A Novel Integrative Expression Vector for Sulfolobus Species
A Novel Integrative Expression Vector for Sulfolobus Species
Journal of Microbiology and Biotechnology. 2014. Nov, 24(11): 1503-1509
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 22, 2014
  • Accepted : July 11, 2014
  • Published : November 28, 2014
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About the Authors
Kyoung-Hwa, Choi
Department of Microbiology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea
Sungmin, Hwang
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA
Naeun, Yoon
Department of Microbiology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea
Jaeho, Cha
Department of Microbiology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea

With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 ( pyrE sso ) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli . For stable expression of the target gene, an α-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene ( saci_1160 ) encoding an α-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase ( gdhAsaci ) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the “pop-in” and “pop-out” method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The α-glucosidase activity was confirmed by the hydrolysis of p NPαG. The pINEX vector should be applicable in delineating gene functions in this organism.
Research on archaea has long been limited by the lack of genetic tools to study gene functions in vivo . The major reason is that the antibiotics used in microbial genetics as selectable markers are readily degraded at the conditions at which various archaea live [14] . Genetic systems were described for hyperthermophilic archaea in the early 2000s and have constantly improved since then. In Thermococcus kodakariaensis , an effective gene deletion system was established by Sato et al. [20 , 21] for which also a complementary expression vector using prototrophic (arginine/citrulline, tryptophan, agmatine, or uracil) or antibiotic (simvastatin or mevinolin) selection markers has been optimized [12 , 19] . Recently, the trials for construction of a stable shuttle vector and selection of targeted mutants from Pyrococcus furiosus have been carried out [8 , 9 , 26] . Hence, nowadays, genetic toolboxes are available for a variety of hyperthermophilic euryarchaeota.
In crenarchaeota, only Sulfolobales genetic systems have been developed. The first shuttle vectors and a gene deletion method were based on the β-galactosidase, LacS, as a selection marker in Sulfolobus solfataricus [2 , 3 , 29] and this method was employed in a number of studies [10 , 15 , 22 , 23 , 25 , 33] . A system for gene deletion mutants including a shuttle vector has also been reported based on the pyrEF selection in S. islandicus [7 , 16] and successfully used in different studies [11 , 30] . The antibiotic simvastatin has also been used to select for the shuttle vector and a knockout system in this strain [31 , 32] . Other Sulfolobus shuttle vectors that rely on pyrEF selection were constructed based on the virus SSV1 and the plasmid pRN1 [3 , 13] . In S. acidocaldarius, pyrEF was employed as a selectable marker to obtain the insertional deletion mutants and persistence of a shuttle vector with a constitutive or inducible promoter for expression [4 , 27] . Recently, we also developed the shuttle vector using the pyrE gene for the expression of foreign genes in S. acidocaldarius . However, the segregrational and structural instability of the plasmid sometimes made it difficult to obtain stable expression of foreign genes [17] .
Therefore, we established an alternative method to express homologous or heterologous target genes, relying on homologous recombination with a chromosome using the pop-in and pop-out method. A novel plasmid vector, pINEX, was generated and included the pyrE gene from S. solfataricus P2 ( pyrE sso ) for selection, a multiple cloning site with histidine tag, and the internal sequences of malE and malG for homologous recombination in S. acidocaldarius , as well as regions of the pGEM-T vector, for the replication in E. coli . For the testing of the successful expression of foreign genes, the malA gene ( saci_1160 ) encoding an α-glucosidase from S. acidocaldarius was selected and examined for the stable expression in this strain.
Materials and Methods
- Strains and Growth Conditions
S. acidocaldarius uracil auxotroph MR31 was provided by Dennis W. Grogan (University of Cincinnati, USA) and aerobically grown at 77℃ in Brock medium supplemented with 0.1% (w/v) tryptone, and 0.005% (w/v) yeast extract containing uracil (50 µg/ml), adjusted to pH 3.0 [18] . After cell transformation, the cells containing pINEX vector were selected on Brock medium supplemented with 0.1% NZ-Amine (Sigma, St. Louis, MO, USA), and 0.2% (w/v) xylose as a carbon and energy source. E. coli strain DH5α was used for the propagation of plasmids and was incubated in Luria-Bertani (LB) medium (1% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl), containing ampicillin (100 µg/ml), at 37℃.
