A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus

Abstract

The pSSVx genetic element from Sulfolobus islandicus REY15/4 is a hybrid between a plasmid and a fusellovirus, able to be maintained in non-integrative form and to spread when the helper SSV2 virus is present in the cells. In this work, the satellite virus was engineered to obtain an Escherichia coli–Sulfolobus solfataricus shuttle vector for gene transfer and expression in S.solfataricus by fusing site-specifically the pSSVx chromosome with an E.coli plasmid replicon and the ampicillin resistance gene. The pSSVx-based vector was proven functional like the parental virus, namely it was able to spread efficiently through infected S.solfataricus cells. Moreover, the hybrid plasmid stably transformed S.solfataricus and propagated with no rearrangement, recombination or integration into the host chromosome. The high copy number of the artificial genetic element was found comparable with that calculated for the wild-type pSSVx in the new host cells, with no need of genetic markers for vector maintenance in the cells and for transfomant enrichment.The newly constructed vector was also shown to be an efficient cloning vehicle for the expression of passenger genes in S.solfataricus. In fact, a derivative plasmid carrying an expression cassette of the lacS gene encoding the β-glycosidase from S.solfataricus under the control of the Sulfolobus chaperonine (thermosome tf55) heat shock promoter was also able to drive the expression of a functional enzyme. Complementation of the β-galactosidase deficiency in a deletion mutant strain of S.solfataricus demonstrated that lacS gene was an efficient marker for selection of single transformants on solid minimal lactose medium.

Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

“…The enzyme activity was also measured quantitatively (as β-galactosidase) at different ODs of a MR31 culture transformed with pJ lacS . Copy numbers were determined simultaneously and it was found that measured β-galactosidase activities correlated well with vector copy numbers (Figure 5E), as previously observed (16,20). It should be noted that the β-galactosidase activity of MR31 transformed with pJ lacS (2–11 U/mg) is much higher than the wild-type β-galactosidase activity of S. solfataricus (0.2 U/mg) (26) and is comparable to the β-galactosidase activity in a viral overexpression system (1.5–5 U/mg) (20).…”

Cellulose Degradation by Sulfolobus solfataricus Requires a Cell-Anchored Endo-β-1-4-Glucanase

“…A Sulfolobus expression vector was constructed, inserting the sso1354 His-tagged coding sequence fused to the putative promoter of the glucose binding protein (glcS) gene into the pMMSV Sulfolobus/E. coli shuttle vector (7). As a first step, an expression cassette containing the glcS promoter and the sso1354 coding sequence was constructed.…”

“…(a) Purification from medium. S. solfataricus GW cells infected with the SSV2 virus were grown in in Brock's basal medium containing 0.1% yeast extract and 0.1% Casamino Acids (mediumYCA) and transformed by electroporation with 50 ng of the pMSSVglcS1354 vector (following the procedure described by Aucelli et al, [7]). Transformed cells were scaled up in Brock's basal medium containing 1g/liter glucose and 1g/liter tryptone up to 50 ml.…”

Structure and function of the primase domain of the replication protein from the archaeal plasmid pRN1

“…Meanwhile, a number of shuttle plasmids based on pRN1-type plasmids have successfully been constructed by several groups [4][5][6].…”