The Ability ofAneurinibacillus migulanus(Bacillus brevis) To Produce the Antibiotic Gramicidin S Is Correlated with Phenotype Variation

Abstract

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.

Synergistic Interaction between Silver Nanoparticles and Membrane-Permeabilizing Antimicrobial Peptides

“…Examples include PGLa plus magainin 2 from Xenopus laevis (18) and ␤-lactam amoxicillin with silver nanoparticles (9), but a combination of silver nanoparticles or silver ions with AMPs has not been tested. We therefore examined commercial polymyxin B, gramicidin A and alamethicin (from Sigma-Aldrich, Buchs, Switzerland), gramicidin S extracted from Aneurinibacillus migulanus (2), and PGLa and magainin 2, which was synthesized in our laboratory. All noncommercial peptides were purified on a reverse-phase-high-performance liquid chromatography system to greater than 94%.…”

Fermentation and Cost-Effective 13 C/ 15 N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

“…Concentrated spore suspensions were stored in sterile water with 30% glycerol at Ϫ20°C. Due to phenotype instability, the spore suspension was always used to first inoculate yeast peptone (YP) medium (18,20) with Bacto tryptone and yeast extract (each 50 g/liter), i.e., under conditions that should promote the development of rough phenotypes (18). Subsequent plating of this culture onto the surface of LBY agar (10 g/liter Bacto tryptone, 10 g/liter yeast extract, 5 g/liter NaCl, and 30 g/liter microbiological agar) was used as a control to check the colony morphology.…”

“…Aneurinibacillus migulanus DSM 5759 was received from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). It had been characterized earlier to consist of an entirely rough phenotype with a convex center, which is capable of GS production (18 2) were prepared separately as 1,000-fold concentrated stocks. NBYS cultures were grown 48 to 50 h and washed with sterile water (8,000 ϫ g at 4°C for 10 min).…”

“…Penultimately, two pentapeptides, one bound as an ester to the active-site serine of the terminal thioesterase domain (TE) and the second bound as a thioester to the adjacent pan-PCP, are ligated to a decapeptidyl-pan-PCP, which is finally cyclized on TE (17). The effectiveness of this unusual way of biosynthesis depends on multiple factors, such as growth temperature, medium composition, and phenotype variability; therefore, GS production is not a trivial task (18).…”

Iridescence of a Marine Bacterium and Classification of Prokaryotic Structural Colors

“…The type of iridescence observed in certain strains of P. aeruginosa was described as a metallic iridescence and has been linked to cell lysis (16,28,80) and/or to the production of quinoline derivatives (9,10,77). A silvery appearance was also recently mentioned in Aneurinibacillus migulanus type III (2). In H. influenzae, colonies of capsulated cells display all spectral colors from red to blue under oblique transmitted light (transillumination).…”