UC San Diego

Recently, a family of bacteriophages has been found to form a nucleus-like replication compartment, called the phage nucleus, which encapsulates the phage DNA and protects it from bacterial host defense systems. Although we have discovered the general replication with the phage nucleus, it is still poorly understood at the molecular level, especially the macromolecule translocations and the key proteins that play roles in these functions. The aim of this thesis is to identify the phage nucleus and its associated proteins in greater detail to shed light specifically on how DNA and mRNA export occurs, as well as the selective protein transport into this structure.

In Chapter 2, we identify a new set of phage nucleus-associated proteins through comprehensive proteomics and biochemistry. Among the identified proteins, now termed ChmB, is further investigated and found to be directly interacting with ChmA, which forms most of the phage nucleus. In addition, it is found to be directly interacting with the portal protein, suggesting that ChmB might be forming pore-like structures to accommodate DNA packaging. This study provides new insights into the composition and functions of the phage nucleus, particularly in protein-protein interactions.

In Chapter 3, we focus on another phage nucleus-associated protein, ChmC. We found that ChmC has structural homology to known RNA binding proteins. We confirmed that ChmC binds to mRNA through its surface-exposed positively charged residues and that it also has phase separation properties. Investigating the samples collected during the infection confirmed the phage mRNA binding, particularly in 5’ regions of the transcripts. When ChmC was knocked down via ddCas13d system, microscopy images showed that the phage nucleus could form, but the replication was arrested at an early stage. Correspondingly, we found that the knockdowns cause a significant reduction in phage bouquet formation as well as phage titers. These results show that ChmC has a critical role in the phage nucleus system, possibly being a part of the mRNA export mechanism after the switch to non-virion RNA Polymerase for transcription.

Chapter 4 describes the other key proteins for the phage nucleus system, such as the portal protein and its potential docking site of octameric assembly of ChmB. We also investigate the other phage nucleus-associated proteins we found by proximity labeling, gp63 and gp64 of phiPA3. Our observations suggest gp63 has a critical role in selective protein transport to the phage nucleus and that gp64 interacts with it to potentially form a complex. Finally, we identify phage proteins that potentially inhibit host cell division, which is required as the jumbo phages require longer time to complete their replication compared to their hosts. These encoded proteins might provide a more comprehensive understanding of the replication mechanism as it is crucial for phages' ability to propagate in bacterial hosts, while potentially providing underlying mechanisms that could be used in therapeutics and the biotech industry.

In conclusion, this thesis provides important insights into the phage nucleus system and the molecular mechanisms behind it. We believe that identifying and characterizing the phage nucleus and its associated proteins can have important implications for developing host-immune system evading phage therapies and allowing broader abilities to prevent severe bacterial infections.