , Proteínas de Xenopus/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Sinergismo Farmacológico , Interações Hidrofóbicas e Hidrofílicas , Magaininas/genética , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Proteínas de Xenopus/genéticaRESUMO
Photobiological processes in nature are usually triggered by nonpeptidic chromophores or by modified side chains. A system is presented in which the polypeptide backbone itself can be conformationally switched by light. An amino acid analogue was designed and synthesized based on a reversibly photoisomerizable diarylethene scaffold. This analogue was incorporated into the cyclic backbone of the antimicrobial peptide gramicidinâ S at several sites. The biological activity of the resulting peptidomimetics could then be effectively controlled by ultraviolet/visible light within strictly defined spatial and temporal limits.
Assuntos
Etilenos/química , Peptídeos Cíclicos/química , Peptidomiméticos/química , Luz , Modelos Moleculares , Conformação Molecular , Fotoquímica , Relação Estrutura-AtividadeRESUMO
The bacterial stress-response peptide TisB in Escherichia coli has been suggested to dissipate the transmembrane potential, such that the depletion of ATP levels induces the formation of dormant persister cells which can eventually form biofilms. We studied the structure and membrane interactions of TisB to find out whether it forms pores or other proton-selective channels. Circular dichroism revealed an amphiphilic α-helical structure when reconstituted in lipid vesicles, and oriented circular dichroism showed that the helix assumes a transmembrane alignment. The addition of TisB to dye-loaded vesicles caused leakage only at very high peptide concentration, notably with a Hill coefficient of 2, which suggests that dimers must be involved. Coarse-grained molecular dynamics simulations showed that membrane binding of monomeric TisB is rapid and spontaneous, and transmembrane insertion is energetically feasible. When TisB oligomers are assembled as transmembrane pores, these channels collapse during the simulations, but transmembrane dimers are found to be stable. Given the pattern of charges on the amphiphilic TisB helix, we postulate that antiparallel dimers could be assembled via a ladder of salt bridges. This electrostatic charge-zipper could enable protons to pass along a wire of trapped water molecules across the hydrophobic membrane.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/fisiologia , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Toxinas Bacterianas/química , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Porosidade , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estresse Fisiológico , Termodinâmica , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
To understand how membrane-active peptides (MAPs) function in vivo, it is essential to obtain structural information about them in their membrane-bound state. Most biophysical approaches rely on the use of bilayers prepared from synthetic phospholipids, i.e. artificial model membranes. A particularly successful structural method is solid-state NMR, which makes use of macroscopically oriented lipid bilayers to study selectively isotope-labelled peptides. Native biomembranes, however, have a far more complex lipid composition and a significant non-lipidic content (protein and carbohydrate). Model membranes, therefore, are not really adequate to address questions concerning for example the selectivity of these membranolytic peptides against prokaryotic vs eukaryotic cells, their varying activities against different bacterial strains, or other related biological issues.Here, we discuss a solid-state (19)F-NMR approach that has been developed for structural studies of MAPs in lipid bilayers, and how this can be translated to measurements in native biomembranes. We review the essentials of the methodology and discuss key objectives in the practice of (19)F-labelling of peptides. Furthermore, the preparation of macroscopically oriented biomembranes on solid supports is discussed in the context of other membrane models. Two native biomembrane systems are presented as examples: human erythrocyte ghosts as representatives of eukaryotic cell membranes, and protoplasts from Micrococcus luteus as membranes from Gram-positive bacteria. Based on our latest experimental experience with the antimicrobial peptide gramicidin S, the benefits and some implicit drawbacks of using such supported native membranes in solid-state (19)F-NMR analysis are discussed.
