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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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Chromosomal map and PCR analysis of the Uridine Monophosphate (UMP) biosynthetic gene cluster in C.bescii DSM 6725 and the spontaneous deletion in pyrFA (JWCB005) locus.(A) A diagram of the pyr operon region with the 878 bp deletion in the pyrFA ORFs. The line below the diagram indicates the length of the deletion. Bent arrows depict primers used for verification of the structure of the chromosome in the JWCB005 (ΔpyrFA) strain. pyrF and pyrE loci indicated as black color filled arrow and black dashed filled arrow, respectively. (B) Gel depicting PCR products of the pyrFA region in wild type (3.44 kb) compared to the ΔpyrFA (2.52 kb) strain amplified by primers (JH020 and FJ298). (C) Gel depicting the 2.66 kb PCR products of pyrE region in wild type and the ΔpyrFA strain by primers (DC326 and DC331). M: 1 kb DNA ladder (NEB).
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pone-0062881-g001: Chromosomal map and PCR analysis of the Uridine Monophosphate (UMP) biosynthetic gene cluster in C.bescii DSM 6725 and the spontaneous deletion in pyrFA (JWCB005) locus.(A) A diagram of the pyr operon region with the 878 bp deletion in the pyrFA ORFs. The line below the diagram indicates the length of the deletion. Bent arrows depict primers used for verification of the structure of the chromosome in the JWCB005 (ΔpyrFA) strain. pyrF and pyrE loci indicated as black color filled arrow and black dashed filled arrow, respectively. (B) Gel depicting PCR products of the pyrFA region in wild type (3.44 kb) compared to the ΔpyrFA (2.52 kb) strain amplified by primers (JH020 and FJ298). (C) Gel depicting the 2.66 kb PCR products of pyrE region in wild type and the ΔpyrFA strain by primers (DC326 and DC331). M: 1 kb DNA ladder (NEB).

Mentions: Here we report the isolation and characterization of new uracil auxotrophic mutant of C. bescii, JWCB005, that contains a deletion in the pyrFA locus (Fig. 1A, Table 1) and allows complementation by the pyrF gene alone. We constructed a replicating shuttle vector based on the smaller of two native C. bescii plasmids, pBAS2 [17], [26], that contains the wild type pyrF allele for uracil prototrophic selection. This plasmid is capable of stable replication and selection in both E. coli and C. bescii. Plasmid DNA was unchanged during transformation and replication in C. bescii and back transformation into E. coli. Transformation with replicating plasmid DNA is an order of magnitude more efficient than transformation with non-replicating plasmids in C. bescii[19], making this vector an important tool to screen transformability of members of this genus. Using similar methods as for C. bescii, we were able to use this plasmid to transform C. hydrothermalis. In addition we demonstrated the utility of this vector as a cloning vector by cloning a portion of the celA gene from C. bescii.


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)

Chromosomal map and PCR analysis of the Uridine Monophosphate (UMP) biosynthetic gene cluster in C.bescii DSM 6725 and the spontaneous deletion in pyrFA (JWCB005) locus.(A) A diagram of the pyr operon region with the 878 bp deletion in the pyrFA ORFs. The line below the diagram indicates the length of the deletion. Bent arrows depict primers used for verification of the structure of the chromosome in the JWCB005 (ΔpyrFA) strain. pyrF and pyrE loci indicated as black color filled arrow and black dashed filled arrow, respectively. (B) Gel depicting PCR products of the pyrFA region in wild type (3.44 kb) compared to the ΔpyrFA (2.52 kb) strain amplified by primers (JH020 and FJ298). (C) Gel depicting the 2.66 kb PCR products of pyrE region in wild type and the ΔpyrFA strain by primers (DC326 and DC331). M: 1 kb DNA ladder (NEB).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643907&req=5

pone-0062881-g001: Chromosomal map and PCR analysis of the Uridine Monophosphate (UMP) biosynthetic gene cluster in C.bescii DSM 6725 and the spontaneous deletion in pyrFA (JWCB005) locus.(A) A diagram of the pyr operon region with the 878 bp deletion in the pyrFA ORFs. The line below the diagram indicates the length of the deletion. Bent arrows depict primers used for verification of the structure of the chromosome in the JWCB005 (ΔpyrFA) strain. pyrF and pyrE loci indicated as black color filled arrow and black dashed filled arrow, respectively. (B) Gel depicting PCR products of the pyrFA region in wild type (3.44 kb) compared to the ΔpyrFA (2.52 kb) strain amplified by primers (JH020 and FJ298). (C) Gel depicting the 2.66 kb PCR products of pyrE region in wild type and the ΔpyrFA strain by primers (DC326 and DC331). M: 1 kb DNA ladder (NEB).
Mentions: Here we report the isolation and characterization of new uracil auxotrophic mutant of C. bescii, JWCB005, that contains a deletion in the pyrFA locus (Fig. 1A, Table 1) and allows complementation by the pyrF gene alone. We constructed a replicating shuttle vector based on the smaller of two native C. bescii plasmids, pBAS2 [17], [26], that contains the wild type pyrF allele for uracil prototrophic selection. This plasmid is capable of stable replication and selection in both E. coli and C. bescii. Plasmid DNA was unchanged during transformation and replication in C. bescii and back transformation into E. coli. Transformation with replicating plasmid DNA is an order of magnitude more efficient than transformation with non-replicating plasmids in C. bescii[19], making this vector an important tool to screen transformability of members of this genus. Using similar methods as for C. bescii, we were able to use this plasmid to transform C. hydrothermalis. In addition we demonstrated the utility of this vector as a cloning vector by cloning a portion of the celA gene from C. bescii.

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