Improved and Versatile Transformation System Allowing Multiple Genetic Manipulations of the Hyperthermophilic Archaeon Thermococcus kodakaraensis

Abstract

We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (⌬pyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (⌬pyrF ⌬trpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (⌬pyrF ⌬trpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (⌬pyrF ⌬trpE ⌬hisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.Recent phylogenetic analysis of living organisms based on rRNA sequences has indicated that hyperthermophiles occupy the deepest and shortest branches in the phylogenetic tree, postulating that the origin and evolution of biological systems may have derived from hyperthermophiles (25). Studies on the unique properties of hyperthermophiles are expected to provide valuable perspectives on the mechanisms that enable them to survive and grow in extreme environments (26). They are also important as potential resources for highly thermostable enzymes (2, 28). Many members of hyperthermophiles belong to the third domain of life, Archaea, along with halophiles and methanogens. Archaea exhibit a mosaic of features from the other two domains, Bacteria and Eucarya; intriguingly, their components for information processing are more closely related to those in eucaryotes than those in bacteria. From these interests, genome projects of various types of hyperthermophilic archaea have been performed, and complete genome sequences of more than 10 species have been determined. The genomes of hyp...

A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection

“…There are three main factors that led to our low number of initial transformants. First, unlike Thermococcus kodakarensis and Pyrococcus furious, which display strong natural competence (Lipscomb et al, 2011;Sato et al, 2003Sato et al, , 2005, electroporation is the only effective way for the exogenous DNA to be introduced into S. islandicus strains. Most of the cells would be killed after electroporation and the relatively lower transformation efficiency [0-5 colonies (mg DNA) 21 for a circular knockout plasmid] usually result in a failure to obtain transformants, particularly when using a gene knockout plasmid for transformation (Deng et al, 2009).…”

A Global Transcriptional Regulator in Thermococcus kodakaraensis Controls the Expression Levels of Both Glycolytic and Gluconeogenic Enzyme-encoding Genes

“…Both glycolytic (starch or maltodextrin) and gluconeogenic (peptides or pyruvate) modes of growth are observed for this archaeon, and the TGM sequences are found on most of the glycolytic and starch-utilizing gene promoters (25). The development of a gene disruption system for T. kodakaraensis (28,29) as well as the availability of genome information (30) makes this archaeon an attractive model organism for the elucidation of the physiological role of unknown gene function in Thermococcales (31)(32)(33).…”

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

“…A genetic manipulation system was developed for Thermococcus kodakarensis (4), and phenotype analyses of the hef mutant strain confirmed that Hef is involved in multiple repair processes, and its especially high sensitivity to mitomycin C suggested that Hef performs a critical function in DNA interstrand cross-link repair (5). The helicase and nuclease activities of the purified T. kodakarensis Hef protein (TkoHef) for forkstructured DNA was also confirmed in vitro (5).…”