Spontaneous induction of cryptic prophages in populations of the model species Corynebacterium glutamicum and Escherichia coli

病毒起源的DNA，包括功能完全的前驱噬菌体、隐蔽噬菌体元件或噬菌体弱智分子，代表着细菌基因组中的一种常见成分。溶源细菌培养的一个特征现象是即使在没有外部触发因素(称为自发原噬菌体诱导，SPI)的情况下，原噬菌体元件也会自发激活。在第一个资助期，我们研究了谷氨酸棒杆菌隐匿前体噬菌体CGP3的SPI。通过对报告菌株的单细胞分析，我们可以证明SOS反应的自发激活是CGP3的SPI导致受影响的谷氨酸杆菌细胞死亡的部分原因。此外，构建并鉴定了谷氨酸杆菌ATCC 13032的无前驱体突变体，该突变体具有几个积极的特征，强调它是这一重要工业有机体代谢工程的良好平台菌株。在目前的提案中，我们计划继续研究SPI及其对细菌种群生理的影响。我们将重点关注两个模式物种：谷氨酸杆菌和大肠杆菌。1)在实验室中，我们将继续以谷氨酰胺为模式物种，研究引发SPI的机制。对单个细胞的时空分析表明，SOS响应是一个重要的触发因素，但仅占SPI的60%左右。因此，在接下来的项目中，我们将重点介绍独立于SOS的机制。这包括表征小核样相关蛋白Lsr2的影响，通过DNA亲和层析识别新的调节因子，以及噬菌体基因组中自发突变的影响。总之，这些数据将增加对SPI的全面洞察，并将用于进一步发展我们在谷氨酸杆菌中SPI的机制模型。2)其次，我们将在实验室建立E.ColiK12作为SPI的模型，并计划对这9个隐含的噬菌体元件进行活性分析，以研究它们在微流控芯片器件中的单细胞水平上的自发诱导的影响。为了能够对不同的报告菌株和突变菌株进行高通量分析，我们将进一步发展我们的微流控系统，包括并行化、动态控制和自动显微镜控制。为了进一步改善后续的图像分析工作流程，我们将实施在线分析，以便对特定感兴趣的区域进行筛选。还将提供这一方法，以支持优先方案内其他几个合作伙伴的项目。在一项长期的进化实验中，我们将使用谷氨酸杆菌和大肠杆菌的无噬菌体变体与野生型菌株比较，对噬菌体元素对宿主生理和稳定性的影响进行基准测试。通过这个拟议的项目，我们将有助于更好地理解SPI的触发机制及其对细菌种群适合性的影响。

DNA of viral origin, including fully functional prophages, cryptic phage elements or phage morons, represents a frequent element in bacterial genomes. A characteristic phenomenon of lysogenic bacterial cultures is the spontaneous activation of prophage elements even in the absence of an external trigger (designated as spontaneous prophage induction, SPI). In the first funding period, we studied the SPI of the cryptic prophage CGP3 of Corynebacterium glutamicum. Using single cell analysis of reporter strains, we could show that the spontaneous activation of the SOS response is partly responsible for SPI of CGP3 leading to death of the affected C. glutamicum cells. Furthermore, a prophage-free variant of C. glutamicum ATCC 13032 was constructed and characterized, which exhibits several positive features emphasizing it as a good platform strain for metabolic engineering of this important industrial organism. In the present proposal, we plan to continue our studies on SPI and its impact on the physiology of bacterial populations. We will focus on two model species: C. glutamicum and Escherichia coli. 1) We will continue to study the mechanisms triggering SPI using C. glutamicum as a well-established model species in our lab. The spatio-temporal analysis of single cells revealed that the SOS response represents a prominent trigger, but only accounts for ~60% of SPI. Consequently, we will now focus on SOS-independent mechanisms in the following project. This includes characterization of the impact of the small nucleoid-associated protein Lsr2, the identification of novel regulatory factors by DNA affinity chromatography, and the influence of spontaneous mutations in phage genomes. Altogether, these data will add to a comprehensive insight in SPI and will be used to further develop our mechanistic model of SPI in C. glutamicum. 2) Second, we will establish E. coli K12 as a model for SPI in our lab and plan to perform an activity profiling of the nine cryptic phage elements to study the influence of their spontaneous induction at the single cell level using time-lapse microscopy in microfluidic chip devices. To enable the high-throughput analysis of different reporter and mutant strains, we will further develop our microfluidic setup regarding parallelization, dynamic control and automated microscope control. To further improve the subsequent image analysis workflow, we will implement an online analysis which enables the screening for specific regions of interest. This methodology will also be provided to support the projects of several other partners within the priority program. In a long-term evolution experiment we will benchmark the impact of phage elements on host physiology and stability using prophage-free variants of C. glutamicum and E. coli in comparison to the wild type strain. With this proposed project we will contribute to a better understanding of the mechanisms triggering SPI and its impact on the fitness of bacterial populations.