- pINEX Vector Construction
For construction of the ectopic integration plasmid, the internal regions of the malE ( saci_1165 ) and malG ( saci_1163 ) genes were amplified using primers SA1165_F/SA1165_R and SA1163_F/SA1163_R, respectively. The oligonucleotide primers used are shown in Table 1 . For the malE region, the forward primer introduced an Apa I restriction site at the 5’-end, whereas the reversed primer was designed to incorporate the multiple restriction enzyme sequence. For the malG region, the forward primer was designed to incorporate the complementary strand of the multiple restriction enzyme sequence with a six-histidine tag sequence of SA1165_R primer, and the reverse primer contained a Sac I restriction site. Two PCR fragments were fused via an overlapping PCR, and the resulting amplified PCR fragment was digested with Apa I and Sac I, and ligated into the pGEM-T vector, yielding plasmid pSM27. To add a selection marker by pyrE gene insertion, a DNA region from 280 nucleotides upstream to 71 nucleotides downstream of the pyrE gene from S. solfataricus P2 ( pyrE sso ) was amplified using the primers KH71 and KH72 [6] . The 770 bp PCR product was digested with Bam HI and cloned into pSM27 to generate pINEX ( Fig. 1 ). The transcription direction of the pyrE gene was confirmed by PCR using primers SA1165_F and pyrEF as well as DNA sequencing.
The oligonucleotide primers used in this study.
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a The restriction enzyme sites are written in bold, and complementary sequences for overlap extension PCR are underlined.
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Map of plasmid pINEX. The unique restriction sites from the multiple cloning sites are indicated outside of the map.
- pINEX-malA Construction
For the construction of pINEX_malA, first, an overexpression cassette controlled by the promoter of gdhA encoding glutamate dehydrogenase ( saci_0155 ) was constructed using a fusion PCR technique. Using the genomic DNA of S. acidocaldarius as a template, the gdhA promoter and N-terminus region containing 403 nucleotides upstream of the gdhA coding sequence was amplified with the primers Gdhsac_F and Gdhfus_R. The forward primer was designed to incorporate a Sac II restriction site, and the reverse primer was designed to incorporate a gdhA leader sequence (15 nucleotides of the gdhA start region) and 15 nucleotides of the malA ( saci_1160 ) start region. PCR was carried out using 2.5 U of PrimeSTAR HS DNA polymerase (Takara, Japan) using the following conditions: 94℃ for 3 min; 30 cycles of 98℃ for 10 sec, 55℃ for 5 sec, and 72℃ for 50 sec; 72℃ for 5 min. The malA gene along with gdhA leader sequence containing 1,906 bp was amplified using the primers MalAfus_F and MalA_R. The forward primer contained 31 nt 5’ overhanging ends (underlined) complementary to the 3’ end of the primer Gdhfus_R, whereas the reverse primer was designed to incorporate a Pst I restriction site. Both fragments were fused by overlap PCR to obtain the fusion gene P gdhA-malA . Overall, a 2,001 bp fragment was purified using a PCR purification kit (ELPIS Biotech, Korea) and cloned into pINEX to generate pINEX_malA. The construction and sequence of pINEX_malA is shown in Fig. 2 . The sequence of the overexpression cassette was confirmed by DNA sequencing.
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Map of plasmid pINEX_malA and the nucleotide sequence of the overexpression cassette of the fusion gene PgdhA-malA.
- Transformation of S. acidocaldarius
S. acidocaldarius MR31 was transformed with plasmids by electroporation, as described previously [6] . The non-methylated or methylated pINEX_malA DNA (1 µg) for the chromosomal integration was electroporated in electrocompetent MR31 using a Gene Pulser II electroporator (Bio-Rad), with input parameters 1.25 kV, 1000 Ω, and 25 µF in 1 mm cuvettes. Following electroporation, an equal volume of 2× recovery solution (1% sucrose, 10 mM MgSO 4 , 0.18% (w/v) β-alanine, and 0.27% (w/v) DL -malic acid, adjusted to pH 4.5 with 10 N NaOH) was immediately added to the pulsed cells, and the mixture was transferred to a 1.5 ml microcentrifuge tube and incubated at 75℃ for 30 min. The cells were then spread on solid medium containing 0.1% (w/v) N-Z-Amine and 0.2% (w/v) xylose without uracil. The plates were sealed in plastic bags to avoid drying-out and incubated for 8 days at 77℃. Single colonies were picked and identified by colony PCR using the primers pyrEF and pyrER, which are specific to gene pyrE .
- Chromosomal Integration of the malA Gene
After the transformation of S. acidocaldarius MR31 by a pINEX_malA plasmid, two steps were needed to carry out the replacement of the malEFG gene by overexpression cassette including the target gene: a pop-in (vector insertion) by one crossover event, and a pop-out recombination ( Fig. 3 A). For this purpose, the colonies that contained the pyrE gene by colony PCR were grown in uracil-free YT medium containing 0.2% xylose to eliminate the false positives as well as induce the second pop-out recombination at this step. PCR amplification using primers Pgdh_F and SA1163_R was performed to check whether the popout recombination resulted in a replacement of the overexpression cassette or wild-type (MR31) genotype recurrence.
