Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning

The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP (1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many of which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against a urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence–function relationships in many phage–bacterial systems.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

ABSTRACTIn most bacteriophages, the genome transport across bacterial envelopes is carried out by the tail machinery. In Podoviridae viruses, where the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins are translocated and assembled into a tube within the periplasm. T7 bacteriophage, a member from the Podoviridae family, infects E. coli gram-negative bacteria. Despite extensive studies, the precise mechanism by which this virus translocates its genome remains unknown. Using cryo-electron microscopy, we have resolved the structure two different assemblies of the T7 bacteriophage DNA translocation complex, built by core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled by interaction with gp16, resulting in a tubular structure with dimensions compatible with traversing the bacterial envelope and a channel that allows DNA passage. The structure of the gp15-gp16 complex also shows the location in gp16 of a canonical transglycosylase motif essential in the bacterial peptidoglycan layer degradation. Altogether these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which helps in the understanding at the molecular level of the mechanism involved in the T7 viral DNA release in the bacterial cytoplasm.SIGNIFICANCE STATEMENTT7 bacteriophage infects E. coli bacteria. During this process, the DNA transverses the bacterial cell wall, but the precise mechanism used by the virus remains unknown. Previous studies suggested that proteins found inside the viral capsid (core proteins) disassemble and reassemble in the bacterial periplasm to form a DNA translocation channel. In this article we solved by cryo-electron microscopy two different assemblies of the core proteins that reveal the steps followed by them to finally form a tube large enough to traverse the periplasm, as well as the location of the transglycosylase enzyme involved in peptidoglycan degradation. These findings confirm previously postulated hypothesis and make experimentally visible the mechanism of DNA transport trough the bacterial wall.

Rapid 40 kb genome construction from 52 parts

ABSTRACTLarge DNA constructs (>10 kb), including small genomes and artificial chromosomes, are invaluable tools for genetic engineering and vaccine development. However, the manufacture of these constructs is laborious. To address this problem, we applied new design insights and modified protocols to Golden Gate assembly. While this methodology is routinely used to assemble 5-10 DNA parts in one-step, we found that optimized assembly permitted >50 DNA fragments to be faithfully assembled in a single reaction. We applied these insights to genome construction, carrying out rapid assembly of the 40 kb T7 bacteriophage genome from 52 parts and recovering infectious phage particles after cellular transformation. The new Golden Gate assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction–modification systems, is also active against BREX. In contrast to the wild–type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R–M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

Transcription polymerase–catalyzed emergence of novel RNA replicons

Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.