Novel anti-phage resistance mechanisms channelled through transcriptional regulation

聚球藻属和原绿球藻属的海洋单细胞蓝藻是世界海洋中重要的初级生产者。它们与高丰度的噬藻体共存可能是由于有效的抗性机制。以前，我们已经表明，多面手（广泛的主机范围）T4-样噬藻体在多个敏感的主机株具有相同的转录程序。由于这个RNA聚合酶家族本身并不编码RNA聚合酶，所以它必须夺取宿主的RNA聚合酶。在分子水平上调节该程序的方法与T4原型噬菌体显著不同，并且仍然未知。此外，我们已经表明，多面手噬藻体附着并进入耐药蓝藻细胞，但不完成感染周期。在抗性蓝藻中停止感染的一个共同特征是噬菌体基因的转录显著减少。这些海洋蓝藻中的绝大多数缺乏已知的抗性机制，如CRISPR-Cas或限制修饰系统。这表明目前未知的细胞内防御机制正在发挥作用。在这个优先计划中，我们的目标是解决如何在敏感的蓝藻中调节海洋T4类噬菌体的噬菌体转录程序的中心问题，并阐明在耐药蓝藻的转录水平上发挥作用的防御机制。我们的具体目标是：（1）确定宿主和噬菌体因子调节噬菌体启动子;（2）阐明噬菌体因子与宿主RNA聚合酶相互作用;（3）研究关键的宿主和噬菌体蛋白在噬菌体转录中的作用;（4）发现蓝藻抗性机制，减少转录。我们将使用T4类噬藻体Syn 9与敏感的聚球藻WH 8109和抗性聚球藻CC 9311作为我们的模型系统，所有这些都可以进行遗传操作。为了实现这些目标，我们将鉴定直接参与噬菌体启动子活性调节和调节转录的噬菌体和宿主蛋白。我们将确定这是通过与噬菌体启动子的直接相互作用，还是通过宿主RNA聚合酶的化学修饰或蛋白质-蛋白质相互作用。我们将产生缺乏这些因子的突变体，以评估对感染过程的影响。此外，我们将确定是什么抑制了抗性蓝藻中的噬菌体转录程序。这项研究将提供新的手段，通过海洋T4-样噬藻体调节其转录程序相对于在T4原型的基本信息。此外，这将为发现在转录水平发挥作用的新的抗病毒防御系统奠定基础。因此，这项研究计划将开辟新的视野，在理解宿主病毒相互作用，在这一生态重要的有机体组。

Marine unicellular cyanobacteria of the genera Synechococcus and Prochlorococcus are important primary producers in the world’s oceans. Their coexistence with high abundances of cyanophages is likely due to effective mechanisms of resistance. Previously, we have shown that generalist (broad host-range) T4-like cyanophages have identical transcriptional programs in multiple sensitive host strains. Since this family of phages does not code for its own RNA polymerase it must usurp that of its hosts. The means through which this program is regulated at the molecule level is significantly different to the T4 archetype phage and remains unknown. In addition, we have shown that generalist cyanophages attach and enter into resistant cyanobacterial cells, yet do not complete the infection cycle. A common feature of halted infections in resistant cyanobacteria is significantly reduced transcription of phage genes. The vast majority of these marine cyanobacteria lack known resistance mechanisms such as CRISPR-Cas or restriction-modification systems. This indicates that presently unknown intracellular defense mechanisms are at play. In this priority program our objectives are to address the central question of how the phage transcriptional program of marine T4-like cyanophages is regulated in sensitive cyanobacteria and to elucidate the mechanisms of defence that function at the transcriptional level in resistant cyanobacteria. Our specific objectives are to: (1) Identify host and phage factors that regulate phage promoters; (2) Elucidate phage factors that interact with the host RNA polymerase; (3) Investigate the role of key host and phage proteins in phage transcription; and (4) Discover cyanobacterial resistance mechanisms that reduce transcription. We will use the T4-like cyanophage, Syn9, with the sensitive Synechococcus WH8109 and resistant Synechococcus CC9311 as our model system, all of which can be genetically manipulated. To achieve these aims we will identify phage and host proteins that are directly involved in regulation of phage promoter activity and modulate transcription. We will determine whether this is through direct interaction with phage promoters or through the chemical modification of, or protein-protein interactions with, the host RNA polymerase. We will generate mutants lacking these factors to assess the impact on the infection process. Furthermore, we will ascertain what inhibits the phage transcriptional program in the resistant cyanobacterium. This study will provide fundamental information on the novel means through which the marine T4-like cyanophages regulate their transcriptional program relative to that in the T4 archetype. Furthermore, this will lay the groundwork for the discovery of new anti-viral defence systems that function at the transcriptional level. As such this research program will open up new horizons in the understanding of host-virus interactions in this ecologically important group of organisms.