title: Augmenting the genetic toolbox for Sulfolobus islandicus with a stringent positive selectable marker for agmatine prototrophy.
abstract: Sulfolobus species have become the model organisms for studying the unique biology of the crenarchaeal division of the archaeal domain. In particular, Sulfolobus islandicus provides a powerful opportunity to explore natural variation via experimental functional genomics. To support these efforts, we further expanded genetic tools for S. islandicus by developing a stringent positive selection for agmatine prototrophs in strains in which the argD gene, encoding arginine decarboxylase, has been deleted. Strains with deletions in argD were shown to be auxotrophic for agmatine even in nutrient-rich medium, but growth could be restored by either supplementation of exogenous agmatine or reintroduction of a functional copy of the argD gene from S. solfataricus P2 into the ΔargD host. Using this stringent selection, a robust targeted gene knockout system was established via an improved next generation of the MID (marker insertion and unmarked target gene deletion) method. Application of this novel system was validated by targeted knockout of the upsEF genes involved in UV-inducible cell aggregation formation.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. cyz@illinois.edu
authors.forename: Changyi
authors.initials: C
authors.lastname: Zhang
authors.forename: Tara E
authors.initials: TE
authors.lastname: Cooper
authors.forename: David J
authors.initials: DJ
authors.lastname: Krause
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Whitaker
mesh_terms: D000376:Agmatine / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods*; D005816:Genetic Complementation Test; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D012641:Selection, Genetic; D016944:Sulfolobus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
chemical_list: D000376:Agmatine; D002262:Carboxy-Lyases; C019722:arginine decarboxylase