Assuntos
Flúor/química , Espectroscopia de Ressonância Magnética/métodos , Membranas/química , Peptídeos/química , Animais , Membrana Eritrocítica/química , Humanos , Marcação por Isótopo , Bicamadas Lipídicas , Lipopolissacarídeos/farmacologia , Lipídeos de Membrana/química , Proteínas de Membrana/química , Modelos Moleculares , Protoplastos/químicaRESUMO
(19)F NMR is a unique tool to examine the structure of fluorine-labeled peptides in their native cellular environment, due to an exquisite sensitivity and lack of natural abundance background. For solid-state NMR analysis, we isolated native membranes from erythrocyte ghosts and bacterial protoplasts and prepared them as macroscopically oriented samples. They showed a high purity and quality of alignment according to (31)P NMR, and the membrane-bound antimicrobial peptide PGLa could be detected by (19)F NMR. The characteristic fingerprint splitting of its (19)F reporter group indicated that the peptide helix binds to the native membranes in a surface alignment, albeit with a higher affinity in the prokaryotic than the eukaryotic system.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/citologia , Membrana Celular/metabolismo , Eritrócitos/citologia , Espectroscopia de Ressonância Magnética , Protoplastos/citologia , Sequência de Aminoácidos , Animais , Halobacterium salinarum/citologia , Humanos , Micrococcus luteus/citologia , Dados de Sequência Molecular , Xenopus laevisRESUMO
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the ultrastructural changes in bacteria induced by antimicrobial peptides (AMPs). Both the beta-stranded gramicidin S and the alpha-helical peptidyl-glycylleucine-carboxyamide (PGLa) are cationic amphiphilic AMPs known to interact with bacterial membranes. One representative Gram-negative strain, Escherichia coli ATCC 25922, and one representative Gram-positive strain, Staphylococcus aureus ATCC 25923, were exposed to the AMPs at sub-MICs and supra-MICs in salt-free medium. SEM revealed a shortening and swelling of the E. coli cells, and multiple blisters and bubbles formed on their surface. The S. aureus cells seemed to burst upon AMP exposure, showing open holes and deep craters in their envelope. TEM revealed the formation of intracellular membranous structures in both strains, which is attributed to a lateral expansion of the lipid membrane upon peptide insertion. Also, some morphological alterations in the DNA region were detected for S. aureus. After E. coli was incubated with AMPs in medium with low ionic strength, the cells appeared highly turgid compared to untreated controls. This observation suggests that the AMPs enhance osmosis through the inner membrane, before they eventually cause excessive leakage of the cellular contents. The adverse effect on the osmoregulatory capacity of the bacteria is attributed to the membrane-permeabilizing action of the amphiphilic peptides, even at low (sub-MIC) AMP concentrations. Altogether, the results demonstrate that both TEM and SEM, as well as appropriate sample preparation protocols, are needed to obtain detailed mechanistic insights into peptide function.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Gramicidina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Membrana Celular/ultraestrutura , Escherichia coli/ultraestrutura , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão , Staphylococcus aureus/ultraestruturaRESUMO
Silver nanoparticles, as well as antimicrobial peptides (AMPs), can be used to fight infectious diseases. Since AMPs are known to permeabilize bacterial membranes and might therefore help silver nanoparticles to access internal target sites, we investigated their combined activities and showed synergistic effects between polymyxin B and silver nanoparticles for gram-negative bacteria.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Nanopartículas Metálicas , Compostos de Prata/farmacologia , Acinetobacter calcoaceticus/efeitos dos fármacos , Sinergismo Farmacológico , Enterobacter/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Proteus/efeitos dos fármacos , Pseudomonas fluorescens/efeitos dos fármacosRESUMO
Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Gramicidina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Enterococcus faecalis/fisiologia , Enterococcus faecium/fisiologia , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Modelos Moleculares , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Peixe-ZebraRESUMO
Many antimicrobial peptides are synthesized non-ribosomally in bacteria, but little is known about their subcellular route of biosynthesis, their mode of intracellular accumulation, or their role in the physiology of the producer cells. Here, we present a comprehensive view on the biosynthesis of gramicidin S (GS) in Aneurinibacillus migulanus, having observed a peripheral membrane localization of its synthetases. The peptide gets accumulated in nano-globules, which mature by fusion into larger granules and end up within vacuolar structures. These granules serve as energy storage devices, as they contain GS molecules that are non-covalently attached to alkyl phosphates and protect them from dephosphorylation and premature release of energy. This finding of a fundamentally new type of high-energy phosphate storage mechanism can explain the curious role of GS biosynthesis in the physiology of the bacterial producer cells. The unknown role of the GrsT protein, which is part of the non-ribosomal GS synthetase operon, can thus be assumed to be responsible for the biosynthesis of alkyl phosphates. GS binding to alkyl phosphates may suggest its general affinity to phosphagens such as ATP and GTP, which can represent the important intracellular targets in pathogenic bacteria.
