Natural transformation of Thermotoga sp. strain RQ7
© Han et al.; licensee BioMed Central Ltd. 2014
Received: 6 November 2013
Accepted: 2 May 2014
Published: 10 May 2014
Thermotoga species are organisms of enormous interest from a biotechnological as well as evolutionary point of view. Genetic modifications of Thermotoga spp. are often desired in order to fully release their multifarious potentials. Effective transformation of recombinant DNA into these bacteria constitutes a critical step of such efforts. This study aims to establish natural competency in Thermotoga spp. and to provide a convenient method to transform these organisms.
Foreign DNA was found to be relatively stable in the supernatant of a Thermotoga culture for up to 6 hours. Adding donor DNA to T. sp. strain RQ7 at its early exponential growth phase (OD600 0.18 ~ 0.20) resulted in direct acquisition of the DNA by the cells. Both T. neapolitana chromosomal DNA and Thermotoga-E. coli shuttle vectors effectively transformed T. sp. strain RQ7, rendering the cells resistance to kanamycin. The kan gene carried by the shuttle vector pDH10 was detected by PCR from the plasmid extract of the transformants, and the amplicons were verified by restriction digestions. A procedure for natural transformation of Thermotoga spp. was established and optimized. With the optimized method, T. sp. strain RQ7 sustained a transformation frequency in the order of 10-7 with both genomic and plasmid DNA.
T. sp. strain RQ7 cells are naturally transformable during their early exponential phase. They acquire DNA from both closely and distantly related species. Both chromosomal DNA and plasmid DNA serve as suitable substrates for transformation. Our findings lend a convenient technical tool for the genetic engineering of Thermotoga spp.
KeywordsThermotoga Natural transformation Natural competence pDH10 pDH12
Thermotoga species are a group of Gram-negative, strictly anaerobic bacteria that grow up to 90°C. Ever since their discoveries in 1980s, they have been attracting great attention due to their extreme life style and the underlining implications in both basic research and biotechnological applications. Most noticeably, they serve as excellent candidates for the production of hydrogen gas, which may be directly used as a clean fuel [1, 2]. Producing biohydrogen using fermentative hyperthermophiles like Thermotoga has many advantages, including independence from light sources, reduced risks of contamination, lower energy requirement for agitation and cooling, and above all, higher hydrogen partial pressure. To fully release the potentials of Thermotoga, one often wishes to modify these bacteria at the genetic level. However, Thermotoga spp. are considered genetically recalcitrant. Their first transformants were isolated only recently after decades of efforts . Making these species more genetically accessible remains a priority for the research community. Effective delivery of recombinant DNA stands as a critical step in any genetic modification attempt dealing with Thermotoga. Previous studies have demonstrated that Thermotoga cells are transformable by liposome-mediated transformation and electroporation [3, 4]. Effective as these methods are, they are labor-intensive and time-consuming. A more convenient approach would be highly desirable if one needs to transform Thermotoga on a daily basis.
Many prokaryotes possess the ability to take up DNA directly from the environment without human intervention, i.e. they are naturally competent. Some bacteria efficiently import DNA from any source, whereas others prefer to acquire homologous DNA through the recognition of specific uptake signal sequences [5, 6]. The process of natural transformation involves the steps of DNA binding, translocation, and recombination. In most competent bacteria, the machinery responsible for DNA binding and translocation is related to Type IV pili (T4P) and Type II secretion systems (T2S) [7, 8]. T4P are retractile appendages that contribute to twitching motility, biofilm formation, adhesion, and conjugation, in addition to natural transformation. A working model proposed for Neisseria gonorrhoeae suggests that exogenous DNA crosses the outer membrane through a pore made of the secretin protein PilQ and transverses the periplasmic space with the aid of the PilE complex and a high affinity DNA-binding protein ComE. One strand of the DNA is then degraded by a nuclease, and the other strand travels across the inner membrane through a channel comprised of the membrane protein ComA [9, 10]. In Gram-positives, DNA is transported across the cell envelope in a similar manner, except that it does not need to overcome the outer membrane barrier. Once in the cytoplasm, the single-stranded DNA has to be protected by single strand DNA binding proteins to avoid degradation by host nucleases. Recent evidence suggests that DNA protection protein DprA conveys incoming ssDNA to RecA, the ubiquitous recombinase responsible for the initiation of homologous recombination [11–13]. Protein ComM influences transformation at a very late stage presumably by affecting recombination of the donor DNA into the chromosome .
