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Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus.

Chung D, Cha M, Farkas J, Westpheling J - PLoS ONE (2013)

Bottom Line: There was no evidence of DNA rearrangement during transformation and replication in C. bescii.A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications.Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. The entire plasmid was cloned into an E. coli cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in E. coli. The wild-type C. bescii pyrF locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105), and the resulting vector was transformed into a new spontaneous deletion mutant in the pyrFA locus of C. bescii that allowed complementation with the pyrF gene alone. Plasmid DNA was methylated in vitro with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in C. bescii. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

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Plasmid map of pDCW129 and verification of its ability to structurally stable maintenance of inserted DNA fragment through transformation and replication in C.bescii.(A) Diagram of pDCW129. A linear DNA fragment containing the CBM3 and linker region derived from celA (Cbes1867) was inserted into pDCW89 shuttle vector. The cross-hatched box corresponds to a 0.68 kb of inserted DNA fragment. All features in pDCW129 are indicated at figure legend in Fig. 2A. The primers and restriction site (EcoRV) used for the construction and verification are indicated. (B) Gel showing the 2.2 kb DNA fragment containing the pyrF cassette and inserted DNA fragment, amplified by using primers DC233 and DC235. Lane 1, total DNA isolated from JWCB005; lane 2, total DNA isolated from C. bescii transformant with pDCW129; lane 3, pCW129 isolated from E. coli. (C) EcoRV restriction digestion analysis of plasmid DNA before and after transformation of C. bescii and back-transformation to E. coli. Lane 1, pDCW129 plasmid DNA isolated from E. coli DH5α; lane 2, 3 and 4, plasmid DNA isolated from three biologically independent E. coli DH5α back-transformed from C. bescii transformants. M: 1 kb DNA ladder (NEB).
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pone-0062881-g004: Plasmid map of pDCW129 and verification of its ability to structurally stable maintenance of inserted DNA fragment through transformation and replication in C.bescii.(A) Diagram of pDCW129. A linear DNA fragment containing the CBM3 and linker region derived from celA (Cbes1867) was inserted into pDCW89 shuttle vector. The cross-hatched box corresponds to a 0.68 kb of inserted DNA fragment. All features in pDCW129 are indicated at figure legend in Fig. 2A. The primers and restriction site (EcoRV) used for the construction and verification are indicated. (B) Gel showing the 2.2 kb DNA fragment containing the pyrF cassette and inserted DNA fragment, amplified by using primers DC233 and DC235. Lane 1, total DNA isolated from JWCB005; lane 2, total DNA isolated from C. bescii transformant with pDCW129; lane 3, pCW129 isolated from E. coli. (C) EcoRV restriction digestion analysis of plasmid DNA before and after transformation of C. bescii and back-transformation to E. coli. Lane 1, pDCW129 plasmid DNA isolated from E. coli DH5α; lane 2, 3 and 4, plasmid DNA isolated from three biologically independent E. coli DH5α back-transformed from C. bescii transformants. M: 1 kb DNA ladder (NEB).

Mentions: To test the use of pDCW89 as a cloning vector, a 0.68 kb DNA fragment containing a carbohydrate binding domain (CBM) and linker region derived from celA (Cbes1867) was cloned into pDCW89 (Fig. 4, pDCW129). Methylated pDCW129 was successfully transformed into JWCB005 at comparable transformation efficiency to pDCW89. Transformation of C. bescii with pDCW129 was initially confirmed by PCR amplification of the region spanning the pyrF cassette and only in the plasmid (Fig. 4B). Total DNA isolated from JWCB018 transformants was used to “back-transform” into E. coli and plasmid DNA isolated from these back-transformants was analyzed by restriction digestion by EcoRV (Fig. 4C) and EcoRI and AatII (data not shown). pDCW129 DNA isolated from the “back transformants” was indistinguishable from the pDCW129 used to transform C. bescii and showed no obvious signs of rearrangement or deletion through transformation and replication in JWCB005.


Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus.

