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.
Highlights
High-throughput PCR tailing for gene disruption has been developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius [1]
The PCR-tailing technique for gene disruption was developed in the thermoacidophilic crenarchaeon S. acidocaldarius [1]; transformation efficiency has not been reported
To develop a gene manipulation system based on PCR tailing, we focused on the possibility of sufficient homologous recombination via double-crossover events with very short homologous regions
Summary
High-throughput PCR tailing for gene disruption has been developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius [1]. Three unmarked gene deletion methodologies, that is, plasmid integration and segregation (PIS), marker replacement and looping out (MRL), and marker insertion and unmarked target gene deletion (MID), have been employed in S. acidocaldarius and S. islandicus [2,3,4] These pop-out recombination-based approaches are effective for multiple gene knockout [5,6,7], but plasmid construction is required. One-step PCR followed by a marker replacement system using the pyrE selection marker flanked by 40–50 bp of homologous regions, for example, 5′ and 3′ flanking regions of the target gene, has been developed in S. acidocaldarius [1] This PCR-tailing method allows for effective, high-throughput gene functional analysis without plasmid construction [1]. This method was not sufficient for repeated gene disruptions
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