A series of new E. coli–Thermococcus shuttle vectors compatible with previously existing vectors

A series of new E. coli–Thermococcus shuttle vectors compatible with previously existing vectors Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replica- tion origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli–T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli–T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression. Keywords Archaea · Hyperthermophiles · Gene cloning and expression · Cloning · Molecular biology · Genetics of extremophiles · Molecular biology of archaea Introduction proteins from thermophiles allow unmatched insight into mechanisms of protein stability (Razvi and Scholtz 2006; Investigating and understanding the ability of cells to sur- Pucci and Rooman 2017). Additionally, enzymes derived vive in hyperthermophilic environments is an increasingly from extremophiles are often functional in conditions where important undertaking in the fields of biochemistry, biotech - mesophilic enzymes would quickly degrade, giving them nology, molecular biology and evolution, to name just a few. enormous potential in biotechnology (Bruins et al. 2001; Biochemists have known the value of thermostable enzymes Haki and Rakshit 2003; Elleuche et al. 2015). In molecular for many decades, particularly in structural studies, where biology, the unusual biology of archaea and their unique genetic systems have opened our eyes to new and intriguing mechanisms of gene regulation, gene transfer, and meta- Communicated by L. Huang. bolic processes (Olsen and Woese 1997; Sato and Atomi 2011; Wagner et al. 2017). Furthermore, the importance of Electronic supplementary material The online version of this thermophiles in evolutionary biology cannot be understated; article (https ://doi.org/10.1007/s0079 2-018-1019-6) contains thermophiles or hyperthermophiles may be at the base of supplementary material, which is available to authorized users. both the bacterial and the archaeal/eukaryal branches of the * Ryan Catchpole tree of life (Boussau et al. 2008; Groussin and Gouy 2011). ryan.catchpole@pasteur.fr As a result, understanding the genetics of such hyperthermo- philes could have far-reaching implications and applications. Unité de Biologie Moléculaire du Gène chez les Unfortunately, the range of genetic tools and techniques Extrêmophiles (BMGE), Département de Microbiologie, Institut Pasteur, 25 Rue du Docteur Roux, 75015 Paris, available is very limited. For example, in the extensively France studied Thermococcales (anaerobic hyperthermophilic Institute for Integrative Biology of the Cell (I2BC), archaea), only a single cloning vector is published (Santan- Microbiology Department, CEA, CNRS, Univ. Paris-Sud, gelo et al. 2008), despite over 175 cultivatable strains having Université Paris-Saclay, Gif-sur-Yvette, France Vol.:(0123456789) 1 3 592 Extremophiles (2018) 22:591–598 been isolated (Lepage et al. 2004; Price et al. 2015) and 26 Lai, University of Canterbury) using primers ‘pTPTK1. complete genomes deposited in the Genbank database. As GA.1’ and ‘pTPTK1.GA.2’ (for primer sequences, see Sup- a result, despite Thermococcus kodakarensis having been plementary Table 2). The HMG-CoA cassette (conferring adopted as a model organism, only a single vector is avail- resistance to mevinolin in T. kodakarensis) was amplified by able for the transformation of, and expression of exogenous PCR from the plasmid pLC70 (kindly gifted by Thomas San- genes in this species. To study the mechanisms of plasmid tangelo, Colorado State University) using primers ‘pTPTK1. maintenance, and to understand the mechanisms of horizon- GA.5’ and ‘pTPTK1.GA.6’. The entire sequence of pTP2 tal gene transfer observed in these extremophilic archaea, it was amplified by PCR from Thermococcus prieurii DNA is important to have genetic tools which allow us to follow using the primers ‘pTPTK1.GA.3’ and ‘pTPTK1.GA.4’. multiple genes, and multiple replicons simultaneously. PCR products were purified, assembled, and used to trans- Hence, we sought to generate a new Escherichia coli–T. form E. coli strain XL1-Blue. Transformants were selected kodakarensis shuttle vector which is compatible with the by growth in the presence of chloramphenicol and confirmed only currently available vector, pLC70. In recent years, our by Sanger sequencing (Beckman Genomics). group has sequenced 43 plasmids from Thermococcales spe- Plasmids pTPTK2 and pTPTK3 were constructed using cies (unpublished data), 29 of which co-exist in the same pTPTK1 as a starting point. Briefly, the pBAD33-pTP2 cells as other plasmids or circular viral genomes, proving backbone of pTPTK1 was amplified by PCR using prim- their compatibility (if plasmid incompatibility exists in ers ‘pTPTK2/3.GA.1’ and ‘pTPTK2/3.GA.2’. For pTPTK2, Thermococcales). This provided a wide selection of poten- the trpE-cassette (conferring tryptophan prototrophy to T. tial origins of replication for use in T. kodakarensis. kodakarensis ΔTK0254 backgrounds) was amplified from We present a series of novel E. coli–T. kodakarensis the plasmid pLC70 using primers ‘pTPTK2.GA.3’ and shuttle vectors based on the small cryptic plasmid pTP2 ‘pTPTK2.GA.4’. For pTPTK3, the gene TK0149 (confer- from T. prieurii, and the E. coli p15A origin of replication. ring agmatine prototrophy to T. kodakarensis ΔTK0149 This plasmid backbone has been developed in combination backgrounds) was amplified from the chromosome of T. with three different markers for selection in T. kodakarensis kodakarensis KOD1 along with its native promoter using strains. Additionally, we show that this plasmid is compat- primers ‘pTPTK3.GA.3’ and ‘pTPTK3.GA.4’. PCR prod- ible with the single published cloning vector for Thermococ- ucts were assembled and sequenced as above. cales, pLC70 (and derivatives thereof). Plasmid pTNAg was constructed by assembling the TK0149 cassette (PCR-amplified from the T.   kodakaren- sis KOD1 chromosome using primers ‘pTNAg.GA.1’ and Materials and methods ‘pTNAg.GA2’) with the ApaI-EcoRV digestion product of pLC70. The resulting plasmid was selected in E. coli by Strains and media growth in the presence of ampicillin and kanamycin and confirmed by Sanger sequencing (Beckman Genomics). Plasmid construction was carried out in Escherichia coli Plasmid pTNTrpE was constructed by blunting and cir- strain XL1-Blue grown at 37 °C in LB medium. Where nec- cularization of the XbaI–EcoRV fragment of pLC70. The essary, media was supplemented with Ampicillin (100 µg/ absence of the HMG-CoA cassette was confirmed by Sanger mL), Kanamycin (40 µg/mL) or Chloramphenicol (20 µg/ sequencing (Beckman Genomics). mL). All archaeal work was carried out in Thermococcus kodakarensis strain TS559 (Santangelo et al. 2010) grown Transformation of T. kodakarensis at 85 °C in either ASW-YT (Sato et al. 2003) or ASW-CH medium with uracil supplementation (10 µg/mL) (Fujikane Transformation was carried out as described previously et  al. 2010). Where necessary, media was supplemented (Sato et al. 2005). Briefly, ~ 5 × 10 late exponential phase with agmatine sulfate (1.0 mM) or mevinolin (10 μM). T. kodakarensis TS559 cells were harvested under anaerobic conditions by centrifugation at 4000×g for 10 min. The cell Plasmid construction pellet was resuspended in 200 µL 0.8×ASW, and 5 µg of plasmid DNA was added. Suspensions were incubated on For a complete list of strains and plasmids used in this study, ice for 60 min, heat shocked at 85 °C for 60 s, then chilled see Supplementary Table 1. on ice for 10 min. 1 mL ASW-YT + agmatine was added, Plasmid pTPTK1 was constructed by Gibson Assembly and the cultures were incubated at 85 °C for 1.5 h. Cells using the NEBuilder HiFi DNA Assembly Master Mix (New were harvested by centrifugation at 4000×g for 3 min and England Biolabs) following the manufacturer’s protocol. used to inoculate 25 mL of selective media. Transformant Briefly, the E. coli p15A origin of replication was amplified cultures were grown at 85 °C for 48 h before being sub- by PCR from the plasmid pBAD33 (kindly gifted by Alicia cultured twice by 1:100 dilution in fresh selective media. 