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Strategy for the integration of malA into the malE and malG loci in the chromosome of S. acidocaldarius. (A) After transformation, the plasmid was integrated into the genome by a first crossover event in the homologous-region fragment. The second step was the pop-out recombination (or excision) event. There are two possibilities: a recombination between the other homologous fragments, resulting in the replacement of the overexpression cassette, or a recombination between the same homologous fragments of the first recombination, which gives the wild-type genotype. (B) To verify the different genotype configurations, PCR amplification was performed with the primers matching Gdhsac_F and SA1163_R regions: primers for identification of pyrE and target gene insertion. Lane M, 1 kb size marker; lane 1, PCR result of MR31 (wt) strain; lane 2, PCR result of integrant transformants (int); lane 3, PCR result of colony obtained from the second single crossover of int transformant (SMmalA).
- Expression and Purification of MalA from S. acidocaldarius
S. acidocaldarius integration mutant SMmalA carrying the MalA overexpression cassette into its chromosome was grown in YT medium containing 0.2% (w/v) sucrose at 77℃ for 24 h. The cells were harvested by centrifugation (8,000 × g for 30 min at 4℃), and resuspended in 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl. The cells were lysed by sonication on ice, and after centrifugation (15,000 × g for 50 min at 4℃), the supernatant was applied to a Ni-NTA af f inity column (GE Healthcare, Freiburg, Germany). The column was washed with wash buf f er (20 mM sodium phosphate buf f er (pH 7.4), 500 mM NaCl, and 80 mM imidazole), and the bound proteins were eluted using elution buffer (20 mM sodium phosphate buffer (pH 7.4), 500 mM NaCl, and 160 mM imidazole). All fractions were collected and dialyzed against the same buffer without imidazole and concentrated using Centricon-10 microfilters (Millipore, Darmstadt, Germany). Protein concentration was determined using the Bradford method [5] with bovine serum albumin as a standard. The molecular mass and purity of the purified MalA were estimated by 12% SDS-PAGE.
- Assay for α-Glucosidase Activity
α-Glucosidase activity was measured by enzymatic determination of D -glucose liberated from maltose, using a glucose oxidase assay kit (Sigma). The reaction mixtures (50 µl) containing 5 mM maltose in 20 mM sodium acetate buffer (pH 5.0) were incubated with 0.1 µg of enzyme at 95℃ for 5 min. One hundred microliters of glucose oxidase assay reagent was added to the mixture and incubated at 37℃ for 30 min. The reaction was stopped by the addition of 100 µl of 12 N H 2 SO 4 , and 200 µl aliquots were measured at 540 nm. The assay for p -nitrophenyl α-glucopyranoside ( p NPαG) hydrolysis was performed in 100 µl of 20 mM sodium acetate buffer (pH 5.0) containing 5 mM p NPαG and 0.1 µg of enzyme. The mixture was incubated at 95℃ f or 5 min and the reaction was stopped using 100 µl of 1 M sodium carbonate. Absorbance of the p -nitrophenol ( p NP) released from p NPαG was determined at 420 nm.
- Characteristics of pINEX
To facilitate the process of gene expression and integration into specific regions of the S. acidocaldarius chromosome, we constructed plasmid pINEX ( Fig. 1 ). It carries the pyrE promoter and structural gene of S. solfataricus as a selection marker and a multiple cloning site (MCS) with unique restriction sites for target gene expression flanked by malE and malG loci, thus enabling excision by host RecA family recombinase. pINEX also contains the β-lactamase coding sequence and ori site from the pGEM-T vector for the cloning and propagation of this plasmid in E. coli . The region including expression sequences, cloned gene(s), and the pyrE cassette can be amplified by PCR and ligated into the regions of homology to cause integration into a specific chromosomal site in an MR31 strain.
- Construction of a Strain Expressing MalA from the Chromosome
pINEX_malA, a plasmid that harbored the malA gene under the constitutive S. acidocaldarius gdhA promoter, was constructed and used to transform the host strain MR31, a uracil auxotroph derived from S. acidocaldarius DSM639. For efficient expression under the gdhA promoter and easy purification by affinity chromatography, the malA gene was fused to five amino acids of glutamate dehydrogenase at the N-terminus and six histidines at the C-terminus ( Fig. 2 ). A transformant exhibiting uracil prototrophy was selected in medium lacking uracil, and the integration of the plasmid into the S. acidocaldarius chromosome was confirmed by colony PCR using pyrE internal primers pyrEF and pyrER, and this led to an amplification of a band corresponded to 400 bp. After PCR verification, selected clones were incubated in uracil-free medium for 2 days at 77℃ to derive the second pop-out recombination. The second crossover event was verified by PCR amplification of a 2,807 bp fragment by using primers Gdhsac_F and SA1163_R ( Fig. 3 B).
