WO2013184089A1 - Restriction/modification polypeptides, polynucleotides, and methods - Google Patents

Restriction/modification polypeptides, polynucleotides, and methods Download PDF

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WO2013184089A1
WO2013184089A1 PCT/US2012/040700 US2012040700W WO2013184089A1 WO 2013184089 A1 WO2013184089 A1 WO 2013184089A1 US 2012040700 W US2012040700 W US 2012040700W WO 2013184089 A1 WO2013184089 A1 WO 2013184089A1
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cbel
dna
haelll
bescii
restriction
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PCT/US2012/040700
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French (fr)
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Janet Westpheling
Daehwan Chung
Jennifer Huddleston
Joel A. Farkas
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University Of Georgia Research Foundation, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria

Definitions

  • Caldicellulosiruptor bescii DSM 6725 (formerly Anaerocellum thermophilum, Yang et al. 2009, Int J Syst Evol Microbiol 60:2011-2015) grows at temperatures up to about 90°C and is the most thermophilic cellulolytic bacterium known.
  • This obligate anaerobe is capable of degrading lignocellulosic biomass including hardwood (e.g., poplar) and grasses with both low lignin (e.g., napier grass and bermuda grass) and high lignin (e.g., switchgrass) content without chemical pretreatment (Yang et al., 2009 Appl Environ Microbiol 75:4762-4769).
  • hardwood e.g., poplar
  • grasses with both low lignin e.g., napier grass and bermuda grass
  • high lignin e.g., switchgrass
  • CBP consolidated bioprocessing
  • the invention provides an isolated polynucleotide comprising the coding of Cbes 2438.
  • the invention can provide a vector that includes such a polynucleotide operably linked to a promoter.
  • the invention can provide a cell that includes such a polynucleotide and/or such a vector.
  • the invention provides an isolated polynucleotide comprising the coding region of Cbes 2437.
  • the invention can provide a vector that includes such a polynucleotide operably linked to a promoter.
  • the invention can provide a cell that includes such a polynucleotide and/or such a vector.
  • the invention provides an isolated polypeptide comprising an amino acid sequence encoded by the coding region of Cbes 2438.
  • the invention provides an isolated polypeptide comprising an amino acid sequence encoded by the coding region of Cbes 2437.
  • the invention provides a method that generally includes incubating a DNA molecule comprising at least one 5'-GGCC-3' sequence with a Cbel polypeptide under conditions effective for the Cbel polypeptide to digest the DNA at the 5'-GG/CC-3' sequence.
  • the invention provides a method that generally includes treating a DNA molecule comprising at least one 5'-GGCC-3' sequence with a M.Cbel polypeptide under condition effective for the M.Cbel polypeptide to methylate at least one C residue of the 5'- GGCC-3' sequence.
  • the invention provides a method that generally includes introducing a polynucleotide into a microbial cell that comprises a thermophile or a hyperthermophile.
  • the method can include treating the DNA with a M.Cbel polypeptide under condition effective for the M.Cbel polypeptide to methylate at least one C residue of the DNA.
  • FIG. 1 Detection of a Haelll-like restriction/modification system in C. bescii.
  • A pDCW68
  • B pATHEOl
  • C pATHE02
  • D pDCW68 isolated from E. coli DH5a incubated with CFE from C. bescii
  • E pATHEOl
  • pATHE02 isolated from C. bescii incubated with CFE from C. bescii
  • F pDCW68 isolated from E. coli treated with M.Haelll and incubated with CFE from C. bescii.
  • Features originating from E. coli are shaded in black and from C.
  • bescii are white; Apr R , apramycin resistant gene cassette; Trp, Cbes 2105-trytophan synthetase a subunit; OriT, origin of transfer for conjugation; pSClOl, low copy replication origin in E. coli; Haelll restriction sites are indicated. All incubation times were as indicated.
  • FIG. 1 Cloning, expression and purification of Cbel.
  • A The region surrounding the location of Cbel in the C. bescii genome.
  • B pDCW72; KanR, kanamycin resistance gene; bacteriophage T7 promoter and T7 terminator; lacl, the gene for the lactose repressor protein; ColEI, origin of replication derived from pBR322
  • C Lane 1 : protein molecular weight standards; lane 2: 15 ng of purified Cbel protein displayed on a 10-20% Tris-HCl gradient gel (CRITERION Precast Gel, Bio-Rad Laboratories; Hercules, CA).
  • FIG. 3 Temperature profile of purified Cbel endonuclease activity.
  • A The 2.558 kb DNA substrate synthesized as described in the Materials and Methods. Haelll cleavage sites are marked by vertical lines and predicted cleavage fragment sizes are indicated below the line.
  • B pDCW72 used to his-tagged expression of Cbel. The substrate was incubated for 10 minutes with 5 ng of protein at the temperatures indicated and the cleavage products were separated on 1.2% agarose gel. The position of the full length undigested fragment and the three major cleavage products derived by digestion with Cbel are indicated by arrows.
  • Figure 4 Phylogram alignment of 46 Haelll-like restriction enzymes.
  • the host organism for each restriction enzyme is indicated as well as the protein name, when available. Otherwise, the GenBank locus tag or accession number is given.
  • the distance scale is indicated by a bar defining the distance for 0.1 amino acid substitution per site.
  • the bracketed organisms represent those containing this new subfamily of Haelll-like enzymes that includes Cbel.
  • FIG. 4 Amino acid identity is shown as shaded areas with the position of the motif within the protein sequence. Motif start site is indicated. Motif 1 : 7.0e-220, motif 2 1.9e-278, motif 3 1.6e- 185. Subgroup sequences shown are Cbel (SEQ ID NO:36); Bhall (SEQ ID NO:37); Haelll (SEQ ID NO:38); Hac_1214 (SEQ ID NO:39); Cthe_2319 (SEQ ID NO:40); HPSH 02550 (SEQ ID NO:41); HMPREF0105 0967 (SEQ ID NO:42); Smon_0161 (SEQ ID NO:43); PRU 0937 (SEQ ID NO:44); HMPREF0573 11018 (SEQ ID NO:45); CUY 2194 (SEQ ID NO:46); Bgr_19490 (SEQ ID NO:47); and GOS 4010239 (SEQ ID NO:48).
  • Caldicellulosiruptor species Total DNA isolated from 7 different species were incubated (-) without or (+) with commercially available Haelll endonuclease at 37°C for 1 hour according to the manufacturer's instructions (NEB).
