Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method.
Organisms belonging to the Crenarchaeota lineage contain three proliferating cell nuclear antigen (PCNA) subunits, while those in the Euryarchaeota have only one, as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandicus, a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. Then, a new knockout scheme, known as marker insertion and target gene deletion (MID), was developed, with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMID-pcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Δpcna1 or Δpcna3) revealed that mutant cells could no longer be propagated, demonstrating that these pcna genes are absolutely required for host cell viability. Because the only prerequisite for this assay is the generation of a MID transformant, this approach can be applied generally to any micro-organisms proficient in homologous recombination.
Zhang
Changyi
C
Archaeal Genetics Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen Biocenter, DK-2200 Copenhagen N, Denmark.|State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
Guo
Li
L
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, PR China.
Deng
Ling
L
Archaeal Genetics Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen Biocenter, DK-2200 Copenhagen N, Denmark.
Wu
Yuanxin
Y
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
Liang
Yunxiang
Y
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
Huang
Li
L
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, PR China.
She
Qunxin
Q
Archaeal Genetics Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen Biocenter, DK-2200 Copenhagen N, Denmark.
D019843:Archaeal Proteins / Q000235:genetics*; D019641:DNA, Archaeal / Q000235:genetics; D055786:Gene Knockout Techniques; D019766:Genes, Archaeal; D020043:Genes, Essential; D054643:INDEL Mutation; D050296:Microbial Viability; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics*; D016944:Sulfolobus / Q000235:genetics* / Q000254:growth & development
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D018809:Proliferating Cell Nuclear Antigen