Project Details
Projekt
Unravelling the intricate regulation of the expression program of T4-like cyanophages
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Microbial Ecology and Applied Microbiology
Microbial Ecology and Applied Microbiology
Term
since 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 464976318
Marine unicellular cyanobacteria are important primary producers in the world’s oceans. Cyanophages are major agents of mortality that impact the abundance, diversity and evolution of their cyanobacterial hosts. A major family of marine cyanophages are the T4-like cyanomyoviruses, which have numerous similarities to the T4 archetype that infects E. coli. As common for dsDNA phages, T4-like cyanophages express their genomes in three expression clusters; early, middle and late, presumably using transcriptional cascades for sequential promoter recognition. They lack their own RNA polymerase and must usurp that of their hosts. Relative to T4, marine cyanophages have different early and middle promoters and lack key proteins known to regulate transcription of these two gene clusters, but have similar late promoters and homologs for late gene regulation. In this priority program project our overarching objective is to address the central question of how the expression program of marine T4-like cyanophages is regulated at the transcriptional and post-transcriptional levels. Our specific objectives are to: (1) Identify host and phage factors that regulate phage promoters; (2) Investigate RNA-binding proteins involved in post-transcriptional regulation; (3) Elucidate phage factors that interact with the host RNA polymerase; and (4) Further develop the genetic tools for marine cyanophage research. We will use the T4-like cyanophage, Syn9, and marine Synechococcus sp. strain WH8109 as our model system. Both host and phage can be genetically manipulated. In the first funding period we made predictions of phage proteins putatively involved in the regulation of the expression program using indirect evidence. We also modified our phage genetic manipulation system for the study of essential genes using a genetic knockdown approach. We have knocked out or knocked down four of these genes and found that all four have clear phenotypes, with three having longer latent periods and two producing fewer phage progeny. We also set up the methods for experimental identification of proteins that bind to phage promoters, to phage RNA, and to host RNA polymerase. In this current funding period these methods will be used to experimentally identify candidate proteins involved in transcriptional and post-transcriptional regulation of Syn9 genome expression. We will knockdown a choice set of candidates, and together with those already under investigation, will assess their influence on phage genome expression and the infection cycle. For a select few, we will delve deeper into the molecular mechanism of their mode of action. Furthermore, we will develop additional genetic tools for marine Synechococcus-cyanophage research. This study will provide fundamental information on the novel means through which marine T4-like cyanophages regulate expression of their genomes. This will open up new horizons for understanding host-virus interactions in this ecologically important system.
DFG Programme
Priority Programmes
Subproject of
SPP 2330:
New concepts in prokaryotic virus-host interactions - from single cells to microbial communities
International Connection
Israel
