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Janet Westpheling - One of the best experts on this subject based on the ideXlab platform.
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overcoming restriction as a barrier to DNA Transformation in caldicellulosiruptor species results in efficient marker replacement
Biotechnology for Biofuels, 2013Co-Authors: Joel Farkas, Daehwan Chung, Janet WestphelingAbstract:Background: Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass. Results: Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA Transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus. Conclusion: Deletion of the cbeI gene removes a substantial barrier to routine DNA Transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of restriction-modification activities in members of this genus suggests a way forward to eliminating restriction as a barrier to DNA Transformation and efficient genetic manipulation of this important group of hyperthermophiles.
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Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii
Journal of Industrial Microbiology & Biotechnology, 2013Co-Authors: Joel Farkas, Daehwan Chung, Minseok Cha, Jennifer Copeland, Philip Grayeski, Janet WestphelingAbstract:Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii , the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD_680) and more efficient DNA Transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii . A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii . Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii , and may be applied broadly across the Caldicellulosiruptor genus.
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methylation by a unique α class n4 cytosine methyltransferase is required for DNA Transformation of caldicellulosiruptor bescii dsm6725
PLOS ONE, 2012Co-Authors: Joel Farkas, Daehwan Chung, Janet Westpheling, Jennifer R Huddleston, Estefania OlivarAbstract:Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA Transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to Transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for Transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA Transformation and the combined frequencies of Transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.
Rainer Haas - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of helicobacter pylori genes essential for gastric colonization
Journal of Experimental Medicine, 2003Co-Authors: Holger Kavermann, Brendan P Burns, Katrin Angermuller, Stefan Odenbreit, Wolfgang Fischer, Klaus Melchers, Rainer HaasAbstract:Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA Transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural Transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.
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topology and membrane interaction of helicobacter pylori comb proteins involved in natural Transformation competence
International Journal of Medical Microbiology, 2003Co-Authors: Dirk Hofreuter, Arno Karnholz, Rainer HaasAbstract:Abstract The human gastric pathogen Helicobacter pylori is naturally competent for genetic Transformation. The H. pylori comB gene cluster encodes the VirB4-homologous ATPase ComB4 and the structural proteins ComB7 – ComB10, which share significant sequence identity to the Agrobacterium tumefaciens virB -encoded type IV secretion system. To study the topology of the ComB7 – 10 proteins, we applied Tn Max transposon mutagenesis by generating fusions of ComB proteins with mature β-lactamase (BlaM) or alkaline phosphatase (PhoA). Our data show that the putative lipoprotein ComB7 is secreted and is found membrane-attached, probably by its lipid anchor. According to our topology mapping ComB8 is a bitopic membrane protein with a short N-terminal portion in the cytoplasm and the remainder of the protein expanding into the periplasmic space. ComB9 was verified as a periplasmic protein, tightly attached to the membrane. The N-terminus of ComB10 is anchored in the cytoplasmic membrane and the major portion of the protein, including a putative coiled-coil domain, is located in the periplasm. Limited protease digestion and protein extraction under different salt and pH conditions confirmed the periplasmic localization and the tight membrane association of the ComB protein complex. A hypothetical model of the ComB DNA Transformation pore in H. pylori is presented.