1 3 Extremophiles (2018) 22:591–598 593 Transformation was confirmed by isolation of plasmid DNA 1990), Phyre2 (Kelley et al. 2015) or HHpred (Söding et al. from 20 to 50 mL of culture (plasmid DNA recovered using 2005) search. The top BLAST hits were aligned using Macherey–Nagel NucleoSpin Plasmid kit) and analysis by MSAprobs v0.9.7 (Liu and Schmidt 2014), gaps removed restriction digestion and gel electrophoresis. using BMGE v1.12 (Criscuolo and Gribaldo 2010), Double transformation of T. kodakarensis TS559 was per- amino acid substitution models determined by ModelFinder formed sequentially, i.e. plasmid-containing cultures were (Kalyaanamoorthy et al. 2017), and phylogenetic trees gen- grown in selective media, and transformed with a second erated with IQ-TREE v1.5.4 (Nguyen et al. 2015). Trees plasmid. Following selection and sub-culturing of double were visualized with iTOL v3.6.1 (Letunic and Bork 2016). transformants, serial dilutions of stationary phase cultures were suspended in a solution of molten 0.7% Gelrite con- taining 10 g/L colloidal sulfur and plated as a top layer on Results solid selective media (under anaerobic conditions). Single colonies (observable by local clearing of the colloidal sulfur) Identification of a suitable origin of replication were used to inoculate selective liquid media. As pLC70 encodes the origin of replication from plasmid Liposome‑mediated transformation pTN1 of Thermococcus nautili (Soler et al. 2007; Santan- gelo et al. 2008), the most obvious place to search for a Transformation was performed using a modification of a plasmid with a compatible origin of replication is the other method previously described (Metcalf et al. 1997). Briefly, co-existing plasmids in T. nautili. However, with pTN2 and cultures of Thermococcales species were grown to late-log pTN3 comprising 13 and 18 kb, respectively, they are less phase in ASW-YT medium. Cells were pelleted at 4000×g than ideal candidates for the design of small, easily manipu- for 10 min under anaerobic conditions, and resuspended lated shuttle vectors. in 0.1 vol of 0.85 M sorbitol (de-gassed and reduced with Phylogenetic analysis of the replication-associated pro- Na S). For each transformation, liposomes were generated tein encoded by pTN1 reveals it to be part of a small fam- by adding 15 μL DOTAP to 150 μL 20 mM HEPES, pH ily of homologous genes encoded by other Thermococcales 7.4, with 2 µg DNA. The liposome suspension was added to plasmids (Fig.  1). One of the rep genes identified in this 1 mL resuspended culture, and incubated at room tempera- analysis is that of the small cryptic plasmid pTP1 from T. ture for 1 h. Cells were pelleted and resuspended in 1 mL prieurii (Gorlas et al. 2013a, b). The rep protein of plasmid ASW-YT medium, then allowed to recover for 2 h at 85 °C. pTP1 (RepTP1, YP_007974239) is 31.3% identical to that Cells were again pelleted and used to inoculate 25 mL selec- of pTN1 (Rep74, ABR10429) at the amino acid level. This tive medium. similarity, combined with the close phylogenetic relatedness of these two species (Gorlas et al. 2014) suggested that the Phylogenetic analysis replication-associated proteins are likely homologs belong- ing to the same family of archaeal plasmids. It is notable Sequences for replication-associated proteins of pTN1 that pTP1 is unusual in structure, likely having acquired its (rep74, ABR10429.1) and pTP2 (repTP2, YP_007974244.1) replication protein via recombination with another mobile were used as query sequences for a BLAST (Altschul et al. element (Gorlas et al. 2013b). Thus, while the other ORFs Fig. 1 a Schematic diagrams AB of pTN1-family plasmids. Predicted ORFs are indicated by gray blocks. ORFs encoding predicted replication-associated proteins are black. In the case of T. prieurii, the complete Rep gene interrupts another Rep-like gene colored in gray with black stripes. b Phylogenetic relation- ship between pTN1-family Rep proteins. Unrooted phyloge- netic tree generated with the full-length Rep proteins of the 4 pTN1-family plasmids 1 3 594 Extremophiles (2018) 22:591–598 of pTP1 differ from the usual organization of pTN1-family activity are limited. The intergenic space between ORF3 plasmids, the Rep protein is clearly homologous to that and ORF4 (Fig. 2) was chosen as it contains a 27-bp non- of pTN1. We, therefore, predicted that co-existing plas- coding region, as well as being at the 3′ termini of both mids from T. prieurii may encode replication origins that ORF3 and ORF4, thus decreasing the chance of disrupting could be compatible with pTN1 (and, therefore, pLC70). upstream regulatory sequences such as promoters. Intrigu- T. prieurii contains three circular extra-chromosomal ele- ingly, linearization of pTP2 in this region resulted in a ~ 1.8- ments—the genome of the virus TPV1 (21,592 bp), and two Kbp fragment, as determined by gel electrophoresis (data small cryptic plasmids, pTP1 and pTP2 (3126 and 2038 bp, not shown), rather than the 2038-bp expected from the pub- respectively) (Gorlas et al. 2013b). Due to its small size and lished sequencing data. Sequencing of the fragment revealed compatibility with pTP1, pTP2 was chosen as a potentially that the linearized pTP2 was missing a tandem repeat corre- suitable origin of replication for shuttle vector construction. sponding to nucleotides 710–907 and 913–1110 of sequence NC_021208.1 (Supplementary Figure 1). It has not been Analysis of pTP2‑rep confirmed whether this fragment was lost in the pTP2 isolate used as a template, or during PCR. While pTP2 is the smallest known Thermococcales plasmid, To minimize the risk of recombination between a pTP2- it appears to have a surprisingly complicated gene structure. derived vector and pLC70 while maintained within a sin- Whereas the pTN1-family of Thermococcales plasmids are gle cell, an E. coli vector backbone and antibiotic-resistant quite simple in structure (usually containing only two ORFs, marker were chosen which are different to those used in one of which encodes the replication-associated protein), generating