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

Abstract

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

Participation of UV-regulated Genes in the Response to Helix-distorting DNA Damage in the Thermoacidophilic Crenarchaeon <i>Sulfolobus acidocaldarius</i>

“…The DNA photolyase-deficient strain DP-1 and its derivatives did not exhibit photoreactivation activity under light conditions (50). To characterize the UV sensitivity of the knockout strains Δ cdc6-2 , Δ tfb3 , Δ rio1 , ΔSaci_0951, and ΔSaci_1302, we examined their growth properties in parallel with the parent strain DP-1 after UV-B irradiation (0, 400, and 800 J m −2 ) (Fig.…”

“…The strains used in the present study are listed in Table S1. The S. acidocaldarius pyrimidine auxotrophic, restriction endonuclease Sua I and DNA photolyase (Phr)-deficient strain DP-1 (Δ pyrE Δ suaI Δ phr ) were constructed from the strain SK-1 (Δ pyrE Δ suaI ) using pop-out recombination (50). DP-1 was then used as the host and parent strain in the present study.…”

“…To grow the pyrimidine auxotrophic strain, 0.02 g L −1 of uracil was added to XT medium (XTU). Solid plate medium was prepared as previously described (50).…”

“…The preparation of a competent cell and the transformation procedure for gene knockout have already been described (50). Gene knockout was conducted using an optimized transformation protocol (50).…”

“…Members of the order Sulfolobales belonging to the class Crenarchaeota are distributed in hot acidic terrestrial environments (39 and references therein). Seven genera belonging to the order Sulfolobales have been described with validly published names: Sulfolobus (6), Acidianus (44), Metallosphaera (23), Stygiolobus (45), Sulfurisphaera (26, 50), Sulfodiicoccus (38), and Saccharolobus (39). Sulfolobus solfataricus , which has been intensively investigated in biochemical and genetic studies, has been reclassified as Saccharolobus solfataricus (39).…”