More than 60 bacterial species have been documented as naturally transformable , which actually represents a minute fraction of all the bacterial species characterized thus far. Natural transformation is therefore considered a rare phenomenon. However, competence genes are wide-spread in bacterial species, even though many of which have not been reported to be naturally competent. Part of the reason is that natural competence usually develops only under certain physiological states that are inducible by environmental factors. Often these conditions are ill-defined and thus cannot be reproduced in laboratory settings. It is very likely that natural competence occurs much more frequently than we observe. For example, this mechanism was only recently noted in such well-studied organisms as Vibrio cholerae, Bacillus cereus, and E. coli. Natural uptake of DNA has not been reported for any bacteria growing optimally at, or above, 80°C, although this ability has been documented for hyperthermophilic archaeon Thermococcus kodakarensis.
Analyses of the published Thermotoga genomes present evidence of frequent lateral gene transfer events between Thermotoga and other bacteria, archaea, and even eukaryotes [20–23]. The chimeric nature of Thermotoga genomes suggests that these bacteria have a remarkable efficiency for acquisition of foreign genes. Although natural competence has not been reported for Thermotoga, putative competence genes have long been noticed in their genomes, including those encoding T2S- and T4P-related proteins . Moreover, mini-plasmids pRQ7, pMC24, and pRKU1 have been discovered in T. sp. strain RQ7 , T. maritima, and T. petrophila RKU-1 , respectively. Therefore, it is possible that Thermotoga spp. are capable of acquiring foreign DNA directly. Searching of the T. maritima genome sequence did not uncover any specific uptake signal sequences . For this reason, if Thermotoga spp. were to acquire DNA naturally, they should do so indiscriminately. The aim of this study was to establish natural competency in Thermotoga spp. and to develop a natural transformation protocol to facilitate the genetic engineering of Thermotoga.
Materials and methods
Strains and cultivation 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. strain RQ7
Isolated from geothermally heated sea floors in Ribeira Quente, the Azores
Caldicellulosiruptor saccharolyticus DSM 8903
Isolated from a thermal pool of Taupo, New Zealand
E. coli DH5α
F-endA1 hsdR17 (rk-, mk+) supE44 thi-1 λ - recA1 gyrA96 relA1 deoR Δ(lacZYA-argF)- U169 ϕ80dlacZΔM15
pUC-derived plasmid, containing a kan cassette for thermostable kanamycin selections
Cryptic miniplasmid from T. sp. strain RQ7
; GenBank: KF798180
pRQ7 cloned between the EcoRI and XbaI sites of pKT1; Apr; Kanr
; GenBank: JN813374
pRQ7 along with its duplicated fragment of 209 bp were cloned between the EcoRI and XbaI sites of pKT1; Apr, Kanr
This study; GenBank: KF798179
Construction of the Thermotoga-E. colishuttle vector pDH12
To construct pDH12, a three-piece ligation strategy was adopted since the coding sequence of the pRQ7 Rep protein conveniently bears a HindIII site about a third way from its start codon. Primers 5′- GGGAATTCTGAAGTACTTATCAAAGGAG-3′ and 5′-CGTTTTTTAAGCTTTTCCCAGC-3′ were used to amplify the N-terminus of the rep gene and its upstream sequence, and the resulting amplicon carries a EcoRI site at its 5′-end. Primers 5′-GGGAAAAGCTTAAAAAACGAATCC-3′ and 5′-GCTCTAGATAATCACACTAACCACATTC-3′ were used to amplify the C-terminus of the gene; the corresponding amplicon carries an XbaI site at its 3′-end. The PCR program for both reactions included 30 cycles of 30 sec at 94°C, 30 sec at 54°C, and 1 min at 72°C. The PCR products were digested with EcoRI-HindIII and HindIII-XbaI, respectively, and were ligated to pKT1, pre-digested with EcoRI-XbaI, to give rise to pDH12. All DNA manipulations followed the standard methods.
Natural transformation of Thermotogaspp
Two slightly different approaches were used. The early stage of the study involved the utilization of a nitrogen gas bottle. Briefly, 1 ml of an overnight culture of the recipient strain was transferred into 50 ml of fresh SVO medium and incubated to various growth stages. One ml of the fresh culture was then transferred to a 25 ml sterile serum bottle filled with N2 gas. At the same time, 50 μg of substrate DNA was added. After 6 h of incubation at 77°C with gentle agitation (100 to 125 rpm), the culture was mixed with hot SVO Gelrite medium and poured into petri dishes, with a supplement of 250 μg/ml kanamycin for selection. The plates were examined after 2 days of incubation at 77°C. Because the purpose of this study is to develop a simple method to transform Thermotoga spp., we intentionally did not apply DNase (to remove untransformed DNA after a designated period of time). This not only simplifies the overall procedure but also maximizes the occurrence of transformation events.
