Novel anti-phage resistance mechanisms channelled through transcriptional regulation

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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.