pLC70. Where pLC70 is comprised of a pUC pTP2 encodes several putative transcriptional regulators, origin of replication in combination with ampicillin- and as well as a transmembrane domain protein (Gorlas et al. kanamycin-resistant genes (derived from pCR2.1-TOPO), a 2013b). The function of these proteins in the maintenance p15A origin of replication was chosen for our shuttle vector, and potential transmission of pTP2-family plasmids, or in combination with a chloramphenicol-resistant gene (both indeed secondary functions, remains unknown. The pre- derived from pBAD33). dicted replication-associated protein of pTP2 is also enig- The range of selectable markers available for T. koda- matic. This protein shares 30–35% sequence identity with a karensis is very limited, comprising genes which com- protein of unknown function found in the genomes of mul- plement strain-specific auxotrophies, e.g. tryptophan or tiple species of methanogenic Euryarchaeota. Both struc- agmatine; or a single antibiotic resistance determinant pro- tural and functional prediction (using Phyre2 and HHpred, duced by HMG-CoA reductase overexpression providing respectively) of these methanogen genes, and of RepTP2 itself reveals the central ~ 120 residues of these proteins to be similar to replication-associated proteins of various mobile elements infecting all three domains of life, e.g. SIRV1 from the archaeon Sulfolobus islandicus; pMV158 from the bac- terium Streptococcus agalactiae; and TYLSCV from the eukaryote Solanum lycopersicum (Supplementary Table 3). The similarity of both RepTP2 and other mobile elements to chromosomally-encoded genes of methanogenic Euryar- chaeota may suggest that these genes are part of integrated pTPTK1(mev) 5489bp pTPTK2 (trp) 5455bp horizontally mobile elements in these species. We were pTPTK3 (agm) 4710bp unable to find any synteny of the genes surrounding these Rep-like proteins, indicating that these proteins may have arisen from multiple different integrated elements, or that the similarity in sequence, structure and predicted function is due to a similarity of function, e.g. a native methanogen protein encoding a replication-associated functionality (heli- case, polymerase, resolvase, etc). MCS region: Generation of shuttle vectors Fig. 2 Plasmid maps of pTPTK1, pTPTK2 and pTPTK3. ORFs/ Plasmid pTP2 encodes five predicted ORFs, three of which genetic elements are indicated by white boxes outlined in black with are overlapping. Therefore, the possible sites where the plas- labels indicating the nature of each element. Gray regions inside the mid could be linearized without disrupting potential gene plasmid map indicate the source and nature of each plasmid region 1 3 Extremophiles (2018) 22:591–598 595 resistance to mevinolin. To maximize the usefulness of an Replication in T. kodakarensis E. coli–T. kodakarensis shuttle vector, we generated three different variants of the pTP2-derived vector, each encod- Following construction of the three plasmids in E. coli, they ing a different selectable marker. were used to transform T. kodakarensis strain TS559 (San- All three vectors contain an identical backbone as tangelo et al. 2010). Initial transformations were performed described above, comprising the 1.8-kb pTP2 sequence, in liquid culture, and each of the three plasmids conferred p15A origin, and chloramphenicol-resistant marker the appropriate prototrophy/resistance (mevinolin resistance (Fig. 2). The following marker cassettes were added for for pTPTK1; tryptophan prototrophy for pTPTK2; agmatine selection in T. kodakarensis: pTPTK1 encodes HMG-CoA prototrophy for pTPTK3). Transformant cultures were able reductase under the constitutive glutamate dehydrogenase to form discreet colonies on solid selective media. Plasmid promoter (sourced from pLC70 (Santangelo et al. 2008)), DNA isolated from transformant cultures (both from origi- conferring resistance to mevinolin; pTPTK2 encodes nal transformations following two sub-cultures, and from anthranilate synthase (TK0254 ≡ trpE) under the control cultures inoculated with single colonies) gave a restriction of the promoter from CDP-alcohol phosphatidyltransferase digestion pattern identical to that observed with plasmids (TK2279) (also sourced from pLC70), conferring trypto- isolated from E. coli suggesting these plasmids were faith- phan prototrophy to trpE mutant backgrounds; pTPTK3 fully replicated in T. kodakarensis (Fig.  3). Additionally, encodes pyruvoyl-dependent arginine decarboxylase re-transformation of E. coli using plasmid DNA isolated (TK0149) under its native promoter, conferring agmatine from T. kodakarensis transformant cultures successfully re- prototrophy to TK0149 mutant backgrounds. introduced the plasmid and chloramphenicol marker. A small multiple cloning site (MCS) site was added to the shuttle vector design (Fig. 2) to aid in the cloning Compatibility with pLC70‑derived plasmids of additional markers and/or genes. A limited number of enzyme recognition sequences were available which nei- To assess the compatibility of pTP2-derived plasmids with ther cut the vector backbone, nor the potential selectable pLC70-derived plasmids, it was necessary to select for both markers; therefore, the MCS comprises 40 bp with sites plasmids in a single culture. However, with pLC70 encod- for ApaI, AvrII, StuI*, NotI, SphI, NruI, PciI and SalI* ing both tryptophan prototrophy and mevinolin-resistant (*StuI is not suitable for cloning in pTPTK2 as there is a markers, it was necessary to modify pLC70 to have dif- site within TK0254. Additionally, although still suitable ferent selectable markers on the co-transforming plas- for cloning, a second SalI site is present at the junction mids. Plasmid pTNAg was generated by deletion of the between the vector and TK0254 cassette, resulting in SalI entire TK0254-PF1848 (TrpE − HMG-CoA) cassette from digestion releasing a 24-bp fragment from the vector). pLC70, and replacement with the TK0149 gene encoding pyruvoyl-dependent arginine decarboxylase (for which T. Fig. 3 Digestion and gel electrophoresis of pTP2-derived plasmids. a isolated from T. kodakarensis. b Plasmids digested with HindIII, rec- Plasmids digested with RruI, recognizing a single site on each plas- ognizing multiple sites on each plasmid. Lane 1: pTPTK1 isolated mid. Lane 1: pTPTK1 isolated from E. coli, lane 2: pTPTK3 isolated from E. coli, lane 2: pTPTK3 isolated from E. coli, lane 3: pTPTK2 from E. coli, lane 3: pTPTK2 isolated from E. coli, lane 4: GeneR- isolated from E. coli, lane 4: GeneRuler 1  kb DNA ladder, lane 5: uler 1  kb DNA ladder, lane 5: pTPTK1 isolated from T. kodakaren- pTPTK1 isolated from T. kodakarensis, lane 6: pTPTK3 isolated sis, lane 6: pTPTK3 isolated from T. kodakarensis, lane 7: pTPTK2 from T. kodakarensis, lane 7: pTPTK2 isolated from T. kodakarensis 1 3 596 Extremophiles (2018) 22:591–598 kodakarensis TS599 is a knockout). Plasmid pTNTrpE was and separately in the T. kodakarensis host. To confirm generated by deletion of PF1848 from pLC70, resulting in that T. kodakarensis cultures were indeed double transfor- a