Construction and transformation of a Thermotoga-E. colishuttle vector
© Han et al; licensee BioMed Central Ltd. 2012
Received: 9 March 2011
Accepted: 6 January 2012
Published: 6 January 2012
Thermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. They may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. A lack of genetic tools has hampered the investigation and application of these organisms. This study aims to develop a genetic transfer system for Thermotoga spp.
Methods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure.
Thermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.
Besides Aquifex, Thermotoga are the only group of bacteria that can grow up to 90°C. Isolates of Thermotoga have been discovered from heated sea floors , continental hot springs , and oil fields . Analysis of their 16S rRNA sequences have positioned Thermotoga spp. to a deep branch of the tree of life, suggesting that these strict anaerobes emerged at an early stage of evolution, when the surface of the Earth was hot and its atmosphere contained little oxygen. Study of the molecular genetics of Thermotoga is expected to shed light on the fundamental questions related to the origin of life as well as the mechanisms of the thermostability of macromolecules under extreme conditions. Most importantly, Thermotoga hydrolyze a number of polysaccharides through fermentative catabolism and produce hydrogen gas as one of the final products . This has stimulated tremendous interest in utilizing these bacteria to produce biomass-based clean energy, especially through metabolic engineering approaches. However, due to the lack of genetic tools, the investigations of Thermotoga are still largely limited to biochemical, genomic, and fermentative studies, as with most hyperthermophiles.
Cryptic mini-plasmids pRQ7, pMC24, and pRKU1 have been identified in T. sp. RQ7 , T. maritima , and T. petrophila RKU-1 , respectively. Although discovered at geologically unrelated locations, the three plasmids are nearly identical. They are extremely small (846 bp) and encode just one apparent open reading frame, presumably the replication protein. Studies of pRQ7 suggest that the plasmid is negatively supercoiled and replicates by a rolling-circle mechanism [5, 8]. Based on pRQ7, two Thermotoga-E. coli shuttle vectors pJY1 (chloramphenicol-resistant) and pJY2 (kanamycin-resistant) have been constructed for expression in T. neapolitana and T. maritima, respectively . Through liposome-mediated transformation, both vectors rendered transient antibiotic resistance to Thermotoga cells in liquid media, but no transformants could be isolated from plates. To date, that report remains the only documented effort of expressing heterologous genes in Thermotoga, out of more than 1200 publications retrieved from PubMed using "Thermotoga" as the key word (last searched December 20, 2011). In fact, genetic manipulation of Thermotoga remains a challenge. To develop a tractable gene transfer system for Thermotoga spp., we systematically examined every aspect pertaining to the cloning and expression of foreign genes in Thermotoga, from plating efficiency to vector stability. We demonstrate that heterologous genes can be introduced to Thermotoga through multiple means, be functionally expressed, and be stably maintained.
Strains and growth conditions
Strains & vectors used in this study
Strain or plasmid
T. neapolitana DSM 4359
Isolated from African continental solfataric springs
T. maritima MSB8
Isolated from geothermally heated sea floors in Italy and the Azores
T. sp. RQ7
Isolated from geothermally heated sea floors in Ribeira Quente, the Azores
F- endA1 hsdR17 (rk-, mk+) supE44 thi-1 λ - recA1 gyrA96 relA1 deoR Δ(lacZYA-argF)- U169 ϕ80dlacZΔM15
A high-copy number E. coli cloning vector containing portions of pBR322 and M13 mp19
Cryptic miniplasmid from T. sp. RQ7
pUC-derived plasmid, containing a kan cassette for thermostable kanamycin selections
pRQ7 DNA cloned between BamHI and EcoRI sites of pUC19; Apr
pRQ7 DNA cloned between EcoRI and XbaI sites of pKT1; Apr; Kanr
This study; GenBank: JN813374
Antibiotics sensitivity tests
One ml of overnight culture was mixed with 25 ml of hot SVO containing 0.3% Gelrite and poured into Petri dishes. Small discs of 7 mm in diameter were cut from Whatman qualitative filter paper and were placed on solidified plates. Various amount of kanamycin (50 ~ 250 μg) was added to the paper discs. After 48 h of anaerobic incubation, sensitive strains would display inhibition zones surrounding the discs. To specify the selective levels of the antibiotic in both liquid and solid media, kanamycin ranging from 50 to 300 μg ml-1 was supplemented, and the proliferation of bacteria was monitored for up to 72 h.