In the late stage of the study, the procedure was optimized as such: cells of 1 ml of the overnight culture of a recipient strain were collected by centrifugation, re-suspended in 200 μl of fresh SVO medium, and were injected into a 100-ml serum bottle containing 10 ml of fresh SVO medium; DNA substrate was added to a final concentration ranging from 5 to 10 μg/ml; the culture-DNA mixture was incubated at 77°C for 4 to 6 h with gentle agitation and then poured into selective plates (with a culture to medium ratio of 1 to 9, v/v). Transformation efficiency was computed as transformants per microgram of DNA, and transformation frequency was expressed as transformants per microgram of DNA per viable cell. The number of viable cells was determined by plating the control culture (no DNA added) with plain SVO solid medium. Plating efficiency was calculated as the ratio of viable cells to the total number of cells counted under microscope with Hausser Bright-Line 3120 Hemacytometer (Horsham, PA, USA).
Stability of DNA in the transformation environment
Although DNA is fairly stable in pure SVO medium, it may not be so in a growing cell culture, where natural transformation is supposed to take place. The extracellular nucleases secreted by the host cells could expedite the degradation of foreign DNA. To take this biological factor into consideration, we next incubated the genomic DNA of C. saccharolyticus in the supernatant of an overnight culture of T. sp. strain RQ7, which contains high levels of free nucleases and is also more acidic (pH 5.5). Indeed, the foreign DNA experienced accelerated degeneration (Figure 2B). Within 3 h of incubation, the DNA fragments were around 6.5 kb or smaller, which was equivalent to 6 h of incubation in the pure growth medium. After 6 h, most DNA fragments were 4 kb or smaller. If DNA is added to a cell culture at an earlier growth stage (as was done in later experiments), its degradation profile is expected to fall between the two extreme scenarios shown here (Figure 2). Therefore, in a practical experiment, heterologous DNA may be incubated with Thermotoga cells for up to 6 h.
Transformation of T. sp. strain RQ7 with T. neapolitanachromosomal DNA
T. sp. strain RQ7 cells were tested at five time points: OD600 of 0.10, 0.13, 0.16, 0.19, and 0.24. The first time point did not yield any Kanr colonies with either the treated sample or the control, probably because cells were not robust enough to be transferred to a N2 bottle, a process inevitably introduces trace amount of oxygen to the system. For the next four time points, the samples treated with the DNA generated significantly more Kanr colonies than the controls did (Figure 3). The colony number of the treated samples peaked to 1088 if DNA was added at a cell density of ~ 0.19. If cells were allowed to grow for another 45 min and reached OD600 of 0.24, the number of Kanr colonies dropped to 700, marking the decline of natural competence. The colony numbers from the controls varied from plate to plate, indicating that the mutation events happened randomly and independently in each N2 bottle rather than in the overnight seed culture or the fresh master culture from where samples were withdrawn.
Transformation of T.sp. strain RQ7 with pDH10
Attempts were made to introduce the Thermotoga-E. coli shuttle vector pDH10 into T. sp. strain RQ7 and T. maritima through the aforementioned natural transformation procedure. The plasmid DNA was prepared from E. coli recombinant strain DH5α/pDH10. A total number of 132 colonies appeared on the transformant plates after 2 days, whereas the control plates had only 20 colonies arising from spontaneous mutations. No colony was observed with the T. maritima samples (including the controls), again indicating that this strain is not naturally transformable.
Optimization of natural transformation
We next devoted our efforts to simplifying the natural transformation procedure. The step of transferring host cultures into a N2 bottle was eliminated, because it was the major source of oxygen invasion. Instead, DNA was added at the time of setting up the fresh culture from the overnight seed. As a precaution of carrying over excessive nucleases from the overnight supernatant, cells were pelleted by centrifugation and were resuspended in fresh SVO medium prior to inoculation. To reduce the amount of DNA used, the total volume of the recipient culture was cut back from 50 ml to 10 ml and the final concentration of substrate DNA was decreased from 50 μg/ml to 5 μg/ml. Considering that cells may have a difficult time to bind DNA if the cell-DNA mixture was constantly shaken, we attempted to transform static T. sp. strain RQ7 cultures but did not get any transformants. Accordingly, the practice of gentle agitation was preserved.
