Institute of Hygiene, University of Münster, Münster, Germany.
Interdisciplinary Center for Clinical Research, Medical Faculty, University of Münster, Münster, Germany.
mSphere. 2020 Jul 15;5(4):e00591-20. doi: 10.1128/mSphere.00591-20.
Colibactin is a nonribosomal peptide/polyketide hybrid natural product expressed by different members of the 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 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 transcription and observed that cultivation in defined minimal media led to increased colibactin expression relative to rich media. Transcription of directly responds to iron availability. We also characterized structural DNA elements inside the colibactin determinant involved in ClbR-dependent regulation, i.e., ClbR binding sites and a variable number of tandem repeats located upstream of We investigated the impact of 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 , 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-myristoyl-d-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. 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 anti-inflammatory 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 regulatory elements involved in colibactin expression and in the growth conditions that 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.
大肠杆菌素是一种非核糖体肽/聚酮化合物的天然产物，由不同成员表达，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仅不完全了解。我们使用 M1/5 菌株作为模型，研究了转录水平上大肠杆菌素决定簇的表达调控，并对位于大肠杆菌素致病性岛本身内的调控元件进行了特征描述。我们测量了 colibactin 的转录，并观察到在限定的基础培养基中培养会导致大肠杆菌素的表达相对于丰富的培养基增加。转录直接响应铁的可用性。我们还描述了 colibactin 决定簇内涉及 ClbR 依赖性调节的结构 DNA 元件，即 ClbR 结合位点和位于 colibactin 上游的可变串联重复数。我们在转录组和蛋白质组水平上研究了过表达或缺失的影响。此外，我们将这些条件下的全局基因调控与过表达或缺失 colibactin 生产通量影响的条件下的全局基因调控进行了比较。将转录组和蛋白质组分析的结果与通过细胞培养测定间接测量大肠杆菌素水平以及通过前大肠杆菌素裂解的第二个产物 N-豆蔻酰-D-天冬酰胺对大肠杆菌素的近似定量相结合，我们证明可变串联重复数在大肠杆菌素表达中起着重要的调节作用。我们确定 ClbR 是迄今为止唯一已知的特异性转录激活因子，对于 colibactin 生产的有效调节是必不可少的。非核糖体肽/聚酮化合物大肠杆菌素可被视为参与肠外感染的细菌毒力因子，也是前致癌剂。尽管如此，尽管具有遗传毒性作用，但大肠杆菌素的表达也可以抑制细菌或肿瘤的生长，并与益生菌的抗炎和镇痛特性相关。尽管已经对这种天然化合物的生物学功能进行了广泛的研究，但我们对大肠杆菌素表达调控的理解仍然远远不够。我们详细研究了参与大肠杆菌素表达的调节元件以及促进大肠杆菌素表达的生长条件的作用。通过这种方式，我们的数据阐明了大肠杆菌素表达涉及的调节机制，并可能支持该有趣的非核糖体肽/聚酮化合物的表达和纯化，以进行进一步的分子表征。