plasmid encoding the TrpE marker alone. Both of these mants, rather than a mixed culture of single transformants, plasmids were able to replicate faithfully in T. kodakarensis cultures were plated to single colonies on solid selective (Supplementary Figure 2), conferring agmatine and trypto- medium. Following incubation, single colonies were sus- phan prototrophy, respectively, to strain TS559. pended in 0.8 × ASW, vortexed vigorously, and serial dilu- Double transformants of T. kodakarensis were generated tions plated again on solid selective medium. Recovered by transforming a strain harboring a pLC70-derived plas- colonies grew well in selective medium, and all gave two mid with a pTP2-derived plasmid, or vice versa. Although plasmids upon DNA extraction and electrophoresis or E. the transformation rate was not quantified, transformation coli transformation. was successful regardless of the nature of the incumbent It should be noted that selection for both tryptophan pro- or incoming plasmid. However, for technical reasons, it is totrophy and mevinolin resistance (ASW-CH + mevinolin) most simple to carry out the double transformation such resulted in slow-growing cultures, likely due to an increased that the incumbent plasmid is one which can be selected in sensitivity of cells to mevinolin in the less-rich ASW-CH rich media, i.e. conferring agmatine prototrophy (pTPTK3 medium over the rich ASW-YT medium. or pTNAg) or mevinolin resistance (pTPTK1 or pLC70), thus ensuring a cell-dense, exponential phase culture can be Host‑range of pTP2‑derived vectors readily produced for the second transformation. Transformation with two plasmids conferred upon cul- To assess the ability of pTP2-derived vectors to replicate in tures the appropriate prototrophies and/or resistance. DNA other Thermococcales species, we attempted to use pTPTK1 extracted from double-transformant cultures indeed gave to transform T. aggregans, T. pacificus, T. siculi, T. celer, T. two plasmid bands upon electrophoresis, and plasmids guaymasensis, T. fumicolans and T. prieurii. As no method produced the appropriate digestion patterns (Fig. 4). Plas- of transformation has been established for these species, we mid extractions were able to transform E. coli to chloram- attempted transformation using the method described for T. phenicol resistance and ampicillin/kanamycin resistance. kodakarensis. This method relies on the natural competence Furthermore, dilutions of plasmid extractions resulted in of T. kodakarensis, and so it is perhaps unsurprising that transformation of E. coli to chloramphenicol resistance we were unable to obtain transformants of these untested while maintaining ampicillin sensitivity, and vice versa, species (only T. kodakarensis gave transformants with this indicating that the two plasmids were replicating faithfully protocol). We then attempted transformation using DNA Fig. 4 Digestion and gel electrophoresis of plasmids from double ble transformant. b Plasmids digested with RruI, recognizing a single transformants. a Plasmids digested with RruI, recognizing a single site on each plasmid. Lane 1: pTNAg isolated from E. coli, lane 2: site on each plasmid. Lane 1: pTNTrpE isolated from E. coli, lane pTPTK2 isolated from E. coli, lane 3: GeneRuler 1  kb DNA ladder, 2: pTPTK1 isolated from E. coli, lane 3: GeneRuler 1  kb DNA lad- lane 4: pTNAg + pTPTK2 isolated from T. kodakarensis double trans- der, lane 4: pTNTrpE + pTPTK1 isolated from T. kodakarensis dou- formant 1 3 Extremophiles (2018) 22:591–598 597 encapsulated in liposomes, as previously described for other tools—represents an important model species. The ability Euryarchaeota such as Methanosarcina (Metcalf et al. 1997) to transform T. kodakarensis with two different and sta- and Methanococcus voltae (Sniezko et al. 1998), as well as ble extra-chromosomal replicons will open up new fields hyperthermophilic bacteria of the genus Thermotoga (Yu of study in these important organisms, e.g. plasmid parti- et al. 2001). Unfortunately, this method did not yield trans- tioning/segregation and the related plasmid compatibility/ formants in any Thermococcus species tested, even T. koda- incompatibility, DNA-binding proteins together with their karensis. Transformation was also attempted of the naturally substrates, alpha-complementation of enzymes, etc. competent Pyrococcus furiosus COM1 strain; however, no Acknowledgements This work was funded by the European Research plasmid-containing transformants were obtained. Council under the European Union’s Seventh Framework Program (FP/2007-2013)/Project EVOMOBIL - ERC Grant Agreement no. Discussion Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco We have established that the cryptic plasmid pTP2 from mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- Thermococcus prieurii encodes an origin of replication tion, and reproduction in any medium, provided you give appropriate which is functional in T. kodakarensis. We have used this credit to the original author(s) and the source, provide a link to the origin of replication to generate a series of E. coli–T. kodaka- Creative Commons license, and indicate if changes were made. rensis shuttle vectors. These vectors replicate stably (under selection) in both T. kodakarensis and E. coli. Importantly, these vectors are compatible with the previously described References E. coli–T. kodakarensis shuttle vector, pLC70 (Santangelo et al. 2008), both in E. coli and in T. kodakarensis, greatly Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410. h t t p s : / / d o i . o rg / 1 0 . 1 0 1 6 / contributing to the range of genetic tools available for Ther- S0022 -2836(05)80360 -2 mococcales. Further work will be necessary to quantify the Boussau B, Blanquart S, Necsulea A et al (2008) Parallel adaptations relative copy numbers of pTN1-derived and pTP2-derived to high temperatures in the Archaean eon. Nature 456:942–945. plasmids when co-maintained in T. kodakarensis; however, https ://doi.org/10.1038/natur e0739 3 Bruins ME, Janssen AEM, Boom RM (2001) Thermozymes and their visual analysis of plasmids by gel electrophoresis suggests applications. Appl Biochem Biotechnol 90:155–186. https ://doi. that they do not differ greatly. org/10.1385/ABAB:90:2:155 The ability of both pTP2-derived (e.g. pTPTK1) and Criscuolo A, Gribaldo S (2010) BMGE (block mapping and gather- pTN1-derived plasmids (e.g. pLC70) to replicate concur- ing with entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC rently and faithfully inside a single T. kodakarensis strain Evol Biol 10:210. https ://doi.org/10.1186/1471-2148-10-210 suggests that pTP2 and pTN1 belong to separate families Elleuche S, Schäfers C, Blank S et al (2015) Exploration of extremo- of plasmids within the Thermococcales. Indeed, sequence philes for high temperature biotechnological processes. 