Translocation of viral DNA across the bacterial host membrane.

• Applicant: Dr. Dorothee Kiefer, since 3/2022
• Subject Area: Biochemistry
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 439129695

Bacteriophages, the viruses of prokaryotes, depend on a bacterial host for propagation. The lytic coliphage T4 and T7 have been extensively studied for their biochemical and genetic processes. Many basic principles of molecular assembly processes and functions of molecular motors originate from phage research. Recent research on microbiomes and phageomes has demonstrated a substantial contribution of phage to human and animal health as well as to ecological systems. Therefore, phage-host interactions are at the forefront of current science.For the infection of a bacterial cell, the phage particle has to recognize its host by adsorbing to distinct components of the cell surface. In the T-phages, this is initiated by the phage tail fibers contacting the receptor molecules exposed at the cell wall. The specific adsorption of the phage induces a conformational change in the phage particle to trigger the release and transfer of the viral DNA into the host cell. Still, only little is known about the molecular mechanisms of this highly dynamic process. For T4 with its contractile tail, the tail’s inner tube is pushed through the cell wall of E. coli. A phage lysozyme domain in the main tube component gp5 locally digests peptidoglycan. This putatively enables the tube tip protein gp5.4 that is firmly bound to the distal end of gp5, to reach the periplasmic side of the cytoplasmic membrane. Although structural data exist for the gp5/5.4 complex, the molecular dynamics of the T4 injection complex and its functional interaction with the host membrane remain unclear. Presumably, a specific contact to a membrane or periplasmic component is required to translocate the viral DNA. In the projected work we aim to elucidate the structure-function dynamics of the T4 needle tip complex by biochemical and biophysical techniques. We want to identify host proteins interacting with the T4 tail tube components during DNA translocation. The second T-phage we focus on, is T7 which depicts a very different way of bringing its genome into the host cell. Phage T7 has no contractile tail, but harbours very versatile proteins in its capsid, the core proteins. The core complex consists of three main subunits, gp14, gp15 and gp16. After adsorption to the host’s cellular surface, T7 swings out the core complex from the capsid and expands it into the host periplasm by a still mysterious mechanism. The core complex proteins presumably provide a transient transporter, the "ejectosome" to translocate the phage genome through the inner and outer host membranes into the bacterial cytoplasm. The core proteins therefore have to be highly dynamic as they have to disassemble from the phage capsid core and reassemble into the lipid environment of the host membrane. In the proposed research we plan to study the core complex assembly and folding of the purified proteins in vitro and their reconstitution into proteoliposomes to be tested for translocation DNA across the lipid bilayer.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Professor Dr. Andreas Kuhn, until 2/2022