  • NEB manufacturer's instructions
  • Plasmid DNA (pUC18) was isolated from E. coli and incubated in vitro with either M.Haelll methyltransferase (NEB) or M.Cbel methyltransferase. After digestion with either Haelll or Cbel (as indicated) fragments were displayed on a 1.2% TAE-agarose gel stained with ethidium bromide. Lanes 1) un-methylated pUC18 DNA; 2) pUC18 treated with M.Haelll; 3) pUC18 treated with M.Cbel are as indicated in each panel; Panel (A) no restriction enzyme added (B) with Haelll for 30 minutes at 37°C or (C) with Cbel for 30 minutes at 75°C. MW: 1 kb DNA ladder (NEB).
  • FIG. 8 Expression, purification, and characterization of M.Cbel.
  • A Physical map of surrounding region of M.Cbel in the C. bescii genome.
  • B Schematic diagram of pDCW73; KanR, kanamycin resistance gene; bacteriophage T7 promoter and T7 terminator; lacl, the gene for the lactose repressor protein; ColEI, origin of replication derived from pBR322
  • C origin of replication derived from pBR322
  • Lane 1 Undigestged unmethylated pDCW 70; Lane 2: Undigested M.Cbel methylated pDCW 70; Lane 3: Digested with purified Cbel of unmethylated pDCW 70; Lane 4: Digested with purified Cbel of M.Cbel methylated pDCW 70; M: 1 kb DNA ladder (NEB).
  • E E
  • pDCW 70 having 0.892 kb region of pyrF and 0.662 kb region of pyrB for homologous recombination. Marker replacement by homologous recombination can occur in the chromosome in the pyrBCF region. Engineered Kpnl site is indicated, and bent arrows depict primers used for verification of transformation.
  • B Electrphoration performance of ApyrBCF strain with unmethylated and M.Cbel methylated pDCW 70.
  • Top plate (Defined + Uracil plates), competent cell after electro pulsing; Middle plate (w/o Uracil plate), transformed with unmethylated pDCW 70; Bottom plate (w/o Uracil plate), transformed with M.Cbel methylated pDCW 70.
  • FIG. 10 Linear order of the three functional groups of M.Cbel. Sequence alignment of three members of Caldicellulosiruptor species and DmtB from Anabaena variabilis, which contain a M.Cbel homologue. Sequences are shown for C. bescii DSM 6725 (SEQ ID NO:51); C kristjanssonii 177R1B (SEQ ID NO:52); C hydrothermalis 108 (SEQ ID NO:53); and Anabaena variabilis (SEQ ID NO:54).
  • the present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii.
  • the discovered restriction enzyme is a Haelll-like restriction enzyme that possesses a thermophilic activity profile.
  • the restriction/modification system also includes a methyltransferase, M.Cbel, that methylates at least one inner cytosine residue in the Cbel recognition sequence (5'-GGCC-3') to m 4 C.
  • the invention provides, in various aspects, isolated Cbel or M.Cbel polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the Cbel or M.Cbel polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a Cbel polypeptide; methods of treating a DNA molecule using a M.Cbel polypeptide; and methods of transforming a Caldicellulosiruptor cell. Despite prior attempts to directly transform members of the Caldicellulosiruptor genus, this is the first report of success.
  • the ability to genetically manipulate these organisms can assist in metabolically engineering members of this genus for, for example, their use in consolidated bioprocessing that produces one or more biofuels and/or one or more bioproducts.
  • certain aspects of the invention can be used to overcome restriction that may assist methods of DNA transformation of Caldicellulosiruptor species using DNA from, for example, homologous and/or heterologous sources.
  • these aspects may be generalized to permit transformation of other thermophilic and/or hyperthermophilic microbes.
  • Caldicellulosiruptor bescii was formerly classified as Anaerocellum thermophilum.
  • the genome of C. bescii was originally annotated when the organism was known as A. thermophilum.
  • the annotations were modified upon reclassification of the organism to replace Athe annotations with Cbes annotations, reflecting the reclassification of the organism. Neither the substantive content of the annotation nor the numerical portion of the annotations changed.
  • the original annotation Athe 2438 is now referred to as Cbes 2438. Nevertheless, Athe annotations and Cbes annotations may be used interchangeably.
  • pATHEOl is now known as pBAL.
  • pATHE02 is now known as pBAS2.
  • Cbel refers to a polypeptide encoded by at least a portion of the coding region of Cbes 2438 and that cleaves DNA at a 5'-GG/CC-3'.
  • Cbel can refer to a 38 kDa polypeptide encoded by a 981 bp coding sequence of Cbes 2438, or a biologically active fragment of such a polypeptide.
  • Biological activity in the context of Cbel, refers to the ability to digest DNA specifically at a 5'- GG/CC-3' recognition site at a temperature from 35°C to 85°C.
  • M.Cbel refers to a polypeptide encoded by at least a portion of the coding region of Cbes 2437 and that, when incubated with DNA at a temperature from 35°C to 85°C methylates a cytosine in the 5'-GG/CC-3' recognition site of Cbel.
  • Methods and “methyltransferase” are synonymous as used herein and may be used interchangeably.
  • the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
  • a potent Haelll-like DNA restriction activity was detected in cell- free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from E. coli as substrate.
  • bescii genome sequence revealed the presence of both a Haelll-like restriction endonuclease (Cbes 2438) and DNA methyltransferase (Cbes 2437).
  • Bes 2438 Haelll-like restriction endonuclease
  • Cbes 2437 DNA methyltransferase
  • restriction/modification activity is widespread in this genus.
  • a phylogenetic analysis based on sequence alignment and conserved motif searches identified features of Cbel distinct from other members of this group and classified Cbel as a member of a novel subfamily of Haelll-like enzymes.
  • the methods described herein also may be used more generally to introduce polynucleotides - whether heterologous or homologous - into such species in order to, for example, achieve overexpression of the polynucleotide, overproduction of at least one polypeptide encoded by the introduced polynucleotide, and an increase in the cellular level of activity of the encoded polypeptide.
  • the methods described herein may be used generally to introduce polynucleotides into other thermophilic species such as, for example, certain Clostridium spp. and/or
  • hyperthermophilic species such as, for example, Thermo anaerobacter spp.
  • thermophilic or hyperthermophilic species - is a prerequisite for their use in consolidated bioprocessing.
  • the activity of host restriction enzymes is a major barrier to the introduction of DNA into cells. Identifying and overcoming restriction systems has allowed the development of genetic systems in previously non-transformable bacteria. Often this is accomplished by in vitro methylation or by in vivo methylation systems in, for example, E. coli.
  • thermophile Bacillus methanolicus plasmids are engineered with fewer Bmel recognition sites and prepared in a dam + E.
  • plasmid DNA is methylated by cell-free extracts to achieve transformation by electroporation (Accetto et al., 2005 FEMS
  • Clostridium perfringens type B transformation can occur only when the transforming plasmid DNA is isolated from a dam + dcm + strain of E. coli (Chen et al., 1996 FEMS Microbiol Lett 140: 185-191).
