Abstract
Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli–T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli–T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.
Highlights
Investigating and understanding the ability of cells to survive in hyperthermophilic environments is an increasingly important undertaking in the fields of biochemistry, biotechnology, molecular biology and evolution, to name just a few
We present a series of novel E. coli–T. kodakarensis shuttle vectors based on the small cryptic plasmid pTP2 from T. prieurii, and the E. coli p15A origin of replication
We have established that the cryptic plasmid pTP2 from Thermococcus prieurii encodes an origin of replication which is functional in T. kodakarensis
Summary
Investigating and understanding the ability of cells to survive in hyperthermophilic environments is an increasingly important undertaking in the fields of biochemistry, biotechnology, molecular biology and evolution, to name just a few. Biochemists have known the value of thermostable enzymes for many decades, in structural studies, where. Despite Thermococcus kodakarensis having been adopted as a model organism, only a single vector is available for the transformation of, and expression of exogenous genes in this species. To study the mechanisms of plasmid maintenance, and to understand the mechanisms of horizontal gene transfer observed in these extremophilic archaea, it is important to have genetic tools which allow us to follow multiple genes, and multiple replicons simultaneously
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