Extraction of DNA from Thermotoga
Plasmid DNA was extracted from Thermotoga using standard alkaline lysis method . For genomic DNA, overnight culture of Thermotoga was extracted with equal volume of phenol: chloroform: isoamyl alcohol (volume ratio 25:24:1) followed by centrifugation at 13,523 g for 5 min to remove cell debris. DNA in the supernatant was precipitated with equal volume of isopropanol, washed once with 70% ethanol, air-dried, and dissolved in 10 mM Tris-EDTA buffer (pH 8.0) containing 20 μg ml-1 RNase.
Construction of pDH10
Transformation and selection methods
Liposome-mediated transformation was conducted as previously described , except that all operations were carried out on the bench top. DOTAP liposomal reagent was purchased from Roche Diagnostics, Indianapolis, IN, USA. To prepare electrocompetent cells, overnight Thermotoga cultures were transferred to 50 ml of SVO liquid media and were allowed to grow until the cell density reached around 0.2. Cells were collected by centrifugation, washed once with cold deionized water and twice with the washing solution (10% glycerol, 0.85 M sucrose) and were resuspended in 500 μl of the same solution. For electroporation, 4 μg of plasmid DNA was mixed with 50 μl of the freshly made competent cells and incubated on ice for 5 minutes prior to introduction to a pre-chilled cuvette of 1 mm gap. The operation settings were 25 μF capacitance, 200 Ω resistance, and 1.5, 1.8, or 2 kV voltage (Gene Pulser Xcell™, Bio-Rad Laboratories, Hercules, CA). After electroporation, 1 ml of fresh SVO liquid medium was added to each cuvette, and the cell suspension was transferred to a N2 serum bottle and incubated at 77°C with gentle rotation for 3 h for recovery. Half of the recovered culture (500 μl) was then mixed with 25 ml of hot SVO solid medium supplemented with 250 μg ml-1 kanamycin, poured to Petri dishes, and incubated in an anaerobic jar to retrieve transformants.
Stability assays of the transformed DNA in Thermotoga and E. coli
Cultures of Thermotoga recombinant strains were transferred every 12 h for 3 days with an inoculum of 2% to a fresh SVO liquid medium in the presence or absence of kanamycin. With each transfer, an aliquot of the cultures was withdrawn and diluted to 10-4. Ten microliters of each diluted sample was then mixed with 10 ml of hot SVO solid medium with or without the antibiotic and was poured into a four-section Petri dish. In order to facilitate comparisons, samples of the same strain (but with different treatments) were arranged to different sections of the same plate. After incubation in an anaerobic jar for 48 h, colonies formed in each section were counted and compared.
To test the stability of pDH10 in E. coli, a liquid culture of DH5α/pDH10 was also transferred six times for every 12 h of growth in plain LB medium (no ampicillin) with 1% inoculum. Samples from each cycle were properly diluted and spread on plain LB plates to separate single colonies. One hundred such colonies were randomly chosen and were tested on LB plates containing 100 μg ml-1 ampicillin. For control purposes, strain DH5α/pKT1 was tested in parallel.
Improved methods for handling Thermotogacultures in an aerobic environment
To facilitate the transfer of single colonies from solid to liquid media under aerobic conditions, we introduced a soft SVO medium by adding 0.075% Gelrite to liquid SVO. Gelrite prevents atmospheric oxygen from penetrating deep into the medium. To transfer cultures from solid to soft SVO, single colonies were picked up from plates by a loop and were pushed down to the bottoms of the test tubes containing soft SVO, where a local anaerobic environment has been created. After 12-24 h of incubation, cultures grown in soft SVO were then transferred to liquid SVO by a syringe. Although the introduction of soft SVO seemed to prolong the overall operation cycle, it ensured maximum viability of Thermotoga cells during the transfer, which eventually allowed us to isolate Thermotoga transformants for the first time (see below). Soft SVO may also serve as an excellent storage medium for Thermotoga. Cultures kept at the bench top for 2 months were still vital and exhibited no growth defects.
Kanamycin is a suitable selection marker for T. sp. RQ7 and T. maritima
We next specified the selective levels of kanamycin in both liquid and solid media. The growth of T. maritima in liquid SVO was completely inhibited by 50 μg ml-1 kanamycin for at least 72 h (Figure 3b). However, spontaneous mutations sometimes caused the cultures to become resistant to the antibiotic, and a complete inhibition over a period of 72 h was only possible when the input amount was increased to 150 μg ml-1. Similar phenomena were also noticed with T. sp. RQ7. On SVO plates, spontaneous mutants of T. maritima and T. sp. RQ7 occasionally appeared after 48 h of incubation when up to 200 μg ml-1 kanamycin was added, but they rarely appeared when the antibiotic concentration was increased to 250 μg ml-1. Based on these observations, for the rest of the study, kanamycin was added at 150 μg ml-1 to liquid media and at 250 μg ml-1 to soft and solid media.