  • Clostridium cellulolyticum can be transformed only if the plasmid DNA is protected from Ccel cleavage using either in vitro or in vivo methylation (Jennert et al, 2000 Microbiology 146(Ptl2):3071-3080).
  • Haelll-like enzymes are a diverse group of proteins with distinct catalytic domains that have in common the ability to cleave the same DNA sequence.
  • the prototype of this group, Haelll was first identified in Haemophilus aegyptius in 1972 (Middleton et al, 1972 J Virol 10:42-50).
  • This enzyme recognizes 5 * -GGCC-3 * and cleaves the DNA between the second G (in the second position) and first C (in the third position) leaving a blunt end.
  • Haelll-like restriction activity is widespread in bacteria and archaea (Roberts et al., 2010 Nucleic Acids Res 38:D234-D236) allowing efficient restriction of foreign DNA.
  • Four- base cutters like Haelll can present a challenge for DNA transfer since the expected frequency of the cleavage sites sequence is greater than the expected frequency of longer recognition sequences.
  • the enzyme identified from C. bescii was named Cbel and its cleavage activity, temperature profile, and thermostability are described.
  • Caldicellulosiruptor Bioinformatic analysis of Cbel and other Haelll isoschizomers reveals a previously unidentified subfamily of this group of restriction endonucleases. The work described herein advances the study of the nature of restriction-modification systems and advances efforts to establish genetic methods for this important group of organisms.
  • a cell-free extract from C. bescii was prepared (Jennert et al., 2000 Microbiology 146(Ptl2):3071-3080) and incubated with pDCW68 DNA (FigurelA), a vector constructed for use in transformation experiments and that had been isolated from E. coli (DH5a dam + dcm + ).
  • the plasmid DNA when incubated with the C. bescii cell-free extract, was completely digested within 10 minutes and had a similar restriction cleavage profile to that of a digest with commercially available Haelll (Figure ID). DNA of the two native C.
  • Organisms that produce restriction enzymes often have a bias against the presence of the recognition sequences of those enzymes in their genomes (Nobusato et al, 2000 Gene 259:89-98; Rocha et al, 2001 Genome Res 11 :946-958).
  • This Haelll-like restriction endonuclease from C. bescii, Cbel. Cloning, expression, and purification of Cbel from Caldicellulosiruptor bescii.
  • a DNA substrate containing three Haelll restriction sites (Figure 3A), which should generate fragments of 1393 bp, 772 bp, 293 bp, and 106 bp when digested, was used in restriction digestion assays.
  • Purified Cbel protein (12-48 ng) was incubated with 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol at pH 7.9, and 100 ng of DNA substrate in 10 ⁇ ( Figure 3 A), and incubated for 10-20 minutes at 75°C.
  • the gene was not toxic to cells grown at 23°C but it was at 37°C, suggesting that the enzyme was less active at low temperature.
  • purified protein was incubated with the DNA substrate at temperatures ranging from 23°C -100°C.
  • the enzyme is optimally active between 75°C and 85°C, exhibiting partial digestion activity at 45°C or below and non-specific activity above 85°C. No activity was detected at 23°C.
  • the optimum temperature for commercially available Haelll activity is 37°C and the enzyme is inactivated by treatment at 80°C for 20 minutes.
  • Cbel is optimally active at 75°C - 85°C and is stable for more than 30 minutes at 75°C (Figure 3B). Cbel did not lose activity after storage for more than one week stored at up to 35°C and was heat-inactivated by incubation at 100°C for 5 minutes.
  • Cbel is an isoschizomer of Haelll.
  • Haelll recognizes a sequence that includes GGCC and cleaves between the G and C leaving a blunt ended fragment.
  • a DNA fragment containing three Haelll recognition/cleavage sites ( Figure 3 A) was used as substrate with purified Cbel enzyme.
  • the cleavage products were either ligated directly to pWSK29 that had been digested with EcoRV leaving a blunt end, or first treated with the Klenow fragment of DNA polymerase prior to ligation.
  • the sequence of the region containing the cloning site revealed the dinucleotide 5'-CC-3' adjacent to the EcoRV site and the dinucleotide 5'-GG-3' adjacent to the EcoRV site, identifying 5'-GG/CC-3' as the Cbel cleavage site.
  • the same result was obtained in eight independent cloning experiments, indicating that Cbel is an isoschizomer of Haelll.
  • Cluster Analysis of Haelll-like proteins reveals that Cbel is a member of a new subfamily of Haelll-like enzymes.
  • Cbel from C. bescii DSM 6725 is an isoschizomer of Haelll (cuts at the same site) which is a Type II restriction enzyme.
  • Type II restriction enzymes and their cognate methyltransferase genes are often adjacent to each other in the chromosome.
  • the locations of the coding region for Cbel (Cbes 2438) and the coding region for the cognate methyltransferase (Cbes 2437) are indicated in FIG. 2A.
  • M.Cbel The coding region of M.Cbel was cloned and expressed shown in FIG. 8. Plasmid DNA (pUC18) was isolated from E. coli and incubated in vitro with either M.Haelll methyltransferase (NEB) or M.Cbel methyltransferase. FIG. 7 shows the result of those digestions. M.Cbel protected pUC18 DNA from digestion by Haelll for 30 minutes at 37°C (middle gel, lane 3) and from digestion by Cbel for 30 Minutes at 75°C (right panel, lane 3).
  • NEB M.Haelll methyltransferase
  • Cbes 2437 had been annotated in the GenBank database as a "D12 class N6 adenine- specific DNA methyltransferase," which would indicate that it is not a methyltransferase that would protect DNA from cleavage. Since Cbel cleaves the sequence 5'-GG/CC-3', a cytosine- specific methyltransferase would be necessary in order to protect the chromosomal DNA from Cbel restriction endonuclease activity.
  • Cbes 2437 prior to our characterization of the expression product of M.Cbel, the expression product of Cbes 2437, Cbes 2437 was incorrectly identified to lead one away from the possibility that it could encode a cognate methyltransferase that would protect chromosomal DNA from digestion by Cbel.
  • Caldicellulosiruptor species may contain the same Haelll-like Cbel/M.Cbel restriction-modification system - i.e., homologues of Cbel and M.Cbel, suggesting that this may be a wide spread system in Caldicellulosiruptor species.
  • M.Cbel is a novel methyltransferase only present in Caldicellulosiruptor species (in C. hydrothermalis the gene locus tag is Calhy 0409 and in C kristjanssonii 177R1B the gene locus tag is Calkr 2088). M.Cbel is also distinctly different both in DNA sequence and protein sequence from M.Haelll and its isoschizomers, the models for 5'-GGCC-3' methylation.
