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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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Comparison of DNA modification status between shuttle vector DNA isolated from E.coli (Lane 1) and C. hydrothermalis transformants (Lane 2) by Restriction analysis.(A) Undigested, (B) Digested with HindIII (4.3 and 3.4 kb cleavage products); (C) Digested with EcoRI (4.6 and 1.9 kb cleavage products); (D) Digested with CbeI (11 cleavage products are expected). M: 1 kb DNA ladder (NEB).
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pone-0062881-g003: Comparison of DNA modification status between shuttle vector DNA isolated from E.coli (Lane 1) and C. hydrothermalis transformants (Lane 2) by Restriction analysis.(A) Undigested, (B) Digested with HindIII (4.3 and 3.4 kb cleavage products); (C) Digested with EcoRI (4.6 and 1.9 kb cleavage products); (D) Digested with CbeI (11 cleavage products are expected). M: 1 kb DNA ladder (NEB).

Mentions: Restriction of transforming DNA is a major and, for C. bescii, apparently an absolute barrier to transformation of DNA from E. coli[19], [30]. Transformation of plasmid DNA from E. coli into C. bescii requires in vitro methylation with an endogenous α-class N4-Cytosine methyltransferase, M.CbeI [19]. To test whether modification by M.CbeI also allowed transformation of other members of this genus, a spontaneous mutation resistant to 5-FOA was isolated in C. hydrothermalis[35], JWCH003 (Table 1). This mutant was a tight uracil auxotroph and was used as a host for plasmid transformation. Unmethylated plasmid DNA isolated from various E. coli hosts failed to transform this mutant but DNA that has been methylated with M.CbeI transformed at a frequency similar to that for C. bescii (typically about 500 transformants per µg of plasmid DNA). Transformants were initially confirmed by PCR amplification of the aac gene contained exclusively on the plasmid (data not shown). As shown in Fig. 3, restriction digestion analysis using HindIII and EcoRI of shuttle vector plasmid DNA isolated from C. hydrothermalis transformants was indistinguishable from that isolated from E. coli (Fig. 3B and 3C) suggesting that is structurally stable in C. hydrothermalis. This further suggests that modification with M.CbeI may have utility in DNA transformation of a variety of Caldicellulosiruptor species and current efforts to isolate deletions of the pyrF locus in a number of other species to test this are in progress. These data also provide evidence that the use of the wild type C. bescii pyrF allele under the control of the ribosomal protein S30EA (Cbes2105) promoter functions in at least one other species and will likely prove to be a useful selection marker for many species. We anticipate that this shuttle vector will facilitate extension of genetic methods to a number of other Caldicellulosiruptor species.


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)

Comparison of DNA modification status between shuttle vector DNA isolated from E.coli (Lane 1) and C. hydrothermalis transformants (Lane 2) by Restriction analysis.(A) Undigested, (B) Digested with HindIII (4.3 and 3.4 kb cleavage products); (C) Digested with EcoRI (4.6 and 1.9 kb cleavage products); (D) Digested with CbeI (11 cleavage products are expected). M: 1 kb DNA ladder (NEB).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062881-g003: Comparison of DNA modification status between shuttle vector DNA isolated from E.coli (Lane 1) and C. hydrothermalis transformants (Lane 2) by Restriction analysis.(A) Undigested, (B) Digested with HindIII (4.3 and 3.4 kb cleavage products); (C) Digested with EcoRI (4.6 and 1.9 kb cleavage products); (D) Digested with CbeI (11 cleavage products are expected). M: 1 kb DNA ladder (NEB).
Mentions: Restriction of transforming DNA is a major and, for C. bescii, apparently an absolute barrier to transformation of DNA from E. coli[19], [30]. Transformation of plasmid DNA from E. coli into C. bescii requires in vitro methylation with an endogenous α-class N4-Cytosine methyltransferase, M.CbeI [19]. To test whether modification by M.CbeI also allowed transformation of other members of this genus, a spontaneous mutation resistant to 5-FOA was isolated in C. hydrothermalis[35], JWCH003 (Table 1). This mutant was a tight uracil auxotroph and was used as a host for plasmid transformation. Unmethylated plasmid DNA isolated from various E. coli hosts failed to transform this mutant but DNA that has been methylated with M.CbeI transformed at a frequency similar to that for C. bescii (typically about 500 transformants per µg of plasmid DNA). Transformants were initially confirmed by PCR amplification of the aac gene contained exclusively on the plasmid (data not shown). As shown in Fig. 3, restriction digestion analysis using HindIII and EcoRI of shuttle vector plasmid DNA isolated from C. hydrothermalis transformants was indistinguishable from that isolated from E. coli (Fig. 3B and 3C) suggesting that is structurally stable in C. hydrothermalis. This further suggests that modification with M.CbeI may have utility in DNA transformation of a variety of Caldicellulosiruptor species and current efforts to isolate deletions of the pyrF locus in a number of other species to test this are in progress. These data also provide evidence that the use of the wild type C. bescii pyrF allele under the control of the ribosomal protein S30EA (Cbes2105) promoter functions in at least one other species and will likely prove to be a useful selection marker for many species. We anticipate that this shuttle vector will facilitate extension of genetic methods to a number of other Caldicellulosiruptor species.

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