ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli
ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli
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DOI：
10.1128/msphere.00591-20
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发表时间：
2020-07-01
期刊：
影响因子：
4.8
通讯作者：
Dobrindt, Ulrich
中科院分区：
文献类型：
--
作者：
Wallenstein, Alexander;Rehm, Nadine;Dobrindt, Ulrich
Colibactin是一种由肠杆菌科不同成员表达的非核糖体肽/聚酮化合物杂合天然产物，其可与真核生物中DNA双链断裂的诱导和细胞周期进程的干扰相关。大肠杆菌素表达的调控特征还不完全清楚。我们使用大肠杆菌菌株M1/5作为一个模型来研究大肠杆菌素决定簇在转录水平上的表达调控，并表征位于大肠杆菌素致病岛本身内的调控元件。我们在体外测量了clbR转录，并观察到在限定的基本培养基中培养导致相对于丰富培养基增加的大肠杆菌素表达。clbR的转录直接响应于铁的可用性。我们还表征了参与ClbR依赖性调节的大肠杆菌素决定簇内的结构DNA元件，即，ClbR结合位点和位于clbR上游的可变数目的串联重复。我们在转录组和蛋白质组水平研究了clbR过表达或缺失的影响。此外，我们比较了在这些条件下的全局基因调控与clbQ过表达或缺失时发生的基因调控，clbQ过表达或缺失会影响大肠杆菌素生产的通量。结合转录组和蛋白质组分析的结果与间接测量colibactin水平的细胞培养试验和colibactin通过第二个产品的colibactin裂解从precolibactin，N-肉豆蔻酰D-天冬酰胺的近似定量，我们证明了可变数目的串联重复序列在colibactin表达中起着重要的调节作用。我们确定ClbR作为迄今为止已知的唯一的转录激活剂，是具体的和必不可少的有效调节colibactin production.IMPORTANCE的非核糖体肽/聚酮杂合colibactin可以被认为是一种细菌的毒力因子参与肠外感染，也是一个procarcinogen。然而，尽管大肠杆菌素具有遗传毒性作用，但其表达也可抑制细菌或肿瘤生长，并与益生菌的抗菌和镇痛特性相关。虽然这种天然化合物的生物学功能已被广泛研究，但我们对大肠杆菌素表达调控的理解仍远未完成。我们详细研究了参与大肠杆菌素表达和促进大肠杆菌素表达的生长条件的元件的作用。通过这种方式，我们的数据揭示了大肠杆菌素表达的调控机制，并可能支持表达和纯化这种有趣的非核糖体肽/聚酮化合物杂合物，以进一步进行分子表征。
Colibactin is a nonribosomal peptide/polyketide hybrid natural product expressed by different members of the Enterobacteriaceae which can be correlated with induction of DNA double-strand breaks and interference with cell cycle progression in eukaryotes. Regulatory features of colibactin expression are only incompletely understood. We used Escherichia coli strain M1/5 as a model to investigate regulation of expression of the colibactin determinant at the transcriptional level and to characterize regulatory elements located within the colibactin pathogenicity island itself. We measured clbR transcription in vitro and observed that cultivation in defined minimal media led to increased colibactin expression relative to rich media. Transcription of clbR directly responds to iron availability. We also characterized structural DNA elements inside the colibactin determinant involved in ClbRdependent regulation, i.e., ClbR binding sites and a variable number of tandem repeats located upstream of clbR. We investigated the impact of clbR overexpression or deletion at the transcriptome and proteome levels. Moreover, we compared global gene regulation under these conditions with that occurring upon overexpression or deletion of clbQ, which affects the flux of colibactin production. Combining the results of the transcriptome and proteome analyses with indirect measurements of colibactin levels by cell culture assays and an approximate quantification of colibactin via the second product of colibactin cleavage from precolibactin, N-myristoylD-asparagine, we demonstrate that the variable number of tandem repeats plays a significant regulatory role in colibactin expression. We identify ClbR as the only transcriptional activator known so far that is specific and essential for efficient regulation of colibactin production.IMPORTANCE The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic antiinflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of elements involved in colibactin expression and in the growth conditionsthat promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization.