The ability of Aneurinibacillus 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.

FIG. 1.

Morphologically distinct colony variants coexist in A. migulanus ATCC 9999^T, which we distinguish as types I through VI.

FIG. 2.

Pure phenotypes of A. migulanus, identified as the R form derived from type V (A), the RC form derived from type I (B), the RP form derived from type II (C), the RT form derived from type III (D), the SC form derived from type IV (E), and the SP form derived from type VI (F). The panels on the left show the homogenous populations on LBY agar plates, and those on the right show individual colonies (magnification, ×7 to ×10).

FIG. 3.

(A and C) Growth curves of phenotype variants of A. migulanus ATCC 9999^T: R (filled circles), RT (open circles), RP (filled squares), RC (open squares), SC (filled triangles), and SP (open triangles). (B and D) Corresponding absolute (g/liter) and specific (g/g DCW) production of GS. The cultures were cultivated either in NBYS sporulation medium (A and B) or in YP medium, which is optimal for GS production (C and D). GS yields were determined at the times indicated by arrows in panels A and C.

FIG. 4.

Bacterial growth (OD₆₆₀) and absolute yield of GS, monitored as a function of time for the pure R phenotype of A. migulanus ATCC 9999^T in YP medium.

FIG. 5.

Representative spore suspensions of different phenotypes of A. migulanus, which were homogeneous in size and refractivity for variant R (A) but heterogeneous in morphology and refractivity for variants RC (B), SC (C), and SP (D).

FIG. 6.

(A) Summary of the cultivation conditions promoting the interconversion of the different phenotypes of A. migulanus. In the reversion process, the R phenotype appears in YP medium (37°C, no agitation) from the SC form of DSM 2895^T (B) and the RC phenotype originates from the SC form of DSM 5668 (C).