Bacillus velezensis LG37: Transcriptome profiling and functional verification of GlnK and NmtA in ammonia (NH4+) assimilation

Abstract

Background In recent years, interest in Bacillus velezensis has increased significantly due to its role in many industrial water bioremediation processes, including probiotics. In this study, we isolated and assessed the transcriptome of Bacillus velezensis LG37 (isolated from aquaculture pond) under different nitrogen sources. Since Bacillus species exhibit heterogeneity, it is worth to investigate the molecular information of LG37 through ammonia nitrogen assimilation, where ammonia’s nitrogen is considered toxic to aquatics. Results Here, a total of 812 differentially expressed genes (DEGs) from the transcriptomic sequencing of LG37 grown minimal medium supplemented with ammonia (treatment) or glutamine (control) were obtained, from which 56 had Fold Change ≥ 2. BLAST NCBI and UniProt databases revealed 27 out of the 56 DEGs were potentially involved in NH 4 + assimilation. Among them, 8 DEGs together with the two-component regulatory system glnK/glnL were randomly selected for validation by quantitative real-time RT-PCR, and the relative expression level showed that glnK/glnL were significantly upregulated that were consistent with the RNA-seq data coupled with six other genes. Moreover, the transcriptome and relative expression analysis showed that the transporter ( amtB ) gene of LG37 was not involved in ammonia transport even in the highest ammonia concentrations. Besides, CRISPR-Cas9 knockout and overexpression LG37 mutants of glnK further evidenced the exclusion of amtB regulation, suggesting involvement of alternative transporter. Additionally, in the transcriptomic data, a novel ammonium transporter nmtA was expressed significantly in increased ammonia concentrations. Subsequently, OE nmtA and Δ nmtA LG37 strains showed a unique expression pattern compared to that of wild-LG37 strain. Conclusion Based on the transcriptome data, regulation of nitrogen related genes were promptly screened from the newly isolated LG37 strain to analyse the key regulating factors during ammonia assimilation. The novel NmtA transporter of LG37 became apparent in ammonia transport instead of AmtB, which transport ammonium nitrogen in other Bacillus strains, using the genomic editing tools. Collectively, this study defines the heterogeneity of B. velezensis LG37 all alone through comprehensive transcriptome analysis subsequently by genome editing techniques, sheds light on the enigmatic mechanisms under different nitrogen sources also reveal the need for further research.