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 considerably due to its ability in many industrial water treatment bioremediation processes that including aquaculture practice as probiotics. In this study, we isolated and assessed the transcriptome genes expression levels under different nitrogen source from a new Bacillus velezensis LG37 strain isolated from the aquaculture pond water. Since the Bacillus species are determined to exhibit heterogeneity, and the underlying molecular mechanisms remain poorly understood in ammonia nitrogen assimilation, where ammonia nitrogen considered as toxic to aquatics.Results Here, a total of 812 differentially expressed genes (DEGs) from the transcriptomic sequencing of LG37 grown with ammonia or glutamine, from which 56 genes obtained with Fold Change ≥ 2 through BLAST NCBI and UniProt database that displayed 27 DEGs potentially involved in NH4+ assimilation. Among them, the relative expression level of the two-component regulatory system glnK/glnL were significantly upregulated in the transcriptomic profile, and the same was authenticated for their consistency by RT-qPCR along with six other genes. In addition, the transcriptome and relative expression analysis showed that the transporter (amtB) gene of LG37 was not in favor of ammonia transport even in the highest ammonia concentrations. Besides, CRISPR-Cas9 knockout (Δ) and overexpression (OE) LG37 mutants of glnK evidenced the exclusion of amtB regulation, suggesting an involvement of alternative transporter. Moreover, the transcriptomic data exhibited a novel ammonium transporter nmtA that expressed significantly in the expanding ammonia concentrations. Subsequently, OEnmtA and ΔnmtA 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, with the aid of the genomic editing tools. The validated NmtA and other transcriptome predicted data would shed a new light on the assimilation mechanism of ammonia, and the isolate can be broadly applicable to facilitate the removal of ammonium nitrogen.