TY - JOUR
T1 - Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus
AU - Deng, Ling
AU - Zhu, Haojun
AU - Chen, Zhengjun
AU - Liang, Yun Xiang
AU - She, Qunxin
N1 - Keywords: Archaea; DNA, Archaeal; Diploidy; Gene Deletion; Genetic Markers; Genetic Vectors; Genotype; Mutation; Oligonucleotides; Plasmids; Recombination, Genetic; Sequence Analysis, DNA; Sulfolobus; beta-Galactosidase
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
U2 - 10.1007/s00792-009-0254-2
DO - 10.1007/s00792-009-0254-2
M3 - Journal article
C2 - 19513584
SN - 1431-0651
VL - 13
SP - 735
EP - 746
JO - Extremophiles
JF - Extremophiles
IS - 4
ER -