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It side with a virus: characterization of two novel plasmids from remains unclear whether this failure in transformation is due Thermococcus prieurii, a host for the spindle-shaped virus TPV1. Appl Environ Microbiol 79:3822–3828. https ://doi.org/10.1128/ to a failure of the plasmid DNA to enter the cell, or a failure AEM.00525 -13 of the plasmid to replicate once inside the cell. Our inclusion Gorlas A, Croce O, Oberto J et al (2014) Thermococcus nautili sp. of T. prieurii in these experiments suggests that, at least for nov., a hyperthermophilic archaeon isolated from a hydrother- this species, the failure is a lack of DNA entry, as T. prieurii mal deep-sea vent. Int J Syst Bacteriol 64:1802–1810. https://doi. org/10.1099/ijs.0.06037 6-0 is the natural host for pTP2. Groussin M, Gouy M (2011) Adaptation to environmental tempera- The range of cultivable and genetically tractable Archaea ture is a major determinant of molecular evolutionary rates in for study as model organisms is extremely limited. Simi- archaea. Mol Biol Evol 28:2661–2674. https ://doi.org/10.1093/ lar limitations exist in the study of hyperthermophilic molbe v/msr09 8 Haki GD, Rakshit SK (2003) Developments in industrially important organisms, which are proving to have great biotechno- thermostable enzymes: a review. Bioresour Technol 89:17–34. logical potential. Thus, T.  kodakarensis—a hyperther- https ://doi.org/10.1016/S0960 -8524(03)00033 -6 mophilic archaeon with an established range of genetic 1 3 598 Extremophiles (2018) 22:591–598 Kalyaanamoorthy S, Minh BQ, Wong TKF et al (2017) ModelFinder: Santangelo TJ, Cubonová L, Reeve JN (2008) Shuttle vector expression fast model selection for accurate phylogenetic estimates. 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A series of new E. coli–Thermococcus shuttle vectors compatible with previously existing vectors

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Life Sciences; Microbiology; Biotechnology; Biochemistry, general; Microbial Ecology
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Abstract

Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replica- tion origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli–T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli–T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression. Keywords Archaea · Hyperthermophiles · Gene cloning and expression · Cloning · Molecular biology · Genetics of extremophiles · Molecular biology of archaea Introduction proteins from thermophiles allow unmatched insight into mechanisms of protein stability (Razvi and Scholtz 2006; Investigating and understanding the ability of cells to sur- Pucci and Rooman 2017). Additionally, enzymes derived vive in hyperthermophilic environments is an increasingly from extremophiles are often functional in conditions where important undertaking in the fields of biochemistry, biotech - mesophilic enzymes would quickly degrade, giving them nology, molecular biology and evolution, to name just a few. enormous potential in biotechnology (Bruins et al. 2001; Biochemists have known the value of thermostable enzymes Haki and Rakshit 2003; Elleuche et al. 2015). In molecular for many decades, particularly in structural studies, where biology, the unusual biology of archaea and their unique genetic systems have opened our eyes to new and intriguing mechanisms of gene regulation, gene transfer, and meta- Communicated by L. Huang. bolic processes (Olsen and Woese 1997; Sato and Atomi 2011; Wagner et al. 2017). Furthermore, the importance of Electronic supplementary material The online version of this thermophiles in evolutionary biology cannot be understated; article (https ://doi.org/10.1007/s0079 2-018-1019-6) contains thermophiles or hyperthermophiles may be at the base of supplementary material, which is available to authorized users. both the bacterial and the archaeal/eukaryal branches of the * Ryan Catchpole tree of life (Boussau et al. 2008; Groussin and Gouy 2011). ryan.catchpole@pasteur.fr As a result, understanding the genetics of such hyperthermo- philes could have far-reaching implications and applications. Unité de Biologie Moléculaire du Gène chez les Unfortunately, the range of genetic tools and techniques Extrêmophiles (BMGE), Département de Microbiologie, Institut Pasteur, 25 Rue du Docteur Roux, 75015 Paris, available is very limited. For example, in the extensively France studied Thermococcales (anaerobic hyperthermophilic Institute for Integrative Biology of the Cell (I2BC), archaea), only a single cloning vector is published (Santan- Microbiology Department, CEA, CNRS, Univ. Paris-Sud, gelo et al. 2008), despite over 175 cultivatable strains having Université Paris-Saclay, Gif-sur-Yvette, France Vol.:(0123456789) 1 3 592 Extremophiles (2018) 22:591–598 been isolated (Lepage et al. 2004; Price et al. 2015) and 26 Lai, University of Canterbury) using primers ‘pTPTK1. complete genomes deposited in the Genbank database. As GA.1’ and ‘pTPTK1.GA.2’ (for primer sequences, see Sup- a result, despite Thermococcus kodakarensis having been plementary Table 2). The HMG-CoA cassette (conferring adopted as a model organism, only a single vector is avail- resistance to mevinolin in T. kodakarensis) was amplified by able for the transformation of, and expression of exogenous PCR from the plasmid pLC70 (kindly gifted by Thomas San- genes in this species. To study the mechanisms of plasmid tangelo, Colorado State University) using primers ‘pTPTK1. maintenance, and to understand the mechanisms of horizon- GA.5’ and ‘pTPTK1.GA.6’. The entire sequence of pTP2 tal gene transfer observed in these extremophilic archaea, it was amplified by PCR from Thermococcus prieurii DNA is important to have genetic tools which allow us to follow using the primers ‘pTPTK1.GA.3’ and ‘pTPTK1.GA.4’. multiple genes, and multiple replicons simultaneously. PCR products were purified, assembled, and used to trans- Hence, we sought to generate a new Escherichia coli–T. form E. coli strain XL1-Blue. Transformants were selected kodakarensis shuttle vector which is compatible with the by growth in the presence of chloramphenicol and confirmed only currently available vector, pLC70. In recent years, our by Sanger sequencing (Beckman Genomics). group has sequenced 43 plasmids from Thermococcales spe- Plasmids pTPTK2 and pTPTK3 were constructed using cies (unpublished data), 29 of which co-exist in the same pTPTK1 as a starting point. Briefly, the pBAD33-pTP2 cells as other plasmids or circular viral genomes, proving backbone of pTPTK1 was amplified by PCR using prim- their compatibility (if plasmid incompatibility exists in ers ‘pTPTK2/3.GA.1’ and ‘pTPTK2/3.GA.2’. For pTPTK2, Thermococcales). This provided a wide selection of poten- the trpE-cassette (conferring tryptophan prototrophy to T. tial origins of replication for use in T. kodakarensis. kodakarensis ΔTK0254 backgrounds) was amplified from We present a series of novel E. coli–T. kodakarensis the plasmid pLC70 using primers ‘pTPTK2.GA.3’ and shuttle vectors based on the small cryptic plasmid pTP2 ‘pTPTK2.GA.4’. For pTPTK3, the gene TK0149 (confer- from T. prieurii, and the E. coli p15A origin of replication. ring agmatine prototrophy to T. kodakarensis ΔTK0149 This plasmid backbone has been developed in combination backgrounds) was amplified