Capsidassembly des Bakteriophagen T4

Der Coliphage T4 ist ein lytischer Bakteriophage, der Escherichia coli befällt. Er wird im Cytoplasma des Wirtes assembliert, indem in drei unabhängigen assembly pathways der Schwanz, die Fibern und das Kapsid zusammengesetzt werden. Der vollständige Phagenpartikel entsteht aus der Zusammensetzung dieser drei Unterstrukturen. Die Phagen-DNA wird ausgehend von concatemerer doppelsträngiger DNA in das Vorkapsid eingebracht. Das Vorkapsid (prehead) wird an der Bakterienmembran assembliert. Zunächst wird ein Ringkomplex aus dem phagencodierten gp20 Protein mit dem Chaperon gp40 an der Membran gebildet. Crosslinking-Experimente zeigten, dass dabei die Membraninsertase YidC beteiligt. ist. Dies bestätigt die Hypothese einer (membranständigen) Chaperone-Aktivität von YidC. Dies bestätigt die Hypothese einer (membranständigen) Chaperone-Aktivität von YidC. Nach dem vollständigen Assembly der Vorköpfe werden diese von der Membran abgelöst und mit DNA bepackt. An diesem hochdynamischen Prozess ist der gp20-gp40 Komplex ebenfalls beteiligt, der zusammen mit dem gp17 Protein den putativen T4 Nanomotor bildet.

The portal protein gp20 of the bacteriophage T4 initiates a membrane-bound phage-assembly complex

Phage are the most abundant forms of life on earth. They populate basically all living spaces from deep sea, hot springs, deserts up to the polar regions and probably even the troposphere. Enterophage are wide spread like are their enterobacterial hosts. The T-phage are particularly well studied since decades; Phage T4 has been discovered and described in the 1940s along with 6 other T-phage isolates. T4 is a lytic phage infesting E. coli B strains.

The phage capsid is icosahedral with a contractile tail and 6 terminal fibers. During phage progeny assembly in the host, the head is assembled in a separate pathway involving several phage-encoded but also some bacterial proteins in a transiently membrane-bound complex. A central player in this assembly complex is the phage gp20 portal protein which, together with its partner phage protein gp40 accumulates at the membrane to form head initiator complexes.

In our T4 project we deeply looked into this assembly complex focussing on the host factors influencing its formation. In cross-linking experiments we found several bacterial chaperones firmly bound to gp 20, among them GroEL and DnaK. Amazingly, also the E coli insertase YidC tightly contacted T4 gp20 and appeared as a cross-link product. This is surprising as YidC normally only assists multispanning inner membrane proteins in their membrane insertion and topological control. Gp20, however does not enter the lipid bilayer but only binds, though tightly, to the cytoplasmic surface of the inner bacterial membrane. The exact role of YidC in the T4 assembly complex formation is still not clear.

Prospective: Isolated gp20 protein builds dodecameric structures which can be visualized in negatively stained preparations in the transmission electron microscope. In the present work we pursue in the T4 project we aim to get higher resolution pictures also with the AFM. In addition, we are planning to determine the influence of other phage related or host proteins on the formation, oligomerization and stability of the T4 gp20 complexes.