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.

Fig. 1

Vector map of the pRN1-based reporter gene shuttle vector pCmalLacS. lacS reporter gene β-glycosidase from Sulfolobus solfataricus; orf56, orf904, orf80, orf90a, orf72: genes of the plasmid pRN1; orf56 and orf80 code for DNA-binding proteins and orf904 for a multifunctional replication protein (Lipps, 2009), pyrE (orotate phosphoribosyltransferase) and pyrF (orotidine-5′-phosphate decarboxylases): used as selection marker in Sulfolobus, bla β-lactamase, ori ColE1 replication origin

Fig. 2

Comparison of the strength of different promoters in a β-galactosidase assay (mean of three replicates, error bars correspond to one standard deviation), names of the constructs are explained in Table 2. ODs of the cultures at sampling: MR31: 0.3, pCLacS: 0.4, pCssodpsLacS: 0.3, pCdpsLacS: 0.6, pClacSLacS: 0.6, pCmalLacS (uninduced) 0.4, pCmalLacS + maltose: 0.4, pCgdhLacS: 0.6, pCsac7dLacS: 0.2

Fig. 3

Expression of the β-galactosidase under the control of the mal promoter. a Growth curves of five different cultures obtained by 1:99 dilution of a preculture into the respective growth media, TP uninduced culture, +mal addition of the indicated amount of maltose, +dex addition of the indicated amount of dextrin at time point 0. The sampling times are indicated by gray arrows. b Measured β-galactosidase activities for the indicated sampling time points in A [early log phase (first arrow), late log phase (second arrow), stationary phase (third arrow) from left to right]

Fig. 4

Influence of different sugars on the basal activity and the inducibility of the mal promoter in β-galactosidase reporter gene assays (mean ± SD). TP tryptone, Mal maltose, Gluc glucose, Dex dextrin, Suc sucrose, Xyl xylose. All sugars were added at 0.2% final concentration. ODs at sampling: TP: 0.3, TP + Mal: 0.2, TP + Gluc: 0.2, TP + Gluc + Mal: 0.2, TP + Dex: 0.2, TP + Dex + Mal: 0.2, TP + Suc: 0.4, TP + Suc + Mal: 0.3, TP + Xyl: 0.5, TP + Xyl + Mal: 0.5

Fig. 5

Determination of the minimal sequence region necessary for inducibility of the mal promoter. a Operon structure, b promoter sequence (the start codon and the engineered NcoI restriction site is in bold). The beginning of the promoter region of each deletion construct is indicated with the number of base pairs remaining above the sequence. Two putative regulatory imperfect palindromic binding sites (18–20 bases) are underlined and the matching bases have an asterisk above the sequence. The region of the predicted BRE-TATA box is double underlined. c β-galactosidase measurements of the deletion constructs (mean ± SD). The activities obtained in the absence of maltose are shown in light bars and the activities in the presence of maltose in dark bars. ODs at sampling: pCmal1LacS both −/+ maltose 0.1, all other constructs: 0.2

Fig. 6

Southern blot for copy number determination of construct pCmalLacS in S. acidocaldarius MR31. The culture shown in lane 1 and 2 was sampled at OD of 0.2, the culture shown in lane 3 and 4 was sampled at an OD of 0.9. In lane 2 and 4 1/9th of the amount in lane 1 and 3 has been loaded onto the gel

Fig. 7

Purification of SSO0287 expressed in S. acidocaldarius by affinity chromatography. The upper panel shows the Coomassie stained gel of the different fractions of the purification procedure. The same samples were used for Western blotting using both, anti-Histag and Streptactin antibodies. ST starting material, FT flow through, W1 and W2 wash steps, E elution