  • methyltransferases can modify DNA in one of three ways to either methylate adenine to N 6 -methyladenine (m 6 A), cytosine to N 4 - methylcytosine (m 4 C), or cytosine to 5-methylcytosine (m 5 C).
  • m 6 A methylate adenine to N 6 -methyladenine
  • m 4 C cytosine to N 4 - methylcytosine
  • m 5 C cytosine to 5-methylcytosine
  • the Cbel cognate methyltransferase (Cbes 2437) does not methylate cytosine to m 5 C as M.Haelll does, but instead methylates cytosine to the more heat-stable m 4 C.
  • M.Cbel is the first example of an a-class (F G G - TRD - DPPY) N 4 -methylcytosine methyltransferase specific for 'GGCC. This may explain why commercially available 'GGCC methyltransferase (M.Haelll) treatment was not successful in protecting the DNA for
  • bescii genome identified a gene encoding a protein homologous to the Haelll endonuclease and an adjacent gene encoding a Type II DNA methyltransferase.
  • the gene for the endonuclease was cloned and expressed in E. coli with a His-tag.
  • Purified enzyme from E. coli was optimally active between 55°C and 85°C and was stable at 35°C for more than a week.
  • the cleavage site of the enzyme was determined to be GG/CC suggesting that it is an isoschizomer of Haelll and we have named this enzyme Cbel.
  • a phylogram of Cbel with other Haelll-like enzymes identified a new subfamily of these enzymes with unique features.
  • thermophilic organisms in E. coli grown at 37°C, but it was expressed efficiently at 23°C. This strategy may be useful for expressing toxic genes derived from thermophilic organisms in E. coli, eliminating the need for complicated highly-regulated expression systems and without the corresponding methyltransferase.
  • thermoformicicum THF T opt 55°C
  • NspLKI Zabaznaya et al, 1999 Biochemistry (Mosc) 64: 189-193) &om Nocardia species LK (T op , 50°C), and Sual (Prangishvili et al, 1985 FEBS Lett 192:57-60) from Sulfolobus acidocaldarius (T opt 82°C).
  • a fourth, Phol from Pyrococcus horikoshii T opt 98°C
  • is commercially available New England Biolabs; Ipswich, MA) but there are no reports on this enzyme in the literature.
  • Cbel Unlike Haelll itself, which is optimally active at 37°C and is inactivated by heating to 80°C, Cbel was optimally active in the range 75°C-85°C and required incubation at 100°C for 5 minutes for inactivation.
  • Cbel isolated from E. coli is thermostable suggests that this feature is due to its conformation, hydrophobic, electrostatic, or other properties rather than by association with other proteins or co factors in C. bescii.
  • Haelll-like enzymes are widespread in both the archaea and bacteria. Genes encoding NgoPII from Neisseria gonorrhoeae, a bacterium, and MthTI from Methanobacterium thermoformicicum, an archaeon, have unexpectedly high similarity (54.5% nucleotide identity) suggesting horizontal gene transfer (Nolling and de Vos, 1992 J Bacteriol 174:5719-5726).
  • a phylogenetic tree based on protein sequence similarity of Haelll-like proteins identified a subgroup that includes four proteins from archaea ⁇ Pyrococcus horikoshii OT3, Sulfolobus islandicus, Sulfolobus acidocaldarius, and Methanothermobacter
  • Haelll-like enzymes are also widespread in both archeael and bacterial thermophiles, such as Clostridium thermocellum ATCC 27405, Methanothermobacter thermautotrophicum THF (Nolling and de Vos, 1992 J Bacteriol 174:5719-5726), Nocardia species LK (Zabaznaya et al, 1999 Biochemistry (Mosc) 64: 189-193), Roseiflexus castenholzii DSM 13941, Sulfolobus islandicus, Sulfolobus acidocaldarius DSM 639 (Prangishvili et al., 1985 FEBS Lett 192:57-60), Pyrococcus horikoshii OT3, and Thermodesulfovibrio yellowstonii DSM 11347.
  • Clostridium thermocellum ATCC 27405 Methanothermobacter thermautotrophicum THF (Nolling and de Vos, 1992 J Bacteriol 174:5719-
  • Clostridium thermocellum (Cthe_2319). Examination of genomic DNA isolated from seven different Caldicellulosiruptor species showed that four of the seven were resistant to Haelll cleavage indicating that Haelll-like restriction-modification systems may be widespread in members of this genus ( Figure 6).
  • Type II restriction endonucleases like Cbel can be a barrier to DNA transformation of several bacterial strains. Thus, successful transformation of such bacterial strains can involve overcoming restriction by the hosts. Approaches include engineering the transforming DNA to contain fewer restriction sites (Gallagher et al, 2008 J Bacteriol. 190(23):7830-7; Cue et al, 1997 Appl Environ Microbiol. 63(4): 1406-20; Purdy et al, 2002 Mol Microbiol. 46(2):439-52), in vitro methylation by purified methyltransferases (Jennert et al., 2000 Microbiology
  • Methylation to m 4 C may be more common than methylation to m 5 C, perhaps because m 5 C may be more readily deaminated to thymine by heat.
  • restriction can be a barrier to transformation of Caldicellulosiruptor by DNA from E. coli and that methylation of a novel aclass Type II cytosine methyltransferase can overcome this barrier. While the apparent transformation frequency may be low, the combined frequencies of transformation and recombination allow maker replacement of chromosomal genes with non-replicating vectors providing an essential tool to generate deletions, gene substitutions, His-tags for protein purification and expression of heterologous proteins to identify genes important for biomass utilization as well as extend substrate utilization and biomass conversion in these organisms.
  • thermostable kanamycin resistance gene previously used for selection of transformants in Thermoanaerobacteria species at 60°C to select transformants in C bescii was complicated by the fact that C bescii, which that grows optimally at 75°C, grows very poorly at or below 70°C. In fact, growth at 60°C increased the spontaneous mutation frequency significantly, from 10 "7 to 10 "5 , making the detection of transformants over this background of spontaneous drug resistance problematic.
  • hph hygromycin phosphotransferase
  • C. bescii cells were plated on 5- fluoroorotic acid (5-FOA).
  • OMP decarboxylase encoded by the pyrF gene in bacteria (ura3 in yeast), converts the pyrimidine analog 5-fluoroorotic acid (5-FOA) to 5-fluorouridine
  • the extent of the deletion was defined by PCR amplification of the pyrBCF region in the mutant ( Figure 9C) and subsequent sequencing of the PCR product. Since mutations in pyrE also lead to uracil auxotrophy and 5FOA resistance, the region around the pyrE locus was amplified from this strain and sequenced to ensure that it was wild type. While the deletion would be expected to affect only the pyrBCF genes, qPCR analysis was performed to monitor expression of the pyrA gene as well as the Cbesl374 open reading frame predicted to encode a uracil xanthine permease. Expression of pyrA and Cbesl374 in the deletion mutant was
  • the ApyrBCF strain was a tight uracil auxotroph and because it contained a deletion, reversion to uracil prototrophy was not a concern making prototrophic selection possible no matter how low the frequency of transformation. Growth of this mutant supplemented with uracil (20 ⁇ ) was indistinguishable from that of the wild type, reaching a cell density of 1.5 x 10 8 in 20 hours.