Transformation of Thermotoga-E. colishuttle vector pDH10
Because most bacteria become competent after a short electric pulse, electroporation was attempted to introduce pDH10 to Thermotoga. Electric pulses of various strengths were applied to both T. sp. RQ7 and T. maritima in the presence of 4 μg of plasmid DNA, and the transformants were selected with embedded growth. When an electric pulse of 2.0 kV was employed, five T. sp. RQ7 and one T. maritima transformants were obtained. A pulse of 1.8 kV resulted in eight T. sp. RQ7 and no T. maritima transformants. When the voltage dropped to 1.5 kV, no transformants were available with either species. These results suggest that the optimal voltage for Thermotoga is around 1.8 to 2.0 kV. In the control experiment, T. sp. RQ7 and T. maritima cells were treated with a pulse of 1.8 kV in the absence of DNA, and no spontaneous mutants were found.
For validation and comparison purposes, pDH10 was also introduced to T. sp. RQ7 and T. maritima through liposome-mediated transformation. Four T. sp. RQ7 and five T. maritima transformants were obtained from 1 μg of plasmid DNA, as opposed to zero colonies from the samples treated with liposomes containing no DNA. All transformants grew well in both soft and liquid selective media, and the presence of the kan gene was also confirmed by PCR.
Transformed DNA was stably maintained in Thermotoga
Percentage of Thermotoga colonies resistant to kanamycin after six transfers*
Resistant colonies (%)
104.92 ± 9.57
SVO + Kan
106.66 ± 3.56
100.96 ± 23.72
SVO + Kan
108.59 ± 7.54
Incorporation of pRQ7 increased the stability of pUC19 derivatives in E. coli
Percentage of E. coli colonies resistant to ampicillin during consecutive transfers*
Number of transfers
96.75 ± 1.30
7.25 ± 5.54
95.5 ± 2.50
1.75 ± 1.48
90 ± 6.04
68.5 ± 6.34
44 ± 11.68
32.25 ± 15.20
Improved method for cultivation of Thermotoga
The success of isolating transformants from solid media is essential to any genetic manipulation attempt. Whereas this is not a concern with aerobic mesophiles like E. coli, this requirement has become a limiting factor for the genetic investigations of many strict anaerobic, hyperthermophilic organisms. One obstacle is the requirement of an anaerobic glove box to handle plates. Since picking up colonies requires great precision, reaching out to a single colony with an inoculation loop or a toothpick through thick gloves has proven to be challenging for many of us. Even though gloveless chambers are commercially available, they are costly to maintain. Rolling tubes or tissue culture flasks in combination to Hungate techniques may serve as alternatives [16, 18–20], but they are prone to cross contaminations due to the narrow openings of these containers. Based on the fact that Thermotoga are fairly oxygen-tolerant, especially when they are not actively growing [1, 17, 21, 22], we prepare Thermotoga solid cultures with an embedded method, independent of an anaerobic chamber or an anoxic gas conduit. Our method sustains ~50% plating efficiency, making it possible to select for Thermotoga transformants among a sizeable population of viable cells. In addition, we developed a soft SVO medium to bridge the transfer of cultures from solid media to liquid media in an aerobic environment. Soft SVO is easy to make and convenient to use, and it also allows the withdrawal of cultures using a syringe.
Transformation and expression of the kangene
A heterologous kan gene has been functionally expressed and stably established in Thermotoga. The kan gene, carried on the shuttle vector pDH10, was introduced to Thermotoga by both electroporation and liposome-mediated transformation. The latter approach yields higher transformation efficiency (4~5 transformants per μg of DNA), but the former has the potential to mass-produce competent cells for future needs. Electrocompetency has been established in Thermotoga in this study, and a transformation efficiency of ~2 transformants per μg of DNA has been observed in RQ7 and ~0.25 in T. maritima. Two factors might have caused the transformation efficiencies to be low: the damages caused by oxygen and the activities of restriction-modification systems. Because the preparation of electrocompetent cells and the transformation procedure were performed under aerobic conditions, a significant portion of the Thermotoga cells would not survive the handling. Performing the experiment in an anaerobic chamber should help to improve the transformation efficiencies, albeit the operations would be cumbersome. In addition, restriction-modification systems have been discovered in Thermotoga, for instance, we have recently characterized a Thermotoga-specific Type II restriction-modification system . Unmethylated foreign DNA will be restricted by host-specific restriction nucleases as soon as they enter Thermotoga cells. Proper methylation of pDH10 prior to transformation is expected to significantly increase the quantity of transformants. Indeed, electrotransformation efficiency in Clostridia has been improved by 104~106 folds by methylating the shuttle vectors .
