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

点击翻译按钮获取中文摘要

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.