Versatile Genetic Tool Box for the Crenarchaeote Sulfolobus acidocaldarius

Abstract

For reverse genetic approaches inactivation or selective modification of genes are required to elucidate their putative function. Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon which grows optimally at 76°C and pH 3. As many antibiotics do not withstand these conditions the development of a genetic system in this organism is dependent on auxotrophies. Therefore we constructed a pyrE deletion mutant of S. acidocaldarius wild type strain DSM639 missing 322 bp called MW001. Using this strain as the starting point, we describe here different methods using single as well as double crossover events to obtain markerless deletion mutants, tag genes genomically and ectopically integrate foreign DNA into MW001. These methods enable us to construct single, double, and triple deletions strains that can still be complemented with the pRN1 based expression vector. Taken together we have developed a versatile and robust genetic tool box for the crenarchaeote S. acidocaldarius that will promote the study of unknown gene functions in this organism and makes it a suitable host for synthetic biology approaches.

Keywords: Sulfolobus; archaea; deletion mutant; expression system; genetics; in-frame deletion.

Figure 1

Construction of the uracil auxotrophic strain MW001. (A) Growth curves of MW001 in medium containing 0.2% NZ-amine and 0 (closed rectangles), 1 (closed triangles), 2.5 (closed circles), 5 (closed diamonds), 10 μg/ml (open rectangles) uracil. (B) PCR analysis of the wild type S. acidocaldarius strain DSM 639 and MW001 using primers 914/915 that amplify 663 bp of the full length pyrE and 340 bp from the deletion mutant pyrE.

Figure 2

Depiction of the different methods to obtain markerless in-frame mutants. (A) Plasmid pSVA406 integrates via single crossover into the genome and by addition of 5-FOA it can be selected for a second single crossover event that leads either to the wild type situation or the aimed add deletion mutant. (B) By using pSVA407 deletion mutants can be obtained via the same method as employed by pSVA406, only that pSVA407 contains the lacS gene in the selection cassette that allows for the easy identification of integrants at the first selection level. (C) pSVA431 allows for double crossover insertion via the sequence of the gene and a downstream region of the gene of interest of the selection cassette. Thereby positives clones should be staining blue. Upon selection on 5-FOA plates the selection cassette will be excised and positive deletion mutants should be white. Goi, gene of interest; ura, pyrEF_sso cassette; amp, ampicillin resistance cassette for selection in E.coli.

Figure 3

UpsE deletion mutants obtained by three different methods. (A) PCR to exemplify the difference in PCR product size obtained by primers 2073/2015 on either MW001 or any of the ΔupsE mutants. (B) As upsE is part of the pili operon that leads to aggregation of UV treated S. acidocaldarius cells (Ajon et al., 2011), an aggregation assay was performed with all the ΔupsE deletion mutants that were either obtained using pSVA406, pSVA407, or pSVA431. A typical result of an UV induced aggregation assay is shown. Aggregation of wild type and deletion mutant were scored before (gray bar) or after UV treatment (black bar). (C) First selection plate colonies from a pSVA407 transformation was sprayed with X-gal. (D) A typical result of a mutant PCR screen with primers 2073/2015 using either plasmid pSVA406 or pSVA431 to obtain the deletion mutant.

Figure 4

Insertion and expression of the S. solfataricus ABC glucose transporter in the S. acidocaldarius genome. (A) Depiction of the genomic integration of the glucose ABC transport operon of S. solfataricus in the upsE locus in the S. acidocaldarius genome. The signal peptide of GlcS, the glusoce binding protein, was exchanged by the MalE signal peptide from S. acidocaldarius. GlcTU are the predicted permase, whereas GlcV is the ATP binding protein in the glucose ABC transporter. (B) Western blot with specific GlcV antibodies on MW001, MW001_445, and S. solfataricus P2 cells. WC, whole cells; CF, cytoplasmic fraction; MF, membrane fraction.

Figure 5

Purification of the genomic c-terminal Strep-Saci1274 fusion protein. (A) Commassie stained SDS-PAGE and (B) immuno blot with STRep-tag antibodies of the purification of Strep-Saci1274 fusion protein from S. acidocaldarius cells using Streptactin affinity material. (C) Commassie SDS-PAGE and an immune blot with a-His-tag antibodies (D) of the purification of Saci1210 which was genomically tagged using His_SELECT material. M, marker; P, insoluble membrane pellet; S, solubilized membrane proteins; FL, flow through; W, wash fraction; E, elution fraction.

Figure 6

Comparison of copper inducible promoter and maltose inducible promoter for expression in MW001. (A) Operon structure of the copper resistance cluster of S. acidocaldarius. Black line indicates the part of the operon that was included into pSVA1673. (B) Schematic representation of the plasmids pCMallacS and pSVA1673 used in (C,D). (C) Direct comparison of LacS expression under control of Pmal (pCMallacS; dark gray) or PcopMA (pSVA1673; light gray) in MW001 cells under non-induced and induced conditions (0.4% maltose or 1 mM Cu, respectively). (D) Specific LacS activity by induction of PcopMA with increasing amounts of Cu in the medium. For each concentration the LacS activity was measured after 2 (light gray) and 4 h (dark gray) of induction.

Figure A1

Codon optimized One-STrEP-tag sequence.