Assuntos
Antibacterianos/biossíntese , Bacillales/metabolismo , Proteínas de Bactérias/genética , Grânulos Citoplasmáticos/metabolismo , Regulação Bacteriana da Expressão Gênica , Gramicidina/biossíntese , Trifosfato de Adenosina/biossíntese , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/metabolismo , Bacillales/genética , Bacillales/ultraestrutura , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Guanosina Trifosfato/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Óperon , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Ligação Proteica , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
Stapling of side chains to stabilize an α-helical structure has been generally associated with an increased uptake of CPPs. Here, we compare four amphiphilic stapled peptides with their linear counterparts in terms of their membrane binding and conformational features in order to correlate these with uptake efficiency and toxicological effects. The impact of lactam stapling was found to vary strongly with regard to the different aspects of peptide-membrane interactions. Nearly all stapled peptides caused less membrane perturbation (vesicle leakage, hemolysis, bacterial lysis) than their linear counterparts. In one case (MAP-1) where stapling enhanced α-helicity in aqueous and lipid environments, leakage was eliminated while cell uptake in HEK293 and HeLa cells remained high, which improved the overall characteristics. The other systems (DRIM, WWSP, KFGF) did not improve, however. The data suggest that cell uptake of amphipathic CPPs correlates with their adopted α-helix content in membranes rather than their helicity in solution.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Lactamas/síntese química , Lactamas/farmacologia , Antibacterianos/metabolismo , Bactérias/efeitos dos fármacos , Células HEK293 , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Lactamas/metabolismo , Membranas Artificiais , Testes de Sensibilidade Microbiana , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/farmacologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
An intrinsic clindamycin-resistant Enterococcus faecalis, the most common single species present in teeth after failed root canal therapy, often possesses acquired tetracycline resistance. In these cases, root canal infections are commonly treated with Ledermix(®) paste, which contains demeclocycline, or the new alternative endodontic paste Odontopaste, which contains clindamycin; however, these treatments are often ineffective. We studied the killing activity of the cyclic antimicrobial peptide gramicidin S (GS) against planktonic and biofilm cells of tetracycline-resistant clinical isolates of E. faecalis. The high therapeutic potential of GS for the topical treatment of problematic teeth is based on the rapid bactericidal effect toward the biofilm-forming, tetracycline-resistant E. faecalis. GS reduces the cell number of planktonic cells within 20-40 min at a concentration of 40-80 µg/mL. It kills the cells of pre-grown biofilms at concentrations of 100-200 µg/mL, such that no re-growth is possible. The translocation of the peptide into the cell interior and its complexation with intracellular nucleotides, including the alarmon ppGpp, can explain its anti-biofilm effect. The successful treatment of persistently infected root canals of two volunteers confirms the high effectiveness of GS. The broad GS activity towards resistant, biofilm-forming E. faecalis suggests its applications for approval in root canal medication.
Assuntos
Aminoácidos , Clorofluorcarbonetos de Metano , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Aminoácidos/síntese química , Aminoácidos/química , Clorofluorcarbonetos de Metano/síntese química , Clorofluorcarbonetos de Metano/química , Dicroísmo Circular , Flúor , Modelos Moleculares , Conformação Proteica , EstereoisomerismoRESUMO
Phenotype instability of bacterial strains can cause significant problems in biotechnological applications, since industrially useful properties may be lost. Here we report such degenerative dissociation for Aneurinibacillus migulanus (formerly known as Bacillus brevis) an established producer of the antimicrobial peptide gramicidin S (GS). Phenotypic variations within and between various strains maintained in different culture collections are demonstrated. The type strain, ATCC 9999, consists of six colony morphology variants, R, RC, RP, RT, SC, and SP, which were isolated and characterized as pure cultures. Correlations between colony morphology, growth, GS production, spore formation, and resistance to their own antimicrobial peptide were established in this study. We found the original R form to be the best producer, followed by RC, RP, and RT, while SC and SP yielded no GS at all. Currently available ATCC 9999(T) contains only 2% of the original R producer and is dominated by the newly described phenotypes RC and RP. No original R form is detected in the nominally equivalent strain DSM 2895(T) (=ATCC 9999(T)), which grows only as SC and SP phenotypes and has thus completely lost its value as a peptide producer. Two other strains from the same collection, DSM 5668 and DSM 5759, contain the unproductive SC variant and the GS-producing RC form, respectively. We describe the growth and maintenance conditions that stabilize certain colony phenotypes and reduce the degree of degenerative dissociation, thus providing a recommendation for how to revert the nonproducing smooth phenotypes to the valuable GS-producing rough ones.