Establishment Of A Sulfolobus Islandicus Genetic Manipulation System And Its Applications

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce a S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation.One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the glycosidase (lacS) gene. Two unmarked gene deletion methods were employed and unmarked lacS mutants were obtained by each method.A new, alternative, recombination mechanism, i.e. marker circularization and integration, was shown to operate in the latter method which did not yield the designed deletion mutation. Subsequently Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complementedΔpyrEFΔlacS mutation after transformation and stably maintained. Thus,a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.After the establishment of the genetic system, Sso0454 (CBP), Sso3071,Sso0693 homologues,TFB2 and TFB3 were tried to knockout in S.islandicus.Sso0454 and Sso3071 homologue disrupted strains were obtained after counter-selection, whereas the other genes knockout cannot be obtained after semi-knockout. Since the above gene knock out strategy both have its advantages and disadvantages.A new gene knockout strategy combining their advantages abolishing their disadvantages was proposed.By using the genetic system, protein expression have also been tried, C-terminal his-tag LacS and ABV polB was successfully expressed and purified. A interesting result was discovered by N-terminal sequencing the LacS protein which contained both N-terminal and C-terminal his-tag, while the translation start site was normal in C-terminal his-tag LacS,it was uncertain in the N-terminal his-tag LacS,this probably caused by the existence of his-tag,which also implies a novel mechanism for translation start site selection in Sulfolobus.The genetic system have also been used to study the CRISPR, which is a recent completely new identified system in microbiology that functions as an immune system to defend the prokaryotes against genetic elements,such as viruses and plasmids. It is an absolutely hot research field. By transforming the CRISPR spacer containing shuttle vectors,some preliminary results have been obtained. To our knowledge, those results are the first experimental results that confirmed the functioning of CRISPR in archaeal, the archaeal CRISPR is targeting at DNA level and the upstream motif of spacer match sequence is required for CRISPR targeting.