  • a non-replicating plasmid was constructed with the wild type copy of the pyrBCF locus but containing an engineered restriction site within the cassette to distinguish it from the chromosomal wild type allele. This plasmid was used to transform the pyrBCF deletion strain selecting marker replacement events that repaired the deletion (strategy diagrammed in Figure 9A).
  • Cbel a potent restriction endonuclease in C. bescii, recognizes and cleaves the same sequence as Haelll, unmethylated DNA at the sequence 5'-GG/CC-3'.
  • Plasmid DNA treated with purified M.Haelll in vitro was partially protected from cleavage by both Haelll and Cbel in vitro ( Figure 7), but no transformants were obtained when this DNA was used in electroporation experiments or added to cells that had been subjected to a procedure to induce natural competence in Mycobacterium and Thermoanaerobaterium species.
  • M.Cbel is a novel a-class N4-Cytosine methyltransferase
  • the region of the chromosome that contains Cbel also contains an open reading frame, Cbes 2437, predicted to encode an adenine specific methyltransferase.
  • This open reading frame was cloned into an E. coli expression vector, pDCW73 ( Figure 8B) that placed a His-tag at the carboxy terminus of the protein allowing purification on a Ni-NTA column.
  • E. coli cells containing this plasmid were viable at 23°C but not 37°C suggesting that expression of M.Cbel was toxic to growing cells. Expression of this methyltransferase was, therefore, performed at 23°C to avoid problems related to toxicity and in E.
  • M.Cbel BL21- CodonPlus(DE3)-RIPL to alleviate problems arising from the significant differences in codon usage between M.Cbel and E. coli proteins.
  • Purified M.Cbel from E. coli was the size predicted from the open reading frame, 33 kDa (Fig 8C). No cleavage of DNA was detected by purified Cbel at 75°C when DNA from E. coli was methylated in vitro by the purified methyltransferase (Fig 8D) and we named this enzyme M.Cbel.
  • To determine the optimal temperature for M.Cbel methyltransferase activity we performed the in vitro methylation reactions with purified M.Cbel at temperatures ranging from 25°C to 100°C and tested the modified DNA for restriction by Cbel. Reactions performed between 65°C and 85°C, the growth temperature range of C. bescii, resulted in the best protection against cleavage by Cbel.
  • pUC18 DNA was methylated in vitro by either M.Cbel or M.Haelll and direct sequencing of the DNA revealed that DNA methylated with M.Cbel showed a higher degree of incorporation of dideoxyguanosine in the 5'-GGCC-3' recognition sequence than DNA methylated with
  • M.Haelll N4-methylcytosine results in an increase in the complementary G (GGCC) signal and this signature (Figure 8E) indicates that M.Cbel methylated DNA contains N4-methylcytosine (m 4 C). M.Haelll methylates the C5 position of cytosine (m 5 C).
  • Amplification of this region in the transformant generated a wild type size product. Digestion with Kpnl resulted in no cleavage of the product generated from the wild type or the ApyrBCF mutant.
  • restriction of DNA from E. coli by host bacteria is often an issue. Restriction/modification of DNA, first recognized as a mechanism of protection against phage infection, varies in effectiveness depending on the activity of restriction endonuclease and the methylation state of the DNA substrate. Methylation of DNA may either facilitate or limit the activity of
  • Transformation of DNA from E. coli to Caldicellulosiruptor bescii is apparently especially sensitive to restriction/modification and here we show that the use of a novel endogenous methyltransferase provided specific modification of DNA from E. coli that allowed efficient transformation.
  • M.Cbel was annotated as a D12 class N6 adenine-specific DNA methyltransferase in GenBank, but our analysis clearly shows that it functions as a cytosine specific
  • methyltransferase Like all known metyltransferases it contains a conserved F G G amino acid motif that facilitates interaction with S-adenosylmethionine, the source of the methyl group in these reactions. M.Cbel also contains a DPPY motif typical of N6-adenine metyltransferases, all of which contain a (D/N)PP(Y/F) motif (Malone et al, 1995 J Mol Biol 253(4):618-32).
  • M.Cbel SPP(Y/F) motif is the hallmark of N4 cytosine metyltransferases active site (Klimasauskas et al., 1989 Nucleic Acids Res 17(23):9823-32), making M.Cbel unusual in that it contains a DPPY motif in the active site (Fig.10). Furthermore, the M.Cbel protein has no reported significant sequence or structural similarity to any characterized N4 cytosine methyltransferase. M.Cbel possesses some similarity to DmtB from Anabaena variabilis ATCC 29413 ( Figure 10), which has been shown to have m4C methyltransferase activity specific to the inner cytosine in the 5'- GGCC-3' recognition sequence.
  • proteins which show 57% amino acid identity, represent a new a-class methyltransferase specific for GGCC sequence, different from the previously characterized ⁇ -class of N4 metyltransferases in hyperthermophiles, M.Sual and M.PhoI, isolated from the archaea Sulfolobus acidocaldarius and Pyrococcus horikoshii OT3, respectively.
  • M.Cbel is the first characterized a-class m4C methyltransferase from a hyperthermophile.
  • Calhy 0409 (88% of protein sequence identity) from C. hydrothermalis 108
  • Calkr 2088 85% of protein sequence identity from C. kristjanssonii 177R1B.
  • M.Haelll-modified DNA (m 5 C) was cleaved at reasonable efficiency by purified Sual, a GGCC specific restriction enzyme completely blocked by m4C methylation at the inner cytosine residue in high concentrations.
  • M.Haelll is also known to have a significant level of promiscuous methylation activity at non-canonical sites and may actually increase restriction activity in vector
  • Efforts to optimize the transformation procedure for C. bescii have included adding cell wall weakening agents (isoniacin or glycine) during cell growth, altering temperature during the preparation of electro-competent cells, changing the composition of the washing and
  • electroporation buffers altering incubation times and temperatures of the cells with DNA prior to electric-pulse, varying the electrical settings during the electric pulse, and altering the composition of the recovery medium and incubation period before plating onto selective medium.
