Genetic and biochemical characterisation of plasmid vesicles & other virus-like elements in Archaea

Abstract

Plasmid vesicles (PVs) are a novel class of vesicles that cross the divide between horizontal gene transfer in extracellular vesicles and targeted trans- fer of nucleic acids by selfishly replicating viruses. The archaeal plasmid pR1SE is enclosed and propagated through PVs containing both host-de- rived and plasmid-encoded proteins. PVs thus display a distinct morphology compared to the extracellular vesicles produced concurrently by the host. Infection of plasmid-free host cells occurs without direct cell-to-cell contact. Dissemination by PVs has only been described for the pR1SE plasmid to date. Its unique characteristics separate it from previously described dissem- ination mechanisms of other non-viral mobile genetic elements. This virus- like lifestyle supports the evolutionary connections between viruses and plas- mids and marks PVs out as potential evolutionary precursors to viruses. This dissertation is part of a continued effort to characterize pR1SE and PVs to better understand this unique interaction between cells and mobile genetic elements. In Chapter I, I describe our successful improvement of the genetic system in Halorubrum lacusprofundi, the only currently known producer of PVs. This system allowed detailed investigations into the PV life cycle by the deletion of targeted genes in the host, as demonstrated in the following chap- ters. We showed that PV stability is negatively affected (Chapter II) when the host machinery responsible for the posttranslational modification of pro- teins with polysaccharides is impaired. Detailed tracking of intracellular pR1SE replication and PV production after infection of a new host revealed strong similarities between the infection cycles of pR1SE and actively repli- cating viruses infecting the same host. This research was expanded to the analysis of the lipid composition of vesicles produced by Hrr. lacusprofundi, and comparison to a related organism Haloferax volcanii (Chapter III). We showed that the vesicles of both organisms are selectively enriched in specific lipid species. This suggests common patterns of physical processes at the cell membrane during the formation of vesicles and membrane-envel- oped viruses in halophilic archaea. Chapter III also includes a description of the archaeal vesiculating GTPase enzyme, which is a critical component of the vesicle formation machinery in H. volcanii. Based on this work, we could show that deletion of this gene also alters the extracellular vesicle formation in Hrr. lacusprofundi (Chapter IV). Finally, we also confirmed that PV pro- duction is independent of the machinery of GTPase-driven production of ex- tracellular vesicles by the host. The results presented in this dissertation provide valuable new insights into the life cycle and host dependency of PVs and set them into a broader con- text with the vesicles that are produced with PVs, and the viruses that infect the same organism. This allows us to better consider the position pR1SE occupies within the total diversity of mobile genetic elements and provides a potential template for future research into unique mobile genetic elements in archaea.