title: Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus.
abstract: Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complemented DeltapyrEFDeltalacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.
authors.affiliation: Huazhong Agricultural University, Wuhan, People's Republic of China. denling@gmail.com
authors.forename: Ling
authors.initials: L
authors.lastname: Deng
authors.forename: Haojun
authors.initials: H
authors.lastname: Zhu
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Chen
authors.forename: Yun Xiang
authors.initials: YX
authors.lastname: Liang
authors.forename: Qunxin
authors.initials: Q
authors.lastname: She
mesh_terms: D001105:Archaea; D019641:DNA, Archaeal / Q000235:genetics; D004171:Diploidy; D017353:Gene Deletion; D005819:Genetic Markers; D005822:Genetic Vectors; D005838:Genotype; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D016944:Sulfolobus / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D019641:DNA, Archaeal; D005819:Genetic Markers; D009841:Oligonucleotides; D001616:beta-Galactosidase