Plating efficiency of T. sp. strain RQ7 cells
Viable cells (×10^8)
Total cells (×10^8)
Plating efficiency (%)
Following the optimized procedure, we measured the transformation efficiency and frequency of T. sp. strain RQ7. The donor DNA was prepared from two sources: the chromosome of T. neapolitana and the plasmid pDH12, which is another Thermotoga-E. coli shuttle vector derived from pKT1 (Figure 1). T. sp. strain RQ7 presented a transformation frequency of (6.03 ± 1.52) × 10-7 (n = 3) with the genomic DNA and a frequency of (1.45 ± 0.02) × 10-7 (n = 2) with the plasmid DNA; for transformation efficiency, the figures were 16.6 ± 3.9 (n = 3) and 19.3 ± 3.8 (n = 2), respectively.
It was hoped that pDH12 would have a higher copy number than pDH10 because the extra sequence upstream of the rep gene may enhance the expression of this gene. However, pDH12 behaved in the same way as pDH10 did. The kan gene of pDH12 can be readily amplified by PCR and validated by restriction digestions, but attempts to identify the transformed plasmid by retransformation or inverse PCR have proven unsuccessful. A better understanding of the replication mechanism of pRQ7 will help to formulate new strategies on improving the copy numbers of pDH10 and pDH12.
Transformability of T. sp. strain RQ7 and T. maritima
It is unclear why T. maritima is not naturally competent while T. sp. strain RQ7 is, even though the two strains are closely related. Because the genome sequence of T. sp. strain RQ7 is not publically available yet, we are unable to compare the putative competence genes across the two genomes. We speculate that the putative competency genes may be differentially regulated between the two strains, or some of the genes may have accumulated mutations leading to altered functionalities in one strain. It is also possible that there are novel natural competence genes that do not exhibit significant homology to known ones and therefore are beyond recognition by sequence comparison. More information should emerge once the genome sequence of T. sp. strain RQ7 is available. Studies on the expression of the putative competence genes should also yield important clues.
Transformation frequency of T.sp. strain RQ7
With our optimized procedure, T. sp. strain RQ7 presented a transformation frequency in the order of 10-7. The chromosomal DNA and the plasmid DNA transformed this strain with comparable frequency, even though the plasmid DNA contained a higher mass ratio of the relevant gene(s), i.e. kanamycin-resistance gene(s). This is probably due to host restriction-modification systems, which selectively degrade DNA of distantly related species. A Type II RM system has been characterized in T. neapolitana and has also been found in T. maritima, T. petrophila, T. sp. strain RQ2, and T. naphthophila. In addition, REBASE has identified a few putative methylases that might be involved in restriction-modification mechanisms in Thermotoga, such as M.TspRQ2ORF1808P in T. sp. strain RQ2 as well as M.TneDDamP and M.TneDORF1590P in T. neapolitana (http://rebase.neb.com). Because restriction nucleases have a low level of sequence conservation in general, it is much harder to predict their presence through bioinformatics analyses than finding methylases. Plasmid incompatibility is unlikely to be the primary reason for the lower than expected transformation frequency associated with pDH12, because plasmids containing the same origin of replication (ori) are more compatible than we previously thought. For instance, two plasmids carrying the same colE1 ori yet different antibiotic markers - kanamycin versus ampicillin - were found to be 100% compatible after five consecutive transfers under just kanamycin selection .
The level of competency observed in T. sp. strain RQ7 is comparable to what reported for Ralstonia solanacearum, whose frequency is in the order of 10-7, but lower than those measured in well-characterized system such as Vibrio cholera and Helicobacter pylori, where a frequency of 10-5 or higher can often be obtained. It did not escape our attention that none of the putative competence genes found in Thermotoga are clustered. In many model systems, competence genes are often co-transcribed, such as the com operons in Bacillus and Streptococcus[15, 38] and the pil operons in Haemophilus and Thermus. The lack of the similar operons in Thermotoga suggests that their relevant molecular machinery either is not well-established or, to a lesser extent, represent a different type of machinery that does not share much similarity to those previously discovered. Nevertheless, the level of competence observed in T. sp. strain RQ7 sufficiently satisfies our engineering needs. As a matter of fact, we have been introducing recombinant DNA into T. sp. strain RQ7 exclusively by natural transformation ever since we discovered this mechanism.
