Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method

Abstract

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 pMIDpcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Dpcna1 or Dpcna3) 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.

Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

“…pMIDherA was constructed for herA disruption in S. islandicus as described previously (38). Three homologous sequence arms, namely, target gene (Tg) arm, integration (In) arm, and looping-out (Out) arm were amplified by PCR using the genomic DNA of S. islandicus as the template, and their respective primers.…”

“…Sulfolobus is also an important model in geomicrobiological study for which genome sequences have been determined for seven strains isolated from hot springs in the United States and Russia (28). Moreover, tools for genetic analysis have been developed for three Sulfolobus species (21), including S. solfataricus, S. acidocaldarius, and S. islandicus, allowing in vivo functional analysis of diverse genes to be conducted (2,12,14,31,36,38).…”

Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

“…DEfficiency of recombination between diverged DNAs, relative to DNAs of identical sequence. (Kurosawa & Grogan, 2005), provides the basis for disrupting other S. acidocaldarius genes (Sakofsky et al, 2011); furthermore, corresponding phenomena have been documented in S. islandicus and S. solfataricus, and developed into effective gene-disruption techniques (Zhang et al, 2010;Maezato et al, 2011).…”

A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection

“…For example, a primary system in Sulfolobus acidocaldarius utilizes uracil selection, in which spontaneous uracil auxotrophs in the pyrEF genes are selected for by resistance to the toxic compound 5-fluoroorotic acid (5-FOA). Transformants of these auxotrophs are then selected for by complementation through addition of a functional pyrEF cassette (Grogan, 2009 allelic replacement and markerless gene deletion, have been constructed in S. islandicus Rey15A and S. acidocaldarius (Ajon et al, 2011;Deng et al, 2009;Li et al, 2011;Sakofsky et al, 2011;Zhang et al, 2010).…”