Although intact plasmid DNA of pDH10 has not been detected in Thermotoga, we favor the idea that pDH10 is autonomous in Thermotoga, because the kan gene is always associated to a plasmid preparation instead of a genomic DNA extract (Figure 4). If pDH10 had been integrated into the chromosome, we would have seen the kan gene appearing more often from the genomic samples than from the plasmid extracts. Our data stated otherwise (Figure 4), suggesting that pDH10 is likely an independent molecule rather than part of the chromosome. Because genomic DNA can include both chromosomal and plasmid DNA, it is not surprising to occasionally obtain a positive signal from a genomic DNA sample, even though the kan gene is carried by a free-living plasmid. We suspect that pDH10 has an extremely low copy number in Thermotoga, because the attempts to detect it by inverse PCR (amplifying pDH10 outward from the kan gene), retransformation (transform E. coli with plasmid extracts of Thermotoga transformants), or Southern blotting (using digoxygenin-labeled probes made from either the kan gene or pRQ7) did not generate any signals. The expected inverse PCR product is ~4 kb, much bigger than the kan gene. This makes the inverse PCR technically more challenging than just amplifying the kan gene.
In spite of the intriguing questions awaited to be answered with these elusive organisms, the door to the genetic manipulation of Thermotoga has been reopened. Ten years after the report of transient expression of heterologous genes in Thermotoga , we confirm that Thermotoga are truly transformable, not only by liposome-mediated transformation, but also by electroporation. Engineered Thermotoga strains are available for the first time.
List of abbreviations
colony forming unit
polymerase chain reaction
Thermotoga sp. RQ7.
We are grateful to Dr. Harald Huber at University of Regensburg, Germany for providing T. sp. RQ7 and T. maritima MSB8 and to Dr. Kenneth Noll at University of Connecticut for sharing his lab protocol for cultivation of Thermotoga with the overlay method. This work was supported by BGSU internal funds to ZX.
- Huber R, Langworthy TA, Konig H, Thomm M, Woese CR, Sleytr UB, Stetter KO: Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 degrees C. Archives of Microbiology. 1986, 144 (4): 324-333. 10.1007/BF00409880.View ArticleGoogle Scholar
- Windberger E, Huber R, Trincone A, Fricke H, Stetter KO: Thermotoga thermarum sp. nov. and Thermotoga neapolitana occurring in African continental solfataric springs. Archives of Microbiology. 1989, 151 (6): 506-512. 10.1007/BF00454866.View ArticleGoogle Scholar
- Takahata Y, Nishijima M, Hoaki T, Maruyama T: Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan. International Journal of Systematic and Evolutionary Microbiology. 2001, 51: 1901-1909. 10.1099/00207713-51-5-1901.View ArticleGoogle Scholar
- Schroder C, Selig M, Schonheit P: Glucose Fermentation to Acetate, CO2 and H2 in the Anaerobic Hyperthermophilic Eubacterium Thermotoga-Maritima - Involvement of the Embden-Meyerhof Pathway. Archives of Microbiology. 1994, 161 (6): 460-470.Google Scholar
- Harriott OT, Huber R, Stetter KO, Betts PW, Noll KM: A Cryptic Miniplasmid from the Hyperthermophilic Bacterium Thermotoga Sp Strain Rq7. Journal of bacteriology. 1994, 176 (9): 2759-2762.Google Scholar
- Akimkina T, Ivanov P, Kostrov S, Sokolova T, Bonch-Osmolovskaya E, Firman K, Dutta CF, McClellan JA: A highly conserved plasmid from the extreme thermophile Thermotoga maritima MC24 is a member of a family of plasmids distributed worldwide. Plasmid. 1999, 42 (3): 236-240. 10.1006/plas.1999.1429.View ArticleGoogle Scholar
- Nesbo CL, Dlutek M, Doolittle WF: Recombination in thermotoga: Implications for species concepts and biogeography. Genetics. 2006, 172 (2): 759-769.View ArticleGoogle Scholar
- Yu JS, Noll KM: Plasmid pRQ7 from the hyperthermophilic bacterium Thermotoga species strain RQ7 replicates by the rolling-circle mechanism. Journal of bacteriology. 1997, 179 (22): 7161-7164.Google Scholar