  • the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • the mineral solution contained the following (per liter): NH 4 C1, 0.25 g; KH 2 P0 4 , 0.33 g; KC1, 0.33 g; MgCl 2 -6H 2 0, 0.33 g; CaCl 2 -2H 2 0, 0.33 g; yeast extract, 0.5 g; casein hydrolysate (enzymatic; US
  • the vitamin solution contained the following (per liter): biotin, 10 mg; folic acid, 10 mg; pyridoxine-HCl, 500 mg; thiamine-HCl, 25 mg; riboflavin, 25 mg; nicotinic acid, 25 mg; calcium pantothenate, 25 mg; vitamin Bi 2 , 500 mg; /?-aminobenzoic acid, 25 mg; lipoic acid, 25 mg.
  • the trace element solution contained the following (per liter): HC1 (25%: 7.7M), 1.0 ml; FeCl 3 -4H 2 0, 2 g; ZnCl 2 , 50 mg; MnCl 2 -4H 2 0, 50 mg; H 3 B0 3 , 50 mg; CoCl 2 -6H 2 0, 50 mg; CuCl 2 -2H 2 0, 30 mg; NiCl 2 -6H 2 0, 50 mg; Na 4 EDTA (tetrasodium salt), 50 mg; (NH 4 ) 2 Mo0 4 , 50 mg; A1K(S0 4 ) 2 - 12H 2 0, 50 mg.
  • the amino acid solution contained the following (per liter): L-alanine, 1.9 g; L-arginine, 3.1 g; L-asparagine, 2.5 g; L-aspartic acid, 1.2; L-glutamic acid, 5.0 g; L-glutamine, 1.2 g; glycine, 5.0 g; L-histidine, 2.5 g; L-isoleucine, 2.5 g; L-leucine, 2.5 g; L-lysine, 2.5 g; L-methionine, 1.9 g; L-phenylalanine, 1.9 g; L-proline, 3.1 g; L-serine, 1.9 g; L-threonine, 2.5 g; L-tryptophan, 1.9 g; L-tyrosine, 0.3 g; L-valine, 1.3 g.
  • the medium was prepared anaerobically under an argon atmosphere, NaHC0 3 (2 g/1) was added, and the mixture was reduced using 3 g/1 cysteine and 1 g/1 Na 2 S. The final pH was 6.4.
  • the medium was filtered sterilized using a 0.22 ⁇ m-pore-size sterile filter (Millipore Filter Corp., Bedford, MA). Cultures were incubated anaerobically overnight at the optimal temperature for each: C. saccharolyticus DSM 8903, 70°C; C. hydrothermalis DSM 18901 , 65°C; C. kristjanssonii DSM 12137, 78°C; C. bescii DSM 6925, 78°C; C.
  • bescii were isolated using the method described by O' Sullivan and Klaenhammer (O'Sullivan and Klaenhammer, 1993 Appl Environ Microbiol 59:2730-2733) with the following modifications: 200 ml of mid-log phase C. bescii cultures were harvested by centrifugation at 3500 x g for 15 minutes and suspended in Lysis buffer (containing 25% sucrose and 25 mg/ml lysozyme to enhance the cell wall degradation).
  • Lysis buffer containing 25% sucrose and 25 mg/ml lysozyme to enhance the cell wall degradation.
  • pDCW68 DNA was isolated from E. coli using a Qiagen Mini-prep Kit. Plasmid constructions.
  • pJHW006 was constructed from pSET152 (GenBank: AJ414670.1) to replace the ColEI origin of replication with the pSClOl origin.
  • the pSClOl origin was amplified from pWSK29 (GenBank: AF016889.1) using primers JH012 with an Xbal site and primer JH013 with a Kpnl site.
  • the pSClOl containing fragment and the pSET vector digested with Xbal and Kpnl were ligated to form pJHW006.
  • Construction of pDCW68 designed for transformation of C. bescii, required three cloning steps as well as overlapping PCR reactions.
  • PCR amplifications were performed using Pfu Turbo DNA polymerase (Agilent Technologies; Santa Clara, CA Technologies).
  • a 3.936 kb PCR product containing the pSClOl replication origin, the apramycin resistance gene and the oriT (origin of transfer) was amplified from pJHW006 using primers DC 176 and DC 165 which contains a BamHI site.
  • a 3.121 kb PCR product containing the pyrBCF region of the C. bescii genome was amplified from chromosomal DNA using primers DC 188 and DC 156 which also contained a BamHI site. The two PCR products were digested with BamHI and ligated to generate a 7.057 kb product.
  • a 0.205-kb PCR product containing the regulatory region of a ribosomal protein (Cbes 2105) was amplified using primers DC 175 containing an Nhel site, and DC 187 using chromosomal DNA as template.
  • a 7.066-kb fragment was amplified from the 7.057 kb product using primers DC 188 and DC 176 and ligated to the 0.205-kb fragment that had been digested by Nhel generating a 7.262-kb product.
  • a 2.067-kb PCR fragment containing the 3' flanking region of the tryptophan synthase, alpha subunit (Cbes 1690) was amplified from chromosomal DNA using primers JF283 and JF287 and joined to a 2.045-kb kb PCR product containing the 5' flanking region of tryptophan synthase, alpha subunit (Cbes 1690) amplified from chromosomal DNA using DC 182 which contained an Aatll site and an overlapping primer JF286 using the high fidelity Pfu DNA polymerase (Agilent Technologies; Santa Clara, CA Technologies).
  • a 4.112- kb product was then generated by overlapping PCR using the two fragments and C.
  • bescii genomic DNA as a template.
  • the 7.262-kb product from the second cloning step was amplified by PCR using DC 180 which contains an Aatll and DC 100.
  • the 7.262-kb product and the overlapping product were digested with Aatll and ligated to yield pDCW68 (11.368 kb).
  • pDCW72 the 0.981 kb Cbel (Cbes 2438) open reading frame was amplified by PCR using primers DC216 and DC217 using C. bescii genomic DNA as template.
  • the PCR product was digested with Ncol and Xhol and ligated to pET24d (Hethke et al, 1996 Nucleic Acids Res 24:2369-2376), which had also been digested with Ncol and Xhol.
  • This vector contains a his-tag sequence that is added to the C-terminus of the expressed protein.
  • the final plasmid was sequenced to confirm that the cloned cbel gene was in frame with the C- terminal His-tag followed by a translation stop codon.
  • a cell free extract of C. bescii was prepared from a 500 ml culture grown to mid- log phase, harvested by centrifugation at 6,000g at 4°C for 15 minutes and resuspended in CelLytic B Cell Lysis Reagent (Sigma- Aldrich; St. Louis, MO) containing a protease inhibitor cocktail (Complete, ED TA- free from Roche; Madison, WI). Extracts were sonicated on ice and then centrifuged at 13,000 rpm for 15 minutes at 4°C. Supernatants were removed and used immediately for enzyme activity assays. Protein concentrations were determined using the Bio- Rad protein assay kit with bovine serum albumin as the standard.