Natural competency has been established in T. sp. strain RQ7. A natural transformation procedure has been developed and optimized for Thermotoga spp.. This study provides an alternative way to transform these genetically recalcitrant yet biotechnologically attractive organisms.
Colony forming unit
Origin of replication
Polymerase chain reaction
Thermotoga sp. RQ7
We thank Dr. Robert Kelly at North Carolina State University for providing C. saccharolyticus. This work was supported by the BGSU Commercialization Catalyst Award and the BGSU Building Strength Award.
- 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. Arch Microbiol. 1994, 161 (6): 460-470.Google Scholar
- Selig M, Xavier KB, Santos H, Schonheit P: Comparative analysis of embden-meyerhof and entner-doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium thermotoga. Arch Microbiol. 1997, 167 (4): 217-232.Google Scholar
- Han D, Norris SM, Xu Z: Construction and transformation of a Thermotoga-E coli shuttle vector. BMC Biotechnol. 2012, 12: 2-10.1186/1472-6750-12-2.View ArticleGoogle 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
- Schwarzenlander C, Averhoff B: Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27. FEBS J. 2006, 273 (18): 4210-4218. 10.1111/j.1742-4658.2006.05416.x.View ArticleGoogle Scholar
- Smith HO, Gwinn ML, Salzberg SL: DNA uptake signal sequences in naturally transformable bacteria. Res Microbiol. 1999, 150 (9–10): 603-616.View ArticleGoogle Scholar
- Dubnau D: Binding and transport of transforming DNA by Bacillus subtilis: the role of type-IV pilin-like proteins–a review. Gene. 1997, 192 (1): 191-198. 10.1016/S0378-1119(96)00804-9.View ArticleGoogle Scholar
- Dubnau D: DNA uptake in bacteria. Annu Rev Microbiol. 1999, 53: 217-244. 10.1146/annurev.micro.53.1.217.View ArticleGoogle Scholar
- Averhoff B, Friedrich A: Type IV pili-related natural transformation systems: DNA transport in mesophilic and thermophilic bacteria. Arch Microbiol. 2003, 180 (6): 385-393. 10.1007/s00203-003-0616-6.View ArticleGoogle Scholar
- Claverys JP, Martin B: Bacterial “competence” genes: signatures of active transformation, or only remnants?. Trends Microbiol. 2003, 11 (4): 161-165. 10.1016/S0966-842X(03)00064-7.View ArticleGoogle Scholar
- Berge M, Mortier-Barriere I, Martin B, Claverys JP: Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands. Mol Microbiol. 2003, 50 (2): 527-536. 10.1046/j.1365-2958.2003.03702.x.View ArticleGoogle Scholar
- Berge M, Moscoso M, Prudhomme M, Martin B, Claverys JP: Uptake of transforming DNA in gram-positive bacteria: a view from streptococcus pneumoniae. Mol Microbiol. 2002, 45 (2): 411-421. 10.1046/j.1365-2958.2002.03013.x.View ArticleGoogle Scholar
- Mortier-Barriere I, Velten M, Dupaigne P, Mirouze N, Pietrement O, McGovern S, Fichant G, Martin B, Noirot P, Le Cam E, Polard P, Claverys JP: A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell. 2007, 130 (5): 824-836. 10.1016/j.cell.2007.07.038.View ArticleGoogle Scholar
- Gwinn ML, Ramanathan R, Smith HO, Tomb JF: A new transformation-deficient mutant of Haemophilus influenzae Rd with normal DNA uptake. J Bacteriol. 1998, 180 (3): 746-748.Google Scholar
- Johnsborg O, Havarstein LS: Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae. FEMS Microbiol Rev. 2009, 33 (3): 627-642. 10.1111/j.1574-6976.2009.00167.x.View ArticleGoogle Scholar
- Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK: Chitin induces natural competence in Vibrio cholerae. Sci. 2005, 310: 1824-1827. 10.1126/science.1120096.View ArticleGoogle Scholar
- Mironczuk AM, Kovacs AT, Kuipers OP: Induction of natural competence in Bacillus cereus ATCC14579. J Microbial Biotechnol. 2008, 1: 226-235. 10.1111/j.1751-7915.2008.00023.x.View ArticleGoogle Scholar
- Tsen SD, Fang SS, Chen MJ, Chien JY, Lee CC, Tsen DH: Natural plasmid transformation in Escherichia coli. J Biomed Sci. 2002, 9 (3): 246-252.Google Scholar
