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

病毒起源的DNA，包括全功能的噬菌体、隐体噬菌体元件或噬菌体白痴，是细菌基因组中常见的元素。溶原性细菌培养的一个特征现象是即使在没有外部触发的情况下，原噬菌体元素也会自发激活（称为自发原噬菌体诱导，SPI）。在第一期资助期内，我们研究了谷氨酸棒状杆菌隐前噬菌体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 SPI导致受影响的谷氨酰胺细胞死亡的部分原因。此外，构建并鉴定了一株无原噬菌体的C. glutamum ATCC 13032，该菌株显示出几个积极的特征，强调它是这一重要工业生物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及其对细菌群体生理的影响。我们将重点关注两个模式物种：谷氨酰胺和大肠杆菌。1)我们将在实验室以谷氨酰胺（C. glutamicum）为模型物种，继续研究SPI的触发机制。单细胞的时空分析表明，SOS反应是一个突出的触发因素，但仅占SPI的约60%。因此，我们现在将在下一个项目中关注与sos无关的机制。这包括表征小核相关蛋白Lsr2的影响，通过DNA亲和层析鉴定新的调节因子，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的全面了解，并将用于进一步开发谷氨酸丙氨酸SPI的机制模型。2)其次，我们将在实验室建立大肠杆菌K12作为SPI的模型，并计划在微流控芯片设备中使用延时显微镜对9种隐体噬菌体元件进行活性分析，研究它们在单细胞水平上自发诱导的影响。为了实现对不同报告菌株和突变菌株的高通量分析，我们将进一步发展我们的微流体装置，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，以筛选感兴趣的特定区域。该方法还将用于支持优先方案内其他几个伙伴的项目。在一个长期的进化实验中，我们将使用谷氨酰胺和大肠杆菌的无噬菌体变体与野生型菌株进行比较，以基准噬菌体元素对宿主生理和稳定性的影响。通过这个项目，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应性的影响。

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.