Abstract
A genome manipulation approach based on double-crossover homologous recombination was developed in the polyploid model organism Thermus thermophilus HB27 without the use of any selectable marker. The method was established and optimized by targeting the megaplasmid-encoded β-glucosidase gene bgl. When linear and supercoiled forms of marker-free suicide vector were used for transformations, the frequencies of obtaining apparent Bgl- mutant were 10- 5 and 10- 3, respectively; while the frequency could reach 10- 2 when transformation with concatemer form of the same vector. All randomly selected Bgl- colonies from the transformations were found to be true bgl knockout mutants. Thus, markerless gene deletion mutants could be constructed in T. thermophilus by the direct selection-free method. The functionality of this approach was further demonstrated by deletion of one chromosomal locus (TTC_0340-0341) as well as by generation of a reporter strain for the phytoene synthase promoter (PcrtB), homozygous mutants of the both targets could also be detected with a frequency of approximately 10- 2. During the genome modification process, heterozygous cells carrying two different alleles at a same locus (e.g., bgl and pyrE) could also be generated. However, in the absence of selection pressure, these strains could rapidly convert to homozygous strains containing only one of the two alleles. This indicated that allele segregation could occur in the heterozygous T. thermophilus cells, which probably explained the ease of obtaining homozygous gene deletion mutants with high frequency (10- 2) in the polyploid genomic background, as after the mutant allele had been introduced to the target region, allele segregation would lead to homozygous mutant cells. This marker-free genome manipulation approach does not require phenotype-based screens, and is applicable in gene deletion and tagging applications.