Insertion of IS4Bsu1 or IS1182Bsu1 into the oligopeptide-permease gene cluster reduces γ-polyglutamic acid production in Bacillus subtilis subsp. natto

Natto, a traditional Japanese fermented soybean product, owes its characteristic stickiness to γ-polyglutamic acid (γ-PGA), synthesized by Bacillus subtilis subsp. natto. In natto production facilities, mutants with reduced γ-PGA production frequently emerge, posing a significant challenge to natto manufacturing. In this study, we isolated and characterized abnormal colony morphology mutants derived from the widely used B. subtilis subsp. natto BEST195 strain. These mutants were classified into types A and B, based on colony morphology and γ-PGA production levels. Notably, type A mutants exhibited a severe reduction in γ-PGA production, primarily due to the insertion of the insertion sequence IS4Bsu1 into comP, a regulator of quorum sensing. By contrast, type B mutants exhibited significantly reduced, but not completely abolished, γ-PGA production. Genomic and molecular analyses identified insertion sequences (IS4Bsu1 or ISBma2-like, designated as IS1182Bsu1 in this study) within the oligopeptide-permease (opp) cluster or mutations in oppA and oppB, which are key components of the Opp peptide transport system, in type B mutants. Based on these findings, we propose a model in which the Opp system positively regulates γ-PGA production by modulating the RapC-ComA-Pi axis and activating DegU-Pi downstream. This study provides insights into the mechanisms of γ-PGA synthesis in B. subtilis subsp. natto, highlighting the role of the Opp system. These results may aid in the development of strategies to improve γ-PGA production and maintain the quality of natto products in industrial settings.