Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius

Abstract

Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli–Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2–0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30–50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L−1 culture. In addition we also determined the promoter strength of several constitutive promoters.

Investigation of the malE Promoter and MalR, a Positive Regulator of the Maltose Regulon, for an Improved Expression System in Sulfolobus acidocaldarius

“…Expression from the promoter of the putative maltose-binding protein Saci_1165 has been shown to be induced in the presence of maltose in the medium (21). Saci_1165 is part of a typical ABC transporter operon that comprises the classical gene components of archaeal ABC transporters (16), as well as genes encoding enzymes involved in carbohydrate degradation.…”

“…However, in the ⌬malR strain, the expression of all the mal locus genes was drastically (3-to 4-fold) reduced, indicating a role for MalR in the activation of mal locus gene expression. Moreover, a reporter gene assay was performed in which LacS (␤-galactosidase) was under the control of the malE promoter on the pRN1-based plasmid pCmalLacS (21). This plasmid was used for the transformation of MW001 and the ⌬malR deletion strain.…”

“…The backbone of all plasmids constructed was pCmalLacS (21) or pSVA1423, a pCmalLacS derivative that contains a copy of Saci_1161 under the control of its own promoter next to the pyrEF cassette, cloned into KpnI and XhoI restriction sites. In these backbone plasmids, the malE promoter was removed with SacII and NcoI and was replaced by the respective mutated promoter.…”

“…acidocaldarius is one of the few genetically tractable crenarchaeal organisms known to date. Knockout and overexpression systems have been established (20,21). The promoter of the putative maltose-binding protein Saci_1165 has been shown to be maltose inducible (21), and very recently, the ABC transporter was confirmed as the maltose transporter in S. acidocaldarius (22).…”

Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids

“…To protect the plasmid DNA from the S. acidocaldarius restriction-modification (R-M) system, SuaI sites (GGCC) were methylated in vivo prior to transformation (13,14). Both vectors express the S. solfataricus ␤-D-glycosidase lacS reporter gene, but they use different promoters, i.e., the tf55␣ gene promoter in the case of pJlacS (15) and a maltose-inducible promoter in pClacS (16).…”