title: The apt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus.
abstract: Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus
  
  
  
  Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA cyz@illinois.edu.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Changyi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Archaea Centre, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Qunxin
authors.identifier: http://orcid.org/0000-0002-4448-6669
authors.initials: Q
authors.lastname: She
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Hongkai
authors.initials: H
authors.lastname: Bi
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Whitaker
mesh_terms: D000228:Adenine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial; D018390:Gene Targeting / Q000379:methods*; D011687:Purines / Q000494:pharmacology*; D012641:Selection, Genetic; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
chemical_list: D011687:Purines; C016588:6-methylpurine; D000228:Adenine Phosphoribosyltransferase