  • pDCW68 DNA (20 ⁇ g), isolated from E. coli DH5a (dam + , dcm + ), was treated with Haelll Methyltransferase (New England Biolabs; Ipswich, MA) according to the suppliers instructions.
  • Haelll Methyltransferase New England Biolabs; Ipswich, MA
  • SAM S-adenosylmethionine
  • Methylated DNA was purified and concentrated using the DNA Clean & ConcentratorTM-25 Kit (Zymo Research; Irvine, CA). The extent of protection was determined using Haelll (New England Biolabs; Ipswich, MA) according to the supplier's instructions.
  • the 2.56 kb fragment containing three Haelll (5'-GGCC-3') sites used in assays with purified Cbel was generated by PCR amplification using primers DC222 and DC224 (Table 1) from pDCW68 template. PCR products were purified and concentrated by Qiaquick PCR Purification Kit (Qiagen; Valencia, CA) prior to use in the restriction assays.
  • Reactions were performed in 60 ⁇ volumes at 75°C using 0.5 ⁇ g to 1.0 ⁇ g of the DNA substrate: pDCW68, methylated pDCW68, pATHE 01 , or pATHE 02.
  • 4 ⁇ g of total cell protein was incubated at 75°C in reaction buffer (10 mM Tris-HCl, pH 6.7, buffer containing 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithioerythritol, 0.01% BSA).
  • Samples (10 ⁇ ) were withdrawn at various time points, mixed with 6 X DNA gel-loading buffer (0.25%
  • Enzyme assays with purified Cbel were carried out in 10 ⁇ reaction volumes with NEBuffer 4 (20 mM Tris-acetate pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol) and 200 ng of DNA substrate. The amount of purified Cbel protein used in each reaction varied depending on the experiment and is indicated.
  • DNA fragments resulting from digestion by either Haelll or Cbel were separated electrophoretically and extracted from the gel matrix using a QIAquick Gel Extraction Kit (Qiagen; Valencia, CA). The products were then cloned into pWSK29 (Wang and Kushner, 1991 Gene 100: 195-199), which had been digested with EcoRV, using the Fast-LinkTM DNA ligation kit (Epicentre Biotechnologies) and sequenced.
  • the washed cells pellets are resuspended in a total volume of 500 ⁇ of ice cold 10%>
  • DNA (0.5-1.0 ⁇ g) is added to competent cells, mixed gently, and incubated for 10 minutes on ice.
  • the mixed competent cells are injected into 10 ml of pre-warmed complex medium (per 1 liter of medium: 20 ml of 5 Ox salts, 2 ml of 500x vitamin mix, 1 ml of lOOOx trace minerals, 40 ml of 25x amino acid solution, 50 ⁇ of 5 mg/ml resazurin, 50 ml of 10% cellobiose, 2.4 ml of 1 M KH 2 P0 4 , 5 ml of 10% yeast extract, and 50 ml of 10% casein hydrolysate) at 75°C, and then incubated at the optimal temperature for the given species overnight.
  • pre-warmed complex medium per 1 liter of medium: 20 ml of 5 Ox salts, 2 ml of 500x vitamin mix, 1 ml of lOOOx trace minerals, 40 ml of 25x amino acid solution, 50 ⁇ of 5
  • the electroporation of mixed competent cells was performed via single electric pulse (1.0 kV, 600 ⁇ , and 25 mF) in 1 mm cuvettes using a Bio-Rad gene Pulser, and then incubated in 10 ml of complex medium at optimal temperature overnight.
  • the 10 ml recovery culture is centrifuged at 3500 x g for 10 minutes and the cell pellet is washed twice with IX AT base salts. After washing, the cells are suspended in 0.28 ml of IX AT base salts. 100 ⁇ of the cell suspension is placed into 4 ml of overlay solution (1.0% agar in water) and the overlay suspension is spread onto an appropriate selective medium. The plates are placed in ajar, degassed, and observed for growth after incubation for four days at 75°C.
  • overlay solution (1.0% agar in water
  • Caldicellulosiruptor species were grown in modified DSMZ 516 medium (Chung et al, 2011 J Ind Microbiol Biotechnol 38: 1867-1877) at a final pH 6.8. Liquid cultures were inoculated with a 1-2% inoculum or with a single colony and then incubated at 75°C overnight in anaerobic culture bottles or Hungate tubes degassed with at least three cycles of vacuum and argon. A solid medium was prepared by mixing an equal volume of liquid medium at a 2X concentration with 1% (wt/vol) Phytagel (Sigma- Aldrich; St. Louis, MO) previously autoclaved to solubilize.
  • C. bescii DSM 6725 was inoculated into 10 ml of modified DSMZ 516 medium and grown anaerobically at 60°C for 24 hours. Cells were harvested at 18,000 x g for five minutes, washed twice with mineral solution (Chung et al., 2011 J Ind Microbiol Biotechnol 38: 1867- 1877), resuspended in 1 ml of mineral solution and plated by mixing 100 ⁇ of cells with 4 ml of 0.3% agar and overlaying onto defined modified DSMZ 516 agar medium (no yeast extract or casein) supplemented with 20 ⁇ uracil and 8 mM 5-FOA (US Biologicals; Swampscott, MA).
  • modified DSMZ 516 agar medium no yeast extract or casein
  • a 1.858 kb fragment containing the pSClOl replication origin was amplified from pDCW68 (Chung et al., 2011 J Ind Microbiol Biotechnol 38: 1867-1877) using primers DC081 and DC230, which contain Kpnl and Aatll sites, respectively.
  • a 4.343 kb fragment containing the apramycin resistance and pyrBCF cassettes was amplified from pDCW68 using primers DC084 and DC232 to which an Aatll and Kpnl site had been added.
  • An additional fragment (1.801 kb) containing DNA sequences not relevant to the experiments described here was amplified using primers DC212 and DC213.
  • pDCW69 (8.014 kb).
  • pDCW70 was constructed by introducing a single nucleotide change (an A to C transversion) in the +978 amino acid of pyrC (Cbes 1376) ORF using "PCR based Site Directed Mutagenesis", using DC 214 and DC 215 primers, to create the Kpnl site (GGTAC/C), in pDCW 69.
  • pDCW73 the 0.837 kb M.Cbel (Cbes 2437) open reading frame was amplified by PCR using primers DC238 and DC239 using C. bescii genomic DNA as template.
  • PCR product was digested with BamHI and Xhol and ligated to pET24d (Hethke et al., 1996 Nucleic Acids Res 24(12):2369-76), which had also been digested with BamHI and Xhol.
  • This vector contains a His-tag sequence that is added to the C-terminus of the expressed protein. All plasmids used in this study were sequenced to confirm their structure.
