沙福芽孢杆菌Spo0A蛋白在锰胁迫生理响应中的调控机制

Regulatory mechanism of Spo0A in the physiological response of Bacillus safensis to manganese stress

摘要:

背景 从锰矿矿坑土壤中分离的沙福芽孢杆菌ST7具有高效的锰适应能力，大量基因参与了锰胁迫的应答，其中转录因子Spo0A (stage 0 sporulation protein A, Spo0A)上调表达，可能参与沙福芽孢杆菌(Bacillus safensis)锰胁迫应答过程的调节。目的 研究spo0A基因在沙福芽孢杆菌ST7响应锰胁迫过程中的调节作用。方法 提取菌株ST7基因组DNA，通过PCR扩增spo0A基因，并克隆至pSWE-Topo Zero Vector载体后测序；采用同源重组单交换法构建spo0A基因缺失突变株Δspo0A，检测野生菌株与突变株在生物量、运动能力、生物膜形成能力、锰氧化能力及芽孢形成能力上的差异，利用RT-qPCR比较芽孢形成相关基因ctc、群体感应相关基因ricR、应激调控相关基因yodB及细胞表面结构相关基因ywcE的表达水平。结果 B. safensis ST7的spo0A基因编码区长804 bp，编码267个氨基酸，无信号肽及跨膜结构域；突变株中该基因后半段被卡那霉素抗性基因取代而失活。锰胁迫下，Δspo0A突变株生长受抑、生物量减少，运动、生物膜形成、锰氧化及芽孢形成能力较野生株分别降低13.5%、85.6%、18.4%和93.36%，ctc、ricR等功能基因表达量下调。结论 spo0A基因可促进沙福芽孢杆菌的鞭毛合成和运动能力，促进细菌生长和生物膜形成，并调控菌株的锰氧化能力和芽孢形成能力。

Abstract:

Background The Bacillus safensis strain ST7, isolated from the soil of manganese mine pits, exhibits efficient manganese adaptability. A large number of genes are involved in the response to manganese stress, of which the gene encoding the transcription factor stage 0 sporulation protein A (Spo0A) is upregulated and may be involved in regulating the manganese stress response process of B. safensis.Objective To investigate the regulatory role of spo0A in B. safensis ST7 to manganese stress.Methods Genomic DNA was extracted from B. safensis ST7. The gene spo0A was amplified via PCR, cloned into the pSWE-Topo Zero Vector, and then sequenced. The spo0A-deleted mutant (Δspo0A) was constructed via the homologous recombination single-exchange method. The biomass, motility, biofilm formation, manganese oxidation, and sporulation were compared between the wild-type strain and the mutant strain. Additionally, RT-qPCR was performed to compare the expression levels of the sporulation-related gene ctc, quorum sensing-related gene ricR, stress regulation-related gene yodB, and cell surface structure-related gene ywcE.Results The coding sequence of spo0A in B. safensis ST7 was 804 bp in length, encoding 267 amino acid residues, without signal peptide or transmembrane domain. In Δspo0A, the partial sequence of spo0A was replaced by the kanamycin resistance gene, which resulted in the inactivation of spo0A. Under manganese stress, Δspo0A exhibited inhibited growth and reduced biomass. Compared with the wild-type strain, Δspo0A showed the decreases of 13.5%, 85.6%, 18.4%, and 93.36% in motility, biofilm formation, manganese oxidation, and sporulation, respectively. Moreover, the mutant presented down-regulated expression levels of functional genes such as ctc and ricR.Conclusion The gene spo0A can promote flagellar synthesis and motility, enhance the growth and biofilm formation, and regulate the manganese oxidation and sporulation of B. safensis.