Bacillus licheniformis global nitrogen homeostatic regulator TnrA is a direct repressor of pgsBCAA transcription in Poly-γ-glutamic acid synthesis

Abstract

Poly-{gamma}-glutamic acid ({gamma}-PGA) is a multifunctional and naturally occurring biopolymer made from D- and L-glutamate as monomers, which is mainly produced by Bacillus. Few reports have been focused on the regulation network of {gamma}-PGA synthesis in recent years. In this study, we have demonstrated that Bacillus licheniformis global nitrogen homeostatic regulator TnrA is a direct repressor of {gamma}-PGA synthase PgsBCAA in {gamma}-PGA synthesis. First, our results confirmed that TnrA repressed {gamma}-PGA synthesis, deficiency of tnrA led to a 22.03% increase of {gamma}-PGA production, and the {gamma}-PGA yield was decreased by 19.02% in the TnrA overexpression strain. Transcriptional level assay illustrated that the {gamma}-PGA synthase gene cluster pgsBCAA transcriptional level were increased in the tnrA deficient strain WX{Delta}tnrA, indicating that {gamma}-PGA synthase PgsBCAA was negatively regulated by TnrA. Furthermore, electrophoretic mobility shift assay (EMSA) and enzyme expression assays confirmed that TnrA directly repressed pgsBCAA expression by binding to pgsBCAA promoter, and the TnrA-binding site \"CGTCGTCTTCTGTTACA\" in the pgsBCAA promoter was identified by sequence and software analysis. Finally, computer analysis confirmed that the transcription regulations of {gamma}-PGA synthase PgsBCAA by TnrA were highly conserved in other well-studied Bacillus species (B. licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens). Collectively, our results implied that TnrA was a direct repressor for pgsBCAA expression in {gamma}-PGA synthesis, and this research provided a novel regulatory mechanism underlying {gamma}-PGA synthesis, and a new approach that deficiency of tnrA increases {gamma}-PGA production.\n\nImportance{gamma}-PGA is an important biopolymer with many applications, which is mainly produced by Bacillus species. Glutamic acid is the precursor for {gamma}-PGA synthesis, which is catalyzed by the {gamma}-PGA synthase PgsBCAA. Previously, the expression of PgsBCAA was reported to be regulated by ComA-ComP and DegS-DegU, DegQ and SwrA systems, however, few researches were focused on the regulation network of {gamma}-PGA synthesis in recent years. In our research, the {gamma}-PGA synthase PgsBCAA was confirmed to be negatively regulated by the nitrogen metabolism regulator TnrA, and the TnrA binding site in the pgsBCAA promoter was identified in B. licheniformis WX-02. Furthermore, computer analysis implied that TnrA-mediated regulation effect on pgsBCAA expression was highly conserved in Bacillus. Collectively, our research provided a novel regulatory mechanism underlying {gamma}-PGA synthesis, and a new approach that deficiency of tnrA increases {gamma}-PGA production.