The apt /6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus

Abstract

Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked ␣-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus. IMPORTANCECurrently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/ 6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains. Diverse Sulfolobus islandicus strains belonging to the hyperthermophilic crenarchaea thrive in geographically isolated populations in hot springs around the world (1). These organisms provide an excellent system for studying microbial evolutionary ecology (2) and may be used as a genetic model system for studying novel molecular mechanisms in the TACK (Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota) lineage of the archaeal domain, which has been hypothesized by some to be the most recent common ancestor of the eukaryotes on the tree of life (3). To date, the genomes of 20 diverse S. islandicus strains have been sequenced (2, 4-6). Versatile genetic tools have been developed for a few representative strains of S. islandicus (6-8), including two efficient plasmid shuttle vectors (9, 10), a set of new selectable markers (8,11,12), and conventional and novel methods of genetic manipulation (13), as well as clustered regularly interspaced short palindromic repeat-Cas-mediated genome editing protocols (14). Nevertheless, the pyrEF/5-f...

The essential genome of the crenarchaeal model Sulfolobus islandicus

“…Two exceptions, topR2 ( M164_1245 ) and apt ( M164_0158 ), were identified to have significant growth defects on plates once they were knocked out (Supplementary Fig. 1c, 2a and 20 ), likely resulting in their under-representation in our transposon library. The third, cdvB3 ( M164_1510 ), a paralog of cdvB , may be incorrectly called essential in our Tn-seq analysis.…”

Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus

“…Based on the lethal incorporation of 6-methylpurine in the purine salvage pathway through TK0664 expression, the TK0664/6-MP system has allowed the deletion of multiple genes in different archaea [ 23 , 45 ]. In this study, we demonstrated the relevance of using this counterselection marker in T. barophilus .…”

Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

“…Homologous recombination (via double-crossover events) efficiencies using linear DNA have been reported in three hyperthermophilic archaea: Thermococcus kodakarensis KOD1, 10 2 colonies/ μ g linear DNA containing 1 kb flanking regions [ 22 ]; P. furiosus COM1 (parent strain DSM 3638), 2.9 × 10 3 colonies/ μ g linear DNA containing 1 kb flanking regions [ 21 ]; S. islandicus M.16.4, 20–30 colonies/ μ g linearized DNA (pC-SsoargD) containing 755 and 671 bp flanking regions [ 10 ] and 10–50 colonies/ μ g linearized DNA (pMID-apt) containing 703 and 617 bp flanking regions [ 23 ]; and S. islandicus REY15A, 10–200 colonies/ μ g linearized DNA (pKL2) containing 1.5 kb flanking regions [ 2 ]. The homologous recombination efficiency reported in our current study (10 2 -10 3 colonies/ μ g DNA) was higher than that of T. kodakarensis and S. islandicus and nearly identical to that of P. furiosus .…”

“…Therefore, the genetic marker system developed in this study will allow versatile genetic manipulation in S. acidocaldarius . Notably, a higher concentration of agmatine was required for cultivation of the S. acidocaldarius argD -deficient strain when compared with other hyperthermophiles [ 10 , 20 , 23 , 28 ].…”