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natural Transformation competence in helicobacter pylori is mediated by the basic components of a type iv secretion system
Molecular Microbiology, 2001Co-Authors: Dirk Hofreuter, Stefan Odenbreit, Rainer HaasAbstract:Dirk Hofreuter, Stefan Odenbreit and Rainer Haas*Max vonPettenkofer Institut fu¨rHygiene und MedizinischeMikrobiologie, Pettenkoferstr. 9a, D-80336 Mu¨nchen,Germany.SummaryHelicobacter pylori (Hp), a Gram-negative bacterialpathogen and aetiologic agent of gastroduodenaldisease in humans, is naturally competent for geneticTransformation. Natural competence in bacteria isusually correlated with the presence of type IV pili ortype IV pilin-like proteins, which are absent in Hp.Instead,werecentlyidentifiedthecomBoperoninHp,carrying four genes tentatively designated as orf2,comB1, comB2 and comB3. We show here that allComB proteins and the 37-amino-acid Orf2 peptidedisplay significant primary sequence and structuralhomology/identity to the basic components of a typeIV secretion apparatus. ComB1, ComB2 and ComB3,now renamed ComB8, ComB9 and ComB10, corre-spond to the Agrobacterium tumefaciens VirB8, VirB9and VirB10 proteins respectively. The peptide Orf2carries a lipoprotein motif and a second cysteineresidue homologous to VirB7, and was thus desig-nated ComB7. The putative ATPase ComB4, encodedby the open reading frame hp0017 of strain 26695,corresponds to virB4 of the A. tumefaciens type IVsecretion system. A Hp comB4 transposon insertionmutantwastotallydefectiveinnaturalTransformation.By complementation of a Hp DcomB deletion mutant,we demonstrate thateach of the proteins fromComB8to ComB10 is absolutely essential for the develop-ment of natural Transformation competence. Theputative lipoprotein ComB7 is not essential, butapparently stabilizes the apparatus and modulatesthe Transformation efficiency. Thus, pathogenic type IHp strains contain two functional independent type IVtransport systems, one for protein translocationencoded by the cag pathogenicity island and one foruptake of DNA by natural Transformation. The lattersystem indicates a possible novel mechanism fornatural DNA Transformation in bacteria.IntroductionHelicobacter pylori (Hp) is one of the most successfulbacterial pathogens, infecting about 50% of the worldpopulation. The persistent chronic infection causes type Bgastritis and, in a certain percentage of infected persons,peptic ulceration. More severe, mucosa-associated lym-phoid tissue (MALT) lymphoma and gastric adenocar-cinoma are also associated with the infection. On thegenetic level, Hp is one of the most diverse bacterialspecies known so far, and this is probably the resultof a high degree of horizontal gene transfer and freerecombination within the species (Suerbaum et al., 1998).This genetic diversity is postulated to result from theadaptation of Hp to its individual host during years andeven decades of infection. Two major types of strainsexist: type I strains, which express a functionalvacuolating cytotoxin (VacA) and carry the cag patho-genicity island (cag-PAI); and type II strains, which lackboth characteristics (Xiang et al., 1995).Hp displays natural competence for genetic transform-ation in vitro (Nedenskov-Sorensen et al., 1990), a featurethat might be the basis for horizontal gene transfer andthe subsequent generation of a high degree of geneticdiversity. To understand the mechanism of naturalTransformation in this species, we screened systematicallyfor the genes involved. Transposon mutagenesis with themini-TnblaM transposon TnMax9 (Hofreuter et al., 1998)yieldedamutationinthecomBlocus,whichconsistsofthegenes comB1 to comB3 preceded by a short open readingframe (ORF), designated orf2. In addition to the comBoperon,theORFHP0333wasidentified(Andoetal.,1999;Smeets et al., 2000a), a homologue of the Haemophilusinfluenzae dprA gene (Karudapuram and Barcak, 1997).By the generation of a corresponding knock-out mutantstrain (Ando et al., 1999; Smeets ., 2000a), hp0333was also shown to be necessary for Transformation.Smeets et al. (2000b) identified a novel competence gene,comH, by screening a Hp mutant library. The ComHprotein carries a putative signal sequence and has noknown orthologues in other bacteria.
Daehwan Chung - One of the best experts on this subject based on the ideXlab platform.
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overcoming restriction as a barrier to DNA Transformation in caldicellulosiruptor species results in efficient marker replacement
Biotechnology for Biofuels, 2013Co-Authors: Joel Farkas, Daehwan Chung, Janet WestphelingAbstract:Background: Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass. Results: Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA Transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus. Conclusion: Deletion of the cbeI gene removes a substantial barrier to routine DNA Transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of restriction-modification activities in members of this genus suggests a way forward to eliminating restriction as a barrier to DNA Transformation and efficient genetic manipulation of this important group of hyperthermophiles.