- Yu JS, Vargas M, Mityas C, Noll KM: Liposome-mediated DNA uptake and transient expression in Thermotoga. Extremophiles. 2001, 5 (1): 53-60. 10.1007/s007920000173.View ArticleGoogle Scholar
- Van Ooteghem SA, Beer SK, Yue PC: Hydrogen production by the thermophilic bacterium Thermotoga neapolitana. Applied Biochemistry and Biotechnology. 2002, 98: 177-189. 10.1385/ABAB:98-100:1-9:177.View ArticleGoogle Scholar
- Sambrook J, Russell DW: The condensed protocols from molecular cloning: a laboratory manual. 2006, Cold Spring Harbor Laboratory PressGoogle Scholar
- Liao H, McKenzie T, Hageman R: Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. Proceedings of the National Academy of Sciences of the United States of America. 1986, 83 (3): 576-580. 10.1073/pnas.83.3.576.View ArticleGoogle Scholar
- Lasa I, Caston JR, Fernandez-Herrero LA, de Pedro MA, Berenguer J: Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8. Molecular microbiology. 1992, 6 (11): 1555-1564. 10.1111/j.1365-2958.1992.tb00877.x.View ArticleGoogle Scholar
- Liao D, Dennis PP: The organization and expression of essential transcription translation component genes in the extremely thermophilic eubacterium Thermotoga maritima. The Journal of biological chemistry. 1992, 267 (32): 22787-22797.Google Scholar
- Maseda H, Hoshino T: Screening and analysis of DNA fragments that show promoter activities in Thermus thermophilus. FEMS Microbiol Lett. 1995, 128 (2): 127-134. 10.1111/j.1574-6968.1995.tb07511.x.View ArticleGoogle Scholar
- Jiang Y, Zhou Q, Wu K, Li XQ, Shao WL: A highly efficient method for liquid and solid cultivation of the anaerobic hyperthermophilic eubacterium Thermotoga maritima. Fems Microbiology Letters. 2006, 259 (2): 254-259. 10.1111/j.1574-6968.2006.00273.x.View ArticleGoogle Scholar
- Van Ooteghem SA, Jones A, Van Der Lelie D, Dong B, Mahajan D: H(2) production and carbon utilization by Thermotoga neapolitana under anaerobic and microaerobic growth conditions. Biotechnol Lett. 2004, 26 (15): 1223-1232.View ArticleGoogle Scholar
- Hermann M, Noll KM, Wolfe RS: Improved Agar Bottle Plate for Isolation of Methanogens or Other Anaerobes in a Defined Gas Atmosphere. Appl Environ Microbiol. 1986, 51 (5): 1124-1126.Google Scholar
- Macy JM, Snellen JE, Hungate RE: Use of syringe methods for anaerobiosis. The American journal of clinical nutrition. 1972, 25 (12): 1318-1323.Google Scholar
- Miller TL, Wolin MJ: A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Applied microbiology. 1974, 27 (5): 985-987.Google Scholar
- Childers SE, Vargas M, Noll KM: Improved Methods for Cultivation of the Extremely Thermophilic Bacterium Thermotoga-Neapolitana. Applied and Environmental Microbiology. 1992, 58 (12): 3949-3953.Google Scholar
- Le Fourn C, Fardeau ML, Ollivier B, Lojou E, Dolla A: The hyperthermophilic anaerobe Thermotoga Maritima is able to cope with limited amount of oxygen: insights into its defence strategies. Environmental microbiology. 2008, 10 (7): 1877-1887. 10.1111/j.1462-2920.2008.01610.x.View ArticleGoogle Scholar
- Xu Z, Han D, Cao J, Saini U: Cloning and characterization of the TneDI restriction: modification system of Thermotoga neapolitana. Extremophiles. 2011, 15 (6): 665-672. 10.1007/s00792-011-0397-9.View ArticleGoogle Scholar
- Mermelstein LD, Papoutsakis ET: In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 1993, 59 (4): 1077-1081.Google Scholar
- Belkin S, Wirsen CO, Jannasch HW: A New Sulfur-Reducing, Extremely Thermophilic Eubacterium from a Submarine Thermal Vent. Applied and Environmental Microbiology. 1986, 51 (6): 1180-1185.Google Scholar
- Grant SG, Jessee J, Bloom FR, Hanahan D: Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proceedings of the National Academy of Sciences of the United States of America. 1990, 87 (12): 4645-4649. 10.1073/pnas.87.12.4645.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.