from the chromosome of T. with three different markers for selection in T. kodakarensis kodakarensis KOD1 along with its native promoter using strains. Additionally, we show that this plasmid is compat- primers ‘pTPTK3.GA.3’ and ‘pTPTK3.GA.4’. PCR prod- ible with the single published cloning vector for Thermococ- ucts were assembled and sequenced as above. cales, pLC70 (and derivatives thereof). Plasmid pTNAg was constructed by assembling the TK0149 cassette (PCR-amplified from the T.   kodakaren- sis KOD1 chromosome using primers ‘pTNAg.GA.1’ and Materials and methods ‘pTNAg.GA2’) with the ApaI-EcoRV digestion product of pLC70. The resulting plasmid was selected in E. coli by Strains and media growth in the presence of ampicillin and kanamycin and confirmed by Sanger sequencing (Beckman Genomics). Plasmid construction was carried out in Escherichia coli Plasmid pTNTrpE was constructed by blunting and cir- strain XL1-Blue grown at 37 °C in LB medium. Where nec- cularization of the XbaI–EcoRV fragment of pLC70. The essary, media was supplemented with Ampicillin (100 µg/ absence of the HMG-CoA cassette was confirmed by Sanger mL), Kanamycin (40 µg/mL) or Chloramphenicol (20 µg/ sequencing (Beckman Genomics). mL). All archaeal work was carried out in Thermococcus kodakarensis strain TS559 (Santangelo et al. 2010) grown Transformation of T. kodakarensis at 85 °C in either ASW-YT (Sato et al. 2003) or ASW-CH medium with uracil supplementation (10 µg/mL) (Fujikane Transformation was carried out as described previously et  al. 2010). Where necessary, media was supplemented (Sato et al. 2005). Briefly, ~ 5 × 10 late exponential phase with agmatine sulfate (1.0 mM) or mevinolin (10 μM). T. kodakarensis TS559 cells were harvested under anaerobic conditions by centrifugation at 4000×g for 10 min. The cell Plasmid construction pellet was resuspended in 200 µL 0.8×ASW, and 5 µg of plasmid DNA was added. Suspensions were incubated on For a complete list of strains and plasmids used in this study, ice for 60 min, heat shocked at 85 °C for 60 s, then chilled see Supplementary Table 1. on ice for 10 min. 1 mL ASW-YT + agmatine was added, Plasmid pTPTK1 was constructed by Gibson Assembly and the cultures were incubated at 85 °C for 1.5 h. Cells using the NEBuilder HiFi DNA Assembly Master Mix (New were harvested by centrifugation at 4000×g for 3 min and England Biolabs) following the manufacturer’s protocol. used to inoculate 25 mL of selective media. Transformant Briefly, the E. coli p15A origin of replication was amplified cultures were grown at 85 °C for 48 h before being sub- by PCR from the plasmid pBAD33 (kindly gifted by Alicia cultured twice by 1:100 dilution in fresh selective media. 1 3 Extremophiles (2018) 22:591–598 593 Transformation was confirmed by isolation of plasmid DNA 1990), Phyre2 (Kelley et al. 2015) or HHpred (Söding et al. from 20 to 50 mL of culture (plasmid DNA recovered using 2005) search. The top BLAST hits were aligned using Macherey–Nagel NucleoSpin Plasmid kit) and analysis by MSAprobs v0.9.7 (Liu and Schmidt 2014), gaps removed restriction digestion and gel electrophoresis. using BMGE v1.12 (Criscuolo and Gribaldo 2010), Double transformation of T. kodakarensis TS559 was per- amino acid substitution models determined by ModelFinder formed sequentially, i.e. plasmid-containing cultures were (Kalyaanamoorthy et al. 2017), and phylogenetic trees gen- grown in selective media, and transformed with a second erated with IQ-TREE v1.5.4 (Nguyen et al. 2015). Trees plasmid. Following selection and sub-culturing of double were visualized with iTOL v3.6.1 (Letunic and Bork 2016). transformants, serial dilutions of stationary phase cultures were suspended in a solution of molten 0.7% Gelrite con- taining 10 g/L colloidal sulfur and plated as a top layer on Results solid selective media (under anaerobic conditions). Single colonies (observable by local clearing of the colloidal sulfur) Identification of a suitable origin of replication were used to inoculate selective liquid media. As pLC70 encodes the origin of replication from plasmid Liposome‑mediated transformation pTN1 of Thermococcus nautili (Soler et al. 2007; Santan- gelo et al. 2008), the most obvious place to search for a Transformation was performed using a modification of a plasmid with a compatible origin of replication is the other method previously described (Metcalf et al. 1997). Briefly, co-existing plasmids in T. nautili. However, with pTN2 and cultures of Thermococcales species were grown to late-log pTN3 comprising 13 and 18 kb, respectively, they are less phase in ASW-YT medium. Cells were pelleted at 4000×g than ideal candidates for the design of small, easily manipu- for 10 min under anaerobic conditions, and resuspended lated shuttle vectors. in 0.1 vol of 0.85 M sorbitol (de-gassed and reduced with Phylogenetic analysis of the replication-associated pro- Na S). For each transformation, liposomes were generated tein encoded by pTN1 reveals it to be part of a small fam- by adding 15 μL DOTAP to 150 μL 20 mM HEPES, pH ily of homologous genes encoded by other Thermococcales 7.4, with 2 µg DNA. The liposome suspension was added to plasmids (Fig.  1). One of the rep genes identified in this 1 mL resuspended culture, and incubated at room tempera- analysis is that of the small cryptic plasmid pTP1 from T. ture for 1 h. Cells were pelleted and resuspended in 1 mL prieurii (Gorlas et al. 2013a, b). The rep protein of plasmid ASW-YT medium, then allowed to recover for 2 h at 85 °C. pTP1 (RepTP1, YP_007974239) is 31.3% identical to that Cells were again pelleted and used to inoculate 25 mL selec- of pTN1 (Rep74, ABR10429) at the amino acid level. This tive medium. similarity, combined with the close phylogenetic relatedness of these two species (Gorlas et al. 2014) suggested that the Phylogenetic analysis replication-associated proteins are likely homologs belong- ing to the same family of archaeal plasmids. It is notable Sequences for replication-associated proteins of pTN1 that pTP1 is unusual in structure, likely having acquired its (rep74, ABR10429.1) and pTP2 (repTP2, YP_007974244.1) replication protein via recombination with another mobile were used as query sequences for a BLAST (Altschul et al. element (Gorlas et al. 2013b). Thus, while the other ORFs Fig. 1 a Schematic diagrams AB of pTN1-family plasmids. Predicted ORFs are indicated by gray blocks. ORFs encoding predicted replication-associated proteins are black. In the case of T. prieurii, the complete Rep gene interrupts another Rep-like gene colored in gray with black stripes. b Phylogenetic relation- ship between pTN1-family Rep proteins. Unrooted phyloge- netic tree generated with the full-length Rep proteins of the 4 pTN1-family plasmids 1 3 594 Extremophiles (2018) 22:591–598 of pTP1 differ from the usual organization of pTN1-family activity are limited. The intergenic space between ORF3 plasmids, the Rep protein is clearly homologous to that and ORF4 (Fig. 2) was chosen as it contains a 27-bp non- of pTN1. We, therefore, predicted that co-existing plas- coding region, as well as