- Characterization of a Strain Expressing MalA
The transformant SMmalA harboring the malA gene was grown in YT medium containing 0.2% sucrose for 24 h. Expression of MalA was examined in the cell lysates. The α-glucosidase activity was examined using maltose and p NPαG as substrates. The cell-free extracts from the SMmalA strain showed enzyme activity, whereas the cellfree extracts from MR31 (which is the control strain) did not show the enzyme activity. The hydrolysis of p NPαG into p NP was detected by the appearance of a yellow color in the reaction mixture, indicating that the expression was successful ( Fig. 4 A). The recombinant MalA was tagged with a 6×His-tag. This enabled us to test purification by His affinity chromatography using this strain. His affinity chromatography of MalA resulted in highly pure MalA in one step ( Fig. 4 B). The purified MalA estimated by SDS-PAGE was approximately 72 kDa, which is in good agreement with the size obtained from the plasmid expression vector pKHmalA [6] . The characteristics of the purified MalA was exactly matched with recombinant MalA expressed from a plasmid-based S. acidocaldarius strain [6] . Therefore, the integrative expression vector pINEX we constructed can be used for the efficient expression of archaeal genes whose protein products are rendered insoluble when expressed in E. coli .
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Enzyme activity measurement (A) and SDS-PAGE analysis (B) of recombinant MalA. The α-glucosidase activity was measured using pNPαG. The yellow color development by pNP produced from the hydrolysis of pNPαG was observed in the S. acidocaldarius SMmalA strain. MalA was purified by His affinity chromatography. Lane M, protein size standards; lane 1, cell-free extract of SMmalA harboring malA; lane 2, flow-through fraction; lane 3, elution fraction with 200 mM imidazole.
Hyperthermophilic archaea have been regarded as a useful organism in the research for cellular aspects of the archaeal kingdom and biotechnology owing to their heat adaptation. In order to understand the physiology, metabolism, and many other cellular aspects, the availability of genetic tools for these organisms is essential. Sulfolobales are the only family for the crenarchaeota that are amenable for genetic manipulation so far. In the last few years, various genetic systems in the Sulfolobales, including S. solfataricus , S. islandicus , and S. acidocaldarius , have been developed and proved to be successful [1 , 24 , 28 , 29] . Although E. coli-Sulfolobus shuttle vectors have been used for promoter studies and adapted for the homologous and heterologous expression of tagged proteins in Sulfolobus spp., the segregational and structural instability of the plasmid sometimes made it difficult to obtain a stable expression of foreign genes.
As a part of the development of expression technology, we constructed the pINEX vector based on pyrE selection for chromosomal gene integration and expression. This plasmid can be utilized as a typical expression vector having a MCS at which the expression cassette including the target gene, along with a strong constitutive or regulated promoter, can be simply inserted into it by specific restriction enzymes. In addition, pINEX was designed to facilitate the process of chromosomal gene integration. Genes cloned in this vector with a pyrE auxotrophic cassette can be integrated into the chromosome by homologous recombination at the malE and malG loci and selected by uracil prototrophy. The integration of the target gene at the malE and malG loci causes the disruption of a malEFG operon in the chromosome, thus preventing the strain from growing in maltose or starch medium [6] . As a result, the integration of the target gene into the chromosome of S. acidocaldarius by transformation of the pINEX vector can also be identified by growth defects in the specific growth conditions. By employing various useful promoters derived from Sulfolobales, the strain can control the regulated or constitutive gene expression. As this plasmid was designed to add a six-histidine tag at the C-terminus of the target gene, the expressed protein can be simply purified by affinity chromatography. To determine whether the expression vector we constructed can work efficiently in S. acidocaldarius , the malA ( saci_1160 ) gene encoding for α-glucosidase was tested. Enzyme activity measurements ( Fig. 4 A) as well as SDS-PAGE analysis ( Fig. 4 B) of the S. acidocaldarius SMmalA strain carrying the malA gene revealed that the plasmid pINEX_malA was successfully integrated into the chromosome of strain MR31, and the integrated malA gene was stably expressed in this strain. These results imply that in addition to being a plasmid expression vector, the pINEX vector can be used for the stable expression of foreign archaeal genes that are not well expressed in the E. coli system.
In conclusion, we constructed a plasmid vector that can genomically express target genes with tag. The expression level of the target genes would be varied depending on the selected promoters, and the His-tag fused at the C-terminus would help the proteins to be easily separated from other proteins in the genomic background. Therefore, vector pINEX might be useful as a tool for the construction of genetically stable and robust S. acidocaldarius strains that express foreign genes.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A01068096) and by the “2013 Post-Doc. Development Program” of Pusan National University.
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