State Key Laboratory of Agricultural Microbiology, College of Fisheries and College of Life Sciences, Huazhong Agricultural University, Wuhan, 430070, Hubei Province, China.
Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Guangdong Provincial Key Laboratory of Waterfowl Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, 510225, Guangdong, China.
BMC Genomics. 2020 Mar 6;21(1):215. doi: 10.1186/s12864-020-6621-1.
In recent years, interest in Bacillus velezensis has increased significantly due to its role in many industrial water bioremediation processes. In this study, we isolated and assessed the transcriptome of Bacillus velezensis LG37 (from an aquaculture pond) under different nitrogen sources. Since Bacillus species exhibit heterogeneity, it is worth investigating the molecular mechanism of LG37 through ammonia nitrogen assimilation, where nitrogen in the form of molecular ammonia is considered toxic to aquatic organisms.
Here, a total of 812 differentially expressed genes (DEGs) from the transcriptomic sequencing of LG37 grown in 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 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 results showed that expression of all the 8 DEGs are consistent with the RNA-seq data. Moreover, the transcriptome and relative expression analysis were consistent with the transporter gene amtB and it is not involved in ammonia transport, even in the highest ammonia concentrations. Besides, CRISPR-Cas9 knockout and overexpression glnK mutants further evidenced the exclusion of amtB regulation, suggesting the involvement of alternative transporter. Additionally, in the transcriptomic data, a novel ammonium transporter mnrA was expressed significantly in increased ammonia concentrations. Subsequently, OEmnrA and ΔmnrA LG37 strains showed unique expression pattern of specific genes compared to that of wild-LG37 strain.
Based on the transcriptome data, regulation of nitrogen related genes was determined in the newly isolated LG37 strain to analyse the key regulating factors during ammonia assimilation. Using genomics tools, the novel MnrA transporter of LG37 became apparent in ammonia transport instead of AmtB, which transports ammonium nitrogen in other Bacillus strains. Collectively, this study defines heterogeneity of B. velezensis LG37 through comprehensive transcriptome analysis and subsequently, by genome editing techniques, sheds light on the enigmatic mechanisms controlling the functional genes under different nitrogen sources also reveals the need for further research.
近年来，由于芽孢杆菌在许多工业水生物修复过程中的作用，人们对其兴趣显著增加。在这项研究中，我们分离并评估了来自水产养殖池塘的芽孢杆菌 LG37 在不同氮源下的转录组。由于芽孢杆菌表现出异质性，因此通过氨氮同化来研究 LG37 的分子机制是值得的，因为以分子氨形式存在的氮被认为对水生生物有毒。
在这里，从在补充有氨（处理）或谷氨酰胺（对照）的最小培养基中生长的 LG37 的转录组测序中获得了总共 812 个差异表达基因（DEGs），其中 56 个具有≥2 的倍数变化。BLAST-NCBI 和 UniProt 数据库显示，56 个 DEGs 中的 27 个可能参与 NH 同化。其中，随机选择 8 个 DEG 和双组分调节系统 GlnK/GlnL 进行定量实时 RT-PCR 验证，结果表明所有 8 个 DEG 的表达与 RNA-seq 数据一致。此外，转录组和相对表达分析与转运蛋白基因 amtB 一致，即使在最高氨浓度下，它也不参与氨的转运。此外，CRISPR-Cas9 敲除和过表达 glnK 突变体进一步证明了 amtB 调节的排除，表明存在替代转运蛋白。此外，在转录组数据中，在增加的氨浓度下，新型铵转运蛋白 mnrA 表达显著。随后，与野生型 LG37 菌株相比，OEmnrA 和ΔmnrA LG37 菌株显示出特定基因的独特表达模式。
基于转录组数据，确定了新分离的 LG37 菌株中与氮相关基因的调控，以分析在氨同化过程中的关键调节因子。使用基因组学工具，LG37 的新型 MnrA 转运蛋白在氨转运中变得明显，而不是在其他芽孢杆菌菌株中运输铵态氮的 AmtB。总的来说，通过综合转录组分析，这项研究定义了 LG37 的芽孢杆菌的异质性，随后通过基因组编辑技术，揭示了控制不同氮源下功能基因的神秘机制，也表明需要进一步研究。