being at the 3′ termini of both mids from T. prieurii may encode replication origins that ORF3 and ORF4, thus decreasing the chance of disrupting could be compatible with pTN1 (and, therefore, pLC70). upstream regulatory sequences such as promoters. Intrigu- T. prieurii contains three circular extra-chromosomal ele- ingly, linearization of pTP2 in this region resulted in a ~ 1.8- ments—the genome of the virus TPV1 (21,592 bp), and two Kbp fragment, as determined by gel electrophoresis (data small cryptic plasmids, pTP1 and pTP2 (3126 and 2038 bp, not shown), rather than the 2038-bp expected from the pub- respectively) (Gorlas et al. 2013b). Due to its small size and lished sequencing data. Sequencing of the fragment revealed compatibility with pTP1, pTP2 was chosen as a potentially that the linearized pTP2 was missing a tandem repeat corre- suitable origin of replication for shuttle vector construction. sponding to nucleotides 710–907 and 913–1110 of sequence NC_021208.1 (Supplementary Figure 1). It has not been Analysis of pTP2‑rep confirmed whether this fragment was lost in the pTP2 isolate used as a template, or during PCR. While pTP2 is the smallest known Thermococcales plasmid, To minimize the risk of recombination between a pTP2- it appears to have a surprisingly complicated gene structure. derived vector and pLC70 while maintained within a sin- Whereas the pTN1-family of Thermococcales plasmids are gle cell, an E. coli vector backbone and antibiotic-resistant quite simple in structure (usually containing only two ORFs, marker were chosen which are different to those used in one of which encodes the replication-associated protein), generating pLC70. Where pLC70 is comprised of a pUC pTP2 encodes several putative transcriptional regulators, origin of replication in combination with ampicillin- and as well as a transmembrane domain protein (Gorlas et al. kanamycin-resistant genes (derived from pCR2.1-TOPO), a 2013b). The function of these proteins in the maintenance p15A origin of replication was chosen for our shuttle vector, and potential transmission of pTP2-family plasmids, or in combination with a chloramphenicol-resistant gene (both indeed secondary functions, remains unknown. The pre- derived from pBAD33). dicted replication-associated protein of pTP2 is also enig- The range of selectable markers available for T. koda- matic. This protein shares 30–35% sequence identity with a karensis is very limited, comprising genes which com- protein of unknown function found in the genomes of mul- plement strain-specific auxotrophies, e.g. tryptophan or tiple species of methanogenic Euryarchaeota. Both struc- agmatine; or a single antibiotic resistance determinant pro- tural and functional prediction (using Phyre2 and HHpred, duced by HMG-CoA reductase overexpression providing respectively) of these methanogen genes, and of RepTP2 itself reveals the central ~ 120 residues of these proteins to be similar to replication-associated proteins of various mobile elements infecting all three domains of life, e.g. SIRV1 from the archaeon Sulfolobus islandicus; pMV158 from the bac- terium Streptococcus agalactiae; and TYLSCV from the eukaryote Solanum lycopersicum (Supplementary Table 3). The similarity of both RepTP2 and other mobile elements to chromosomally-encoded genes of methanogenic Euryar- chaeota may suggest that these genes are part of integrated pTPTK1(mev) 5489bp pTPTK2 (trp) 5455bp horizontally mobile elements in these species. We were pTPTK3 (agm) 4710bp unable to find any synteny of the genes surrounding these Rep-like proteins, indicating that these proteins may have arisen from multiple different integrated elements, or that the similarity in sequence, structure and predicted function is due to a similarity of function, e.g. a native methanogen protein encoding a replication-associated functionality (heli- case, polymerase, resolvase, etc). MCS region: Generation of shuttle vectors Fig. 2 Plasmid maps of pTPTK1, pTPTK2 and pTPTK3. ORFs/ Plasmid pTP2 encodes five predicted ORFs, three of which genetic elements are indicated by white boxes outlined in black with are overlapping. Therefore, the possible sites where the plas- labels indicating the nature of each element. Gray regions inside the mid could be linearized without disrupting potential gene plasmid map indicate the source and nature of each plasmid region 1 3 Extremophiles (2018) 22:591–598 595 resistance to mevinolin. To maximize the usefulness of an Replication in T. kodakarensis E. coli–T. kodakarensis shuttle vector, we generated three different variants of the pTP2-derived vector, each encod- Following construction of the three plasmids in E. coli, they ing a different selectable marker. were used to transform T. kodakarensis strain TS559 (San- All three vectors contain an identical backbone as tangelo et al. 2010). Initial transformations were performed described above, comprising the 1.8-kb pTP2 sequence, in liquid culture, and each of the three plasmids conferred p15A origin, and chloramphenicol-resistant marker the appropriate prototrophy/resistance (mevinolin resistance (Fig. 2). The following marker cassettes were added for for pTPTK1; tryptophan prototrophy for pTPTK2; agmatine selection in T. kodakarensis: pTPTK1 encodes HMG-CoA prototrophy for pTPTK3). Transformant cultures were able reductase under the constitutive glutamate dehydrogenase to form discreet colonies on solid selective media. Plasmid promoter (sourced from pLC70 (Santangelo et al. 2008)), DNA isolated from transformant cultures (both from origi- conferring resistance to mevinolin; pTPTK2 encodes nal transformations following two sub-cultures, and from anthranilate synthase (TK0254 ≡ trpE) under the control cultures inoculated with single colonies) gave a restriction of the promoter from CDP-alcohol phosphatidyltransferase digestion pattern identical to that observed with plasmids (TK2279) (also sourced from pLC70), conferring trypto- isolated from E. coli suggesting these plasmids were faith- phan prototrophy to trpE mutant backgrounds; pTPTK3 fully replicated in T. kodakarensis (Fig.  3). Additionally, encodes pyruvoyl-dependent arginine decarboxylase re-transformation of E. coli using plasmid DNA isolated (TK0149) under its native promoter, conferring agmatine from T. kodakarensis transformant cultures successfully re- prototrophy to TK0149 mutant backgrounds. introduced the plasmid and chloramphenicol marker. A small multiple cloning site (MCS) site was added to the shuttle vector design (Fig. 2) to aid in the cloning Compatibility with pLC70‑derived plasmids of additional markers and/or genes. A limited number of enzyme recognition sequences were available which nei- To assess the compatibility of pTP2-derived plasmids with ther cut the vector backbone, nor the potential selectable pLC70-derived plasmids, it was necessary to select for both markers; therefore, the MCS comprises 40 bp with sites plasmids in a single culture. However, with pLC70 encod- for ApaI, AvrII, StuI*, NotI, SphI, NruI, PciI and SalI* ing both tryptophan prototrophy and mevinolin-resistant (*StuI is