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Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii
Journal of Industrial Microbiology & Biotechnology, 2013Co-Authors: Joel Farkas, Daehwan Chung, Minseok Cha, Jennifer Copeland, Philip Grayeski, Janet WestphelingAbstract:Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii , the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD_680) and more efficient DNA Transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii . A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii . Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii , and may be applied broadly across the Caldicellulosiruptor genus.
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methylation by a unique α class n4 cytosine methyltransferase is required for DNA Transformation of caldicellulosiruptor bescii dsm6725
PLOS ONE, 2012Co-Authors: Joel Farkas, Daehwan Chung, Janet Westpheling, Jennifer R Huddleston, Estefania OlivarAbstract:Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA Transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to Transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for Transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA Transformation and the combined frequencies of Transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.
Steven H Seifert - One of the best experts on this subject based on the ideXlab platform.
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DNA uptake sequence mediated enhancement of Transformation in neisseria gonorrhoeae is strain dependent
Journal of Bacteriology, 2010Co-Authors: Paul M Duffin, Steven H SeifertAbstract:Natural Transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing the nonpalindromic 10- or 12-nucleotide sequence 5′-ATGCCGTCTGAA-3′ (additional semiconserved nucleotides are underlined), termed the DNA uptake sequence (DUS10 or DUS12). Here we investigated the effects of the DUS on Transformation and DNA uptake for several laboratory strains of N. gonorrhoeae. We found that all strains showed efficient Transformation of DUS containing DNA (DUS10 and DUS12) but that the level of Transformation with DNA lacking a DUS (DUS0) was variable in different strains. The DUS-enhanced Transformation was 20-fold in two strains, FA1090 and FA19, but was approximately 150-fold in strains MS11 and 1291. All strains tested provide some level of DUS0 Transformation, and DUS0 Transformation was type IV pilus dependent. Competition with plasmid DNA revealed that Transformation of MS11 was enhanced by the addition of excess plasmid DNA containing a DUS while FA1090 Transformation was competitively inhibited. Although FA1090 was able to mediate much more efficient Transformation of DNA lacking a DUS than was MS11, DNA uptake experiments showed similar levels of uptake of DNA containing and lacking a DUS in FA1090 and MS11. Finally, DNA uptake was competitively inhibited in both FA1090 and MS11. Taken together, our data indicate that the role of the DUS during DNA Transformation is variable between strains of N. gonorrhoeae and may influence multiple steps during Transformation.
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differential roles of homologous recombination pathways in neisseria gonorrhoeae pilin antigenic variation DNA Transformation and DNA repair
Molecular Microbiology, 1998Co-Authors: Ian J Mehr, Steven H SeifertAbstract:Neisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA-dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high-frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA Transformation. This suggests that a RecF-like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA Transformation and DNA repair but not in pilin antigenic variation.
Joel Farkas - One of the best experts on this subject based on the ideXlab platform.
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overcoming restriction as a barrier to DNA Transformation in caldicellulosiruptor species results in efficient marker replacement
Biotechnology for Biofuels, 2013Co-Authors: Joel Farkas, Daehwan Chung, Janet WestphelingAbstract:Background: Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass. Results: Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA Transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus. Conclusion: Deletion of the cbeI gene removes a substantial barrier to routine DNA Transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of restriction-modification activities in members of this genus suggests a way forward to eliminating restriction as a barrier to DNA Transformation and efficient genetic manipulation of this important group of hyperthermophiles.
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Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii
Journal of Industrial Microbiology & Biotechnology, 2013Co-Authors: Joel Farkas, Daehwan Chung, Minseok Cha, Jennifer Copeland, Philip Grayeski, Janet WestphelingAbstract:Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii , the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD_680) and more efficient DNA Transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii . A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii . Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii , and may be applied broadly across the Caldicellulosiruptor genus.
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methylation by a unique α class n4 cytosine methyltransferase is required for DNA Transformation of caldicellulosiruptor bescii dsm6725
PLOS ONE, 2012Co-Authors: Joel Farkas, Daehwan Chung, Janet Westpheling, Jennifer R Huddleston, Estefania OlivarAbstract:Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA Transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to Transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for Transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA Transformation and the combined frequencies of Transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.