Chung D, Cha M, Farkas J, Westpheling J - PLoS ONE (2013)

Plasmid map of pDCW129 and verification of its ability to structurally stable maintenance of inserted DNA fragment through transformation and replication in C.bescii.(A) Diagram of pDCW129. A linear DNA fragment containing the CBM3 and linker region derived from celA (Cbes1867) was inserted into pDCW89 shuttle vector. The cross-hatched box corresponds to a 0.68 kb of inserted DNA fragment. All features in pDCW129 are indicated at figure legend in Fig. 2A. The primers and restriction site (EcoRV) used for the construction and verification are indicated. (B) Gel showing the 2.2 kb DNA fragment containing the pyrF cassette and inserted DNA fragment, amplified by using primers DC233 and DC235. Lane 1, total DNA isolated from JWCB005; lane 2, total DNA isolated from C. bescii transformant with pDCW129; lane 3, pCW129 isolated from E. coli. (C) EcoRV restriction digestion analysis of plasmid DNA before and after transformation of C. bescii and back-transformation to E. coli. Lane 1, pDCW129 plasmid DNA isolated from E. coli DH5α; lane 2, 3 and 4, plasmid DNA isolated from three biologically independent E. coli DH5α back-transformed from C. bescii transformants. M: 1 kb DNA ladder (NEB).
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Related In: Results  -  Collection

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pone-0062881-g004: Plasmid map of pDCW129 and verification of its ability to structurally stable maintenance of inserted DNA fragment through transformation and replication in C.bescii.(A) Diagram of pDCW129. A linear DNA fragment containing the CBM3 and linker region derived from celA (Cbes1867) was inserted into pDCW89 shuttle vector. The cross-hatched box corresponds to a 0.68 kb of inserted DNA fragment. All features in pDCW129 are indicated at figure legend in Fig. 2A. The primers and restriction site (EcoRV) used for the construction and verification are indicated. (B) Gel showing the 2.2 kb DNA fragment containing the pyrF cassette and inserted DNA fragment, amplified by using primers DC233 and DC235. Lane 1, total DNA isolated from JWCB005; lane 2, total DNA isolated from C. bescii transformant with pDCW129; lane 3, pCW129 isolated from E. coli. (C) EcoRV restriction digestion analysis of plasmid DNA before and after transformation of C. bescii and back-transformation to E. coli. Lane 1, pDCW129 plasmid DNA isolated from E. coli DH5α; lane 2, 3 and 4, plasmid DNA isolated from three biologically independent E. coli DH5α back-transformed from C. bescii transformants. M: 1 kb DNA ladder (NEB).
Mentions: To test the use of pDCW89 as a cloning vector, a 0.68 kb DNA fragment containing a carbohydrate binding domain (CBM) and linker region derived from celA (Cbes1867) was cloned into pDCW89 (Fig. 4, pDCW129). Methylated pDCW129 was successfully transformed into JWCB005 at comparable transformation efficiency to pDCW89. Transformation of C. bescii with pDCW129 was initially confirmed by PCR amplification of the region spanning the pyrF cassette and only in the plasmid (Fig. 4B). Total DNA isolated from JWCB018 transformants was used to “back-transform” into E. coli and plasmid DNA isolated from these back-transformants was analyzed by restriction digestion by EcoRV (Fig. 4C) and EcoRI and AatII (data not shown). pDCW129 DNA isolated from the “back transformants” was indistinguishable from the pDCW129 used to transform C. bescii and showed no obvious signs of rearrangement or deletion through transformation and replication in JWCB005.

Bottom Line: There was no evidence of DNA rearrangement during transformation and replication in C. bescii.A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications.Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. The entire plasmid was cloned into an E. coli cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in E. coli. The wild-type C. bescii pyrF locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105), and the resulting vector was transformed into a new spontaneous deletion mutant in the pyrFA locus of C. bescii that allowed complementation with the pyrF gene alone. Plasmid DNA was methylated in vitro with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in C. bescii. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

Show MeSH
Related in: MedlinePlus