Living organisms have developed specific defence mechanisms to counteract hostile environmental conditions. Alkylation stress response mechanisms also occur in Mycobacterium tuberculosis (MTB) the pathogen responsible for tuberculosis. The effect of alkylating agents on the cellular growth of MTB was investigated using methyl methanesulfonate (MMS) as methyl donor demonstrating that limited doses of alkylating agents might affect MTB cell viability. A global investigation of Mycobacterium smegmatis response to alkylating stress was then pursued by differential proteomics to identify the most affected cellular pathways. Quantitative analysis of proteomic profiles demonstrated that most of the proteins upregulated in the presence of alkylating agents are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we identified the GlmU bifunctional enzyme as a possible factor involved in biofilm production. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the M. smegmatis defence mechanisms.

中文翻译：

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双功能蛋白GlmU是耻垢分枝杆菌中烷基化应激诱导生物膜形成的关键因素。

活生物体已开发出专门的防御机制来抵抗敌对的环境条件。烷基化应激反应机制也发生在结核分枝杆菌的致病菌结核分枝杆菌（MTB）中。使用甲磺酸甲酯（MMS）作为甲基供体，研究了烷基化剂对MTB细胞生长的影响，表明有限剂量的烷基化剂可能会影响MTB细胞的活力。然后，通过差异蛋白质组学进行了耻垢分枝杆菌对烷基化应激反应的全球调查，以确定受影响最大的细胞途径。蛋白质组学概况的定量分析表明，在烷基化剂存在下上调的大多数蛋白质都参与生物膜的形成和/或细胞壁的生物合成。量身定制的实验证实，在压力条件下，耻垢分枝杆菌会通过增加生物膜的形成引发物理防御机制。在上调的蛋白质中，我们确定了GlmU双功能酶是参与生物膜生产的可能因素。有条件的缺失和过表达的glmU突变体的实验表明，GlmU的下调降低了耻垢分枝杆菌产生生物膜的能力，而酶的过表达增加了生物膜的形成。这些结果得到了两种不同抑制剂对GlmU乙酰转移酶活性的抑制的支持，表明该酶参与了耻垢分枝杆菌的防御机制。我们将GlmU双功能酶鉴定为参与生物膜生产的可能因素。有条件的缺失和过表达的glmU突变体的实验表明，GlmU的下调降低了耻垢分枝杆菌产生生物膜的能力，而酶的过表达增加了生物膜的形成。这些结果得到了两种不同抑制剂对GlmU乙酰转移酶活性的抑制的支持，表明该酶参与了耻垢分枝杆菌的防御机制。我们将GlmU双功能酶鉴定为参与生物膜生产的可能因素。有条件的缺失和过表达的glmU突变体的实验表明，GlmU的下调降低了耻垢分枝杆菌产生生物膜的能力，而酶的过表达增加了生物膜的形成。这些结果得到了两种不同抑制剂对GlmU乙酰转移酶活性的抑制的支持，表明该酶参与了耻垢分枝杆菌的防御机制。