Subtilosin B: A New Derivative of Subtilosin A Isolated from Barnacle-Associated Bacillus sp. HJ9

다국어 초록 (Multilingual Abstract)

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a promising class of antimicrobial agents to combat antibiotic resistance. Subtilosin A, a cyclic peptide produced by Bacillus subtilis, possesses a unique structure stabilized by three thioether bridges formed through radical SAM enzyme-mediated linkages. It selectively targets Gram-positive pathogens with narrow-spectrum activity. However, inconsistent minimum inhibitory concentration (MIC) values across studies indicate the existence of uncharacterized structural variants. In this study, we isolated a strain Bacillus sp. HJ9 from the barnacle Balanus albicostatus collected from intertidal mudflats in Songdo, Incheon, Republic of Korea. Whole genome sequencing and antiSMASH analysis confirmed the presence of a subtilosin A biosynthetic gene cluster (BGC) in the HJ9 genome. Through large-scale cultivation followed by organic solvent extraction, column chromatography, and RP-HPLC, a new derivative subtilosin B (1) was successfully isolated and characterized. Comprehensive structural elucidation using MALDI-TOF-MS, HR-ESI-MS, multidimensional NMR spectroscopy (HMBC, TOCSY, HSQC), CD spectroscopy, FT-IR, and Marfey's method revealed that subtilosin B (1) is an Nε-acetylated derivative of subtilosin A at the lysine-2 residue. The mass difference of 42.01 Da and modified cosine similarity score of 0.7436 between MS/MS fragmentation patterns confirmed the close structural relationship between the two compounds. Notably, CD analysis indicated that acetylation does not significantly alter the overall three-dimensional conformation of the peptide. Antimicrobial assays demonstrated that subtilosin B (1) retains the narrow spectrum activity characteristic of the subtilosin family with moderate antimicrobial activity. The absence of an acetyltransferase gene within the BGC suggests involvement of extra-cluster enzymatic machinery. This study provides the first experimental evidence of post-translational modification diversity within the subtilosin family and expands understanding of natural product chemical space in RiPP biosynthesis. Keywords: subtilosin, RiPPs, Bacillus subtilis, post-translational modification, antimicrobial peptide, biosynthetic gene cluster

목차 (Table of Contents)

• ABSTRACT Ⅴ
• LIST OF TABLES Ⅷ
• LIST OF FIGURES Ⅸ
• LIST OF ABBREVIATIONS Ⅺ
• 1. INTRODUCTION 13
• 2. EXPERIMENTAL SECTION 18
• 3. RESULTS AND DISCUSSION 27
• 4. REFERENCES 56
• 국문 초록 63