Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus.
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.
Deng
Ling
L
Huazhong Agricultural University, Wuhan, People's Republic of China. denling@gmail.com
Zhu
Haojun
H
Chen
Zhengjun
Z
Liang
Yun Xiang
YX
She
Qunxin
Q
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
D019641:DNA, Archaeal; D005819:Genetic Markers; D009841:Oligonucleotides; D001616:beta-Galactosidase