- Cava F, Hidalgo A, Berenguer J: Thermus thermophilus as biological model. Extremophiles. 2009, 13 (2): 213-231. 10.1007/s00792-009-0226-6.View ArticleGoogle Scholar
- Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD, Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D, Heidelberg J, Sutton GG, Fleischmann RD, Eisen JA, White O, Salzberg SL, Smith HO, Venter JC, Fraser CM: Evidence for lateral gene transfer between archaea and bacteria from genome sequence of thermotoga maritima. Nature. 1999, 399 (6734): 323-329. 10.1038/20601.View ArticleGoogle Scholar
- Nesbo CL, Doolittle WF, Mongodin EF, Nelson KE: Outside forces helped shape the Thermotoga metagenome. Microbe. 2006, 1 (5): 235-241.Google Scholar
- Noll KM, Thirangoon K: Interdomain transfers of sugar transporters overcome barriers to gene expression. Methods Mol Biol. 2009, 532: 309-322. 10.1007/978-1-60327-853-9_18.View ArticleGoogle Scholar
- Zhaxybayeva O, Swithers KS, Lapierre P, Fournier GP, Bickhart DM, DeBoy RT, Nelson KE, Nesbo CL, Doolittle WF, Gogarten JP, Noll KM: On the chimeric nature, thermophilic origin, and phylogenetic placement of the thermotogales. Proc Natl Acad Sci U S A. 2009, 106 (14): 5865-5870. 10.1073/pnas.0901260106.View ArticleGoogle Scholar
- Harriott OT, Huber R, Stetter KO, Betts PW, Noll KM: A cryptic miniplasmid from the hyperthermophilic bacterium thermotoga Sp strain Rq7. J Bacteriol. 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
- Belkin S, Wirsen CO, Jannasch HW: A new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent. Appl Environ Microbiol. 1986, 51 (6): 1180-1185.Google Scholar
- 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. Arch Microbiol. 1986, 144 (4): 324-333. 10.1007/BF00409880.View ArticleGoogle Scholar
- Sissons CH, Sharrock KR, Daniel RM, Morgan HW: Isolation of cellulolytic anaerobic extreme thermophiles from new zealand thermal sites. Appl Environ Microbiol. 1987, 53 (4): 832-838.Google Scholar
- Grant SG, Jessee J, Bloom FR, Hanahan D: Differential plasmid rescue from transgenic mouse DNAs into escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990, 87 (12): 4645-4649. 10.1073/pnas.87.12.4645.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. Mol Microbiol. 1992, 6 (11): 1555-1564. 10.1111/j.1365-2958.1992.tb00877.x.View ArticleGoogle Scholar
- Van Ooteghem SA, Beer SK, Yue PC: Hydrogen production by the thermophilic bacterium thermotoga neapolitana. Appl Biochem Biotechnol. 2002, 98: 177-189.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
- Velappan N, Sblattero D, Chasteen L, Pavlik P, Bradbury AR: Plasmid incompatibility: more compatible than previously thought?. Protein Eng Des Sel. 2007, 20 (7): 309-313. 10.1093/protein/gzm005.View ArticleGoogle Scholar
- Mercier A, Bertolla F, Passelegue-Robe E, Simonet P: Influence of DNA conformation and role of comA and recA on natural transformation in Ralstonia solanacearum. Can J Microbiol. 2009, 55 (6): 762-770. 10.1139/W09-025.View ArticleGoogle Scholar
- Lo Scrudato M, Blokesch M: The regulatory network of natural competence and transformation of Vibrio cholerae. PLoS Genet. 2012, 8 (6): e1002778-10.1371/journal.pgen.1002778.View ArticleGoogle Scholar
- Lin EA, Zhang XS, Levine SM, Gill SR, Falush D, Blaser MJ: Natural transformation of helicobacter pylori involves the integration of short DNA fragments interrupted by gaps of variable size. PLoS Pathog. 2009, 5 (3): e1000337-10.1371/journal.ppat.1000337.View ArticleGoogle Scholar
- Londono-Vallejo JA, Dubnau D: comF, a bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases. Mol Microbiol. 1993, 9 (1): 119-131. 10.1111/j.1365-2958.1993.tb01674.x.View ArticleGoogle Scholar
- Friedrich A, Prust C, Hartsch T, Henne A, Averhoff B: Molecular analyses of the natural transformation machinery and identification of pilus structures in the extremely thermophilic bacterium thermus thermophilus strain HB27. Appl Environ Microbiol. 2002, 68 (2): 745-755. 10.1128/AEM.68.2.745-755.2002.View ArticleGoogle Scholar
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