Development Of The Gene Knockout System For The Thermoacidophile Archaeaon Sulfolobus Acidocaldarius

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but an efficient gene knockout system remains to be developed.This problem has consistently hampered the further genetic studies of this model organism.We were interested in developing gene knockout system for the model archaeon Sulfolobus acidocaldarius. Since an effecient selection marker remained to be tested for S. acidocaldarius, we aimed to obtain a pyrEF deletion mutant and use it as the host to test whether genetic expression of pyrEF complementation could be used as a selection marker for gene knockout analysis for this model archaeon. We chose to construct a pyrEF deletion mutant via homologous recombination. The E. coli vector pUC19 was used to construct the knockout plasmid. The homologous sequence arms were amplified by PCR from S. acidocaldarius, both of which were then inserted into pUC19, giving the pyrEF deletion plasmid pEFD. After transforming S. acidocaldarius with pEFD, pyrEF mutants appeared as 5-FOA resistant colonies. Screening 121 colonies revealed that one of the mutants contains 963-bp deletion in the pyrEF genes, retaining only 122-bp pyrE and 155-bp pyrF in the mutant allele. This mutant is designated as DEF-963 and used as the host for testing genetic systems.To test the usefulness of the pyrEF marker in generating gene knockout mutants, the beta-bgaS gene was deleted from DEF-963 by the so-called markerless gene deletion protocol. The marker gene pyrEF was amplified from S. solfataricus by PCR and the homologous sequence arms, from S. acidocaldarius, all of which were then inserted into pUC19, giving the bgaS deletion plasmid pSD. Then pSD was used to transform DEF-963, and the transformants formed colonies on a uracil-free medium whereas the mutant failed to do so. Analyzing the bgaS mutant allele in 4 transformants by PCR indicated that 2 of them contain the bgaS allele of the expected size, suggesting that they constitute the pyrEF bgaS double mutants. To verify these deletion mutations, the genome of the mutant was analyzed using Southern hybridization and only the expected mutant gene alleles are hybridized by the probes. These results indicate that the obtained mutant is indeed pyrEF bgaS double deletion mutant. Furthermore, the mutant does not grow up in a lactose minimal medium and this is in contrast to the wild-type strain, which does grow in the same medium, albeit at a much reduced growth rate comparing to that in a rich medium.Taking together, my results indicate that as for haloarchaea, constructing gene deletion mutants using pyrE as the selection marker has been proven an efficient and effective method for S. acidocaldarius. Furthermore, the obtained double mutant, is finally designated as DEF-964, will allow us to use two selection markers for developing versatile genetic systems that are not currently available to this model organism.