not suitable for cloning in pTPTK2 as there is a markers, it was necessary to modify pLC70 to have dif- site within TK0254. Additionally, although still suitable ferent selectable markers on the co-transforming plas- for cloning, a second SalI site is present at the junction mids. Plasmid pTNAg was generated by deletion of the between the vector and TK0254 cassette, resulting in SalI entire TK0254-PF1848 (TrpE − HMG-CoA) cassette from digestion releasing a 24-bp fragment from the vector). pLC70, and replacement with the TK0149 gene encoding pyruvoyl-dependent arginine decarboxylase (for which T. Fig. 3 Digestion and gel electrophoresis of pTP2-derived plasmids. a isolated from T. kodakarensis. b Plasmids digested with HindIII, rec- Plasmids digested with RruI, recognizing a single site on each plas- ognizing multiple sites on each plasmid. Lane 1: pTPTK1 isolated mid. Lane 1: pTPTK1 isolated from E. coli, lane 2: pTPTK3 isolated from E. coli, lane 2: pTPTK3 isolated from E. coli, lane 3: pTPTK2 from E. coli, lane 3: pTPTK2 isolated from E. coli, lane 4: GeneR- isolated from E. coli, lane 4: GeneRuler 1  kb DNA ladder, lane 5: uler 1  kb DNA ladder, lane 5: pTPTK1 isolated from T. kodakaren- pTPTK1 isolated from T. kodakarensis, lane 6: pTPTK3 isolated sis, lane 6: pTPTK3 isolated from T. kodakarensis, lane 7: pTPTK2 from T. kodakarensis, lane 7: pTPTK2 isolated from T. kodakarensis 1 3 596 Extremophiles (2018) 22:591–598 kodakarensis TS599 is a knockout). Plasmid pTNTrpE was and separately in the T. kodakarensis host. To confirm generated by deletion of PF1848 from pLC70, resulting in that T. kodakarensis cultures were indeed double transfor- a plasmid encoding the TrpE marker alone. Both of these mants, rather than a mixed culture of single transformants, plasmids were able to replicate faithfully in T. kodakarensis cultures were plated to single colonies on solid selective (Supplementary Figure 2), conferring agmatine and trypto- medium. Following incubation, single colonies were sus- phan prototrophy, respectively, to strain TS559. pended in 0.8 × ASW, vortexed vigorously, and serial dilu- Double transformants of T. kodakarensis were generated tions plated again on solid selective medium. Recovered by transforming a strain harboring a pLC70-derived plas- colonies grew well in selective medium, and all gave two mid with a pTP2-derived plasmid, or vice versa. Although plasmids upon DNA extraction and electrophoresis or E. the transformation rate was not quantified, transformation coli transformation. was successful regardless of the nature of the incumbent It should be noted that selection for both tryptophan pro- or incoming plasmid. However, for technical reasons, it is totrophy and mevinolin resistance (ASW-CH + mevinolin) most simple to carry out the double transformation such resulted in slow-growing cultures, likely due to an increased that the incumbent plasmid is one which can be selected in sensitivity of cells to mevinolin in the less-rich ASW-CH rich media, i.e. conferring agmatine prototrophy (pTPTK3 medium over the rich ASW-YT medium. or pTNAg) or mevinolin resistance (pTPTK1 or pLC70), thus ensuring a cell-dense, exponential phase culture can be Host‑range of pTP2‑derived vectors readily produced for the second transformation. Transformation with two plasmids conferred upon cul- To assess the ability of pTP2-derived vectors to replicate in tures the appropriate prototrophies and/or resistance. DNA other Thermococcales species, we attempted to use pTPTK1 extracted from double-transformant cultures indeed gave to transform T. aggregans, T. pacificus, T. siculi, T. celer, T. two plasmid bands upon electrophoresis, and plasmids guaymasensis, T. fumicolans and T. prieurii. As no method produced the appropriate digestion patterns (Fig. 4). Plas- of transformation has been established for these species, we mid extractions were able to transform E. coli to chloram- attempted transformation using the method described for T. phenicol resistance and ampicillin/kanamycin resistance. kodakarensis. This method relies on the natural competence Furthermore, dilutions of plasmid extractions resulted in of T. kodakarensis, and so it is perhaps unsurprising that transformation of E. coli to chloramphenicol resistance we were unable to obtain transformants of these untested while maintaining ampicillin sensitivity, and vice versa, species (only T. kodakarensis gave transformants with this indicating that the two plasmids were replicating faithfully protocol). We then attempted transformation using DNA Fig. 4 Digestion and gel electrophoresis of plasmids from double ble transformant. b Plasmids digested with RruI, recognizing a single transformants. a Plasmids digested with RruI, recognizing a single site on each plasmid. Lane 1: pTNAg isolated from E. coli, lane 2: site on each plasmid. Lane 1: pTNTrpE isolated from E. coli, lane pTPTK2 isolated from E. coli, lane 3: GeneRuler 1  kb DNA ladder, 2: pTPTK1 isolated from E. coli, lane 3: GeneRuler 1  kb DNA lad- lane 4: pTNAg + pTPTK2 isolated from T. kodakarensis double trans- der, lane 4: pTNTrpE + pTPTK1 isolated from T. kodakarensis dou- formant 1 3 Extremophiles (2018) 22:591–598 597 encapsulated in liposomes, as previously described for other tools—represents an important model species. The ability Euryarchaeota such as Methanosarcina (Metcalf et al. 1997) to transform T. kodakarensis with two different and sta- and Methanococcus voltae (Sniezko et al. 1998), as well as ble extra-chromosomal replicons will open up new fields hyperthermophilic bacteria of the genus Thermotoga (Yu of study in these important organisms, e.g. plasmid parti- et al. 2001). Unfortunately, this method did not yield trans- tioning/segregation and the related plasmid compatibility/ formants in any Thermococcus species tested, even T. koda- incompatibility, DNA-binding proteins together with their karensis. Transformation was also attempted of the naturally substrates, alpha-complementation of enzymes, etc. competent Pyrococcus furiosus COM1 strain; however, no Acknowledgements This work was funded by the European Research plasmid-containing transformants were obtained. Council under the European Union’s Seventh Framework Program (FP/2007-2013)/Project EVOMOBIL - ERC Grant Agreement no. Discussion Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco We have established that the cryptic plasmid pTP2 from mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- Thermococcus prieurii encodes an origin of replication tion, and reproduction in any medium, provided you give appropriate which is functional in T. kodakarensis. We have used this credit to the original author(s) and the source, provide a link to the origin of replication to generate a series of E. coli–T. kodaka- Creative Commons license, and indicate if changes were made. rensis shuttle vectors. These vectors replicate stably (under selection) in both T. kodakarensis and E. coli. 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ExtremophilesSpringer Journals

Published: Mar 1, 2018

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