原文链接：https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-024-02493-z

AbstractBackground

To effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process.

Results

Transcriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R−M− without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained.

Conclusions

The enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.

摘要

背景

为了有效地将质粒导入芽孢杆菌属物种并在芽孢杆菌底盘菌株中进行遗传操作，优化转化方法至关重要。这些方法旨在延长感受态时间并增强细胞膜的通透性以促进外源DNA的进入。尽管已经探索了各种策略，但很少有研究深入研究与增强感受态相关的代谢物和途径。此外，具有非功能性限制修饰系统的衍生芽孢杆菌菌株在转化外源DNA方面表现出更高的效率，但对限制修饰系统对转化过程的调控缺乏更多的探索。

结果

通过转录组学比较，发现了Bacillus subtilis 168在Spizizen转化条件下，BsuMI甲基化修饰组执行的感受态形成机制及调控途径，推测为细胞对碳源的优先选择和利用碳源时对特定代谢途径的偏好。发现细胞利用糖酵解途径利用环境葡萄糖，同时降低该途径对其他磷酸化前体的需求。这些ATP-底物竞争性代谢途径的减弱，使得更多的ATP底物在感受态形成过程中被分配到信号转导因子ComP的自磷酸化中，从而增加了关键调控蛋白ComK的表达水平。ComK的表达上调了宿主细胞中淀粉和蔗糖负调控因子SacX的表达，增强了对葡萄糖作为主要碳源的偏好。限制-修饰系统中主要蛋白BsuMI的甲基化修饰基团与氧化磷酸化途径中关键酶的功能修饰相关。BsuMI甲基化修饰基团的缺失导致细胞色素氧化酶亚基表达降低，从而导致氧化磷酸化途径减弱，促进细胞糖酵解速率，进而改善ATP分子向感受态形成区的分配。基于构建的不包含原生限制-修饰系统的菌株B. subtilis 168-R−M−，成功建立了野生型芽孢杆菌菌株遗传转化平台。利用该平台获得了较高的质粒转化效率，与对照组相比显著提高了63倍，并且获得了芽孢杆菌种间通用性的提高。

结论

归纳了限制-修饰体系中功能蛋白BsuMI增强感受态形成的机制及调控途径，为进一步研究提供参考。建立了一个有效的转化平台，克服了野生型芽孢杆菌菌株DNA转化的障碍。

图1 不同芽孢杆菌菌株质粒转化示意图

图2  具有低效R-M系统的枯草芽孢杆菌菌株的生长曲线和转化效率。分析了枯草芽孢杆菌168中的R-M系统，并在此基础上构建了低效R-M系统菌株。进一步确定了这些菌株的生长曲线和质粒转化。A 枯草芽孢杆菌168中R-M系统的分析结果。B 菌株在LB培养基中的生长曲线。C Spizizen转化中GM1和GM2培养的生长曲线。D 构建菌株中质粒pUC980-2的转化。质粒修饰：从大肠杆菌DH5α中提取的质粒用其天然甲基化系统进行修饰，并视为“M+”组。从 pUC980-2 和 pHT43 中删除天然 168 R-M 系统的识别序列“CTCGAG”（XhoI 识别位点），得到质粒 pUC980-2-DXhoI 和 pHT43-DXhoI。此外，从大肠杆菌 110 中提取的未甲基化质粒被归类为“M-”，并与正常甲基化（M+）质粒进行比较。所有实验均进行了三次生物学重复。所有组中 p < 0.05，除了菌株 168-R− 和 168-R−M− 之间的 pUC980-2-DXhoI(M+) 和 pUC980-2-DXhoI(M-) 转化结果不显著。ns 不显著

图3 对三株菌株在Spizizen条件和LB培养基中的差异基因转录组分析及“SRMvsSR”组和“SRMvsLRM”组KEGG差异基因富集情况分析。样品名称中的前缀代表不同的培养条件：“L”表示LB培养；“S”表示Spizizen转化条件。A Spizizen条件下三株菌株不同基因的Venn图。Spizizen转化条件下差异基因的筛选标准为“|log2(FoldChange)| > 0,padj < 0.05”。B “SRMvsSR”组独立差异基因的KEGG富集情况，padj < 0.05。C 氧化磷酸化途径中富集的13个基因的具体表达倍数变化。D Spizizen转化和LB培养下各菌株不同基因的Venn图。筛选标准为“|log2(FoldChange)| > 2, padj < 0.05”。E “SRMvsLRM”中独立差异基因的KEGG富集度，padj < 0.05。F 氧化磷酸化途径中富集的10个基因的具体表达倍数变化

表1 氧化磷酸化途径富集基因比较

图4 Spizizen转化过程中感受态形成及BsuMI调控的推测机制，结合三株菌株差异基因的分析结果，我们提出了感受态形成过程中的代谢流和BsuMI甲基化亚基的调控作用。A 两种培养条件下三株菌株在淀粉和蔗糖代谢中富集的不同基因的热图。B 两种培养条件下三株菌株在磷酸转移酶系统中富集的不同基因的热图。C 沉默manP基因的菌株的质粒转化。所有实验均进行了三次生物学重复。p < 0.05。D Spizizen转化过程中感受态形成及BsuMI调控的推测机制。

图5 基于DNA甲基化修饰模拟模型的野生芽孢杆菌转化平台构建，利用具有低效R-M系统的重组菌株B. subtilis 168-R-M-模拟野生芽孢杆菌DNA甲基化修饰，并基于这些模拟模型建立高效的遗传转化平台。A：Bacillus. amyloliquefaciens 205的R-M系统分析。B：Bacillus. licheniformis 702的R-M系统分析。C：用于构建DNA甲基化修饰模拟模型的质粒图谱。D：DNA甲基化修饰模拟模型菌株的生长曲线。E：野生芽孢杆菌菌株的遗传转化。利用代表不同甲基化修饰的不同宿主菌株获得的pUC980-2来转移野生型芽孢杆菌菌株。pUC980-2(DH5α)（具有大肠杆菌DH5α中的常规DNA甲基化修饰）、pUC980-2(EC135)（常用菌株大肠杆菌EC135到甲基化修饰质粒用于野生型芽孢杆菌转化）、从重组菌株168-R-M--P43-205Ms和168-R-M--P43-702Ms获得的pUC980-2(205Ms)和​​pUC980-2(702Ms)（甲基化修饰环境模拟模式受到野生芽孢杆菌的影响）。所有实验均进行了三次生物学重复。所有组中p < 0.05

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