  • M.Cbel Purification of M.Cbel was similar to the method described by Chung et al. (Chung et al, 2011 J Ind Microbiol Biotechnol 38: 1867-1877).
  • BL21-CodonPlus(DE3)-RILP cells (Agilent Technologies; Santa Clara, CA Technologies), containing pDCW73, was used for M.Cbel protein expression.
  • Cells were grown at 23°C in LB broth supplemented with kanamycin (25 ⁇ / ⁇ 1) and chloramphenicol (50 ⁇ ) to OD 60 o 0.7 and induced by addition of 0.5 mM isopropyl b-D-l-thiogalactopyranoside (IPTG) at 23°C overnight.
  • IPTG isopropyl b-D-l-thiogalactopyranoside
  • His-tagged (carboxy terminus) M.Cbel was purified as described previously (Chung et al, 2011 J Ind Microbiol Biotechnol 38: 1867-1877) except for the use of a His-Spin Protein MiniprepTM (Zymo Research; Irvine, CA). Protein concentration was determined by the Bio-Rad protein assay using bovine serum albumin (BSA) as the standard. Purified protein was displayed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 1996 Comput Appl Biosci 12(4):357-8) and stained with Coomassie brilliant blue G-250 as described (Sedmak and Grossberg, 1977 Anal Biochem 79(l-2):544-52). Protein purity was determined to be >98%.
  • BSA bovine serum albumin
  • DNA isolated from E. coli DH5a was treated with either M.Cbel or M.Haelll methyltransferase (NEB).
  • 50 ng of purified M.Cbel was incubated with 50 mM Tris-HCl, 50 mM NaCl, 80 ⁇ S-adenosylmethionine (SAM, Samuelson and Xu, 2002 J Mol Biol 319(3):673-83), 10 mM Dithiothreitol (DTT) at pH 8.5 and 20 ⁇ g of DNA substrate in 400 ⁇ reaction, and incubate for two hours at 78°C.
  • SAM S-adenosylmethionine
  • DTT Dithiothreitol
  • the M.Haelll methylation reaction was performed according supplier's instructions. To allow complete methylation, an additional 10 units of M.Haelll and 80 ⁇ SAM was added to the reaction every four hours of incubation at 37°C for a total of 12 hours. Methylated DNAs were purified and concentrated by Phenol/Chloroform extraction and ethanol precipitation. The extent of protection was determined by cleavage using Haelll and Notl (NEB) restriction enzymes according to the supplier's instructions.
  • NEB Haelll and Notl
  • CFE bescii cell free extracts
  • Digested DNA was displayed by agarose gel electrophoresis and visualization using ethidium bromide staining.
  • Automatic sequencing was performed using primers M13F(-20) and M13R(-20) in an ABI automated PRISM big-dye-terminator system (Macrogen, Inc.; Rockville, MD). Sequences were analyzed using the Chromas Lite v2.01 (Technelysium Pty Ltd.) and ABI chromatograms were compared by aligning the Sequencing traces and using SeqDoc (Crowe, 2005 BMC
  • ApyrBCF (ApyrBCF) culture was inoculated into 500 ml of fresh medium, and incubated at 78°C to mid- log phase (O.D 6 oo - 0.1 or 2 x 10 7 cells/ml). The cultures were cooled to room temperature for 1 hour, harvested by centrifugation (5000 x g, 15 minutes) at 25°C and washed twice with 250 ml of pre-chilled 10% sucrose. After the final wash, the cell pellets were resuspended in a total volume of 1 ml of pre-chilled 10% sucrose and aliquots of 50 ⁇ were freeze-dried in
  • Plasmid DNA (0.5-1.0 ⁇ g) was added to cells, gently mixed and incubated in 10% sucrose for 15 minutes at room temperature.
  • Electrotrans formation of the cell/DNA mixture was performed via single electric pulse (1.8 kV, 600 ⁇ , and 25 ⁇ ) in a pre-chilled 1 mm cuvette using a Bio-Rad gene Pulser. After pulsing, cells were incubated overnight at 75°C in 10 ml modified DSMZ 516 medium supplemented with 20 ⁇ of uracil, harvested by centrifugation (at 5000 x g for 20 minutes) and resuspended in 1 ml of lx base salt. A cell suspension (100 microliters) was plated onto defined medium without uracil.
  • a solid defined modified DSMZ 516 medium (no yeast extract and casein) was prepared by mixing an equal volume of 2X liquid medium with 1% (wt/vol) previously autoclaved Phytagel (Sigma-Aldrich; St. Louis, MO). Both solutions were maintained at 95°C prior to mixing and immediately poured into petri dishes. Transformation mixtures were incubated overnight at 78°C in 10 ml modified DSMZ 516 medium supplemented with uracil.
  • Cells were harvested by centrifugation, resuspended in 1 ml of lx base salt (Chung et al, 201 1 J Ind Microbiol Biotechnol 38: 1867-1877), (100 microliters) mixed with 4 ml of soft agar (0.3 % agar), that had been melted at 100°C and cooled in a 45°C heating block and plated onto defined medium without uracil. Plates were incubated in anaerobic jars at 75°C for three to four days.
  • lx base salt Choung et al, 201 1 J Ind Microbiol Biotechnol 38: 1867-1877
  • 4 ml of soft agar 0.3 % agar
  • DNA from uracil prototrophic transformants was used to amplify the chromosomal region using primers DC 163 and DC 188 which anneal outside the regions of the pyrBCF fragment contained on pDCW70. PCR products of this locus amplified from the wild type, the deletion mutant and the
  • RNA extraction and RT-qPCR analyses Total RNA was extracted using an RNeasy Mini kit (Qiagen; Valencia, CA) and stored at -80°C. RNA was treated with RNase-free DNase (Qiagen; Valencia, CA) according to manufacturer's instructions. cDNA was then prepared using the Affinity Script quantitative PCR (qPCR) cDNA synthesis kit (Agilent Technologies; Santa Clara, CA Technologies). All quantitative reverse transcription-PCR (RT-qPCR) experiments were carried out with an

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Abstract

The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

Description

RESTRICTION/MODIFICATION POLYPEPTIDES, POLYNUCLEOTIDES, AND
METHODS
GOVERNMENT FUNDING
The present invention was made with government support under Grant No. DE-AC05- 00OR22725, awarded by the U.S. Department of Energy, BioEnergy Science Center. The Government has certain rights in this invention.
BACKGROUND
Caldicellulosiruptor bescii DSM 6725 (formerly Anaerocellum thermophilum, Yang et al. 2009, Int J Syst Evol Microbiol 60:2011-2015) grows at temperatures up to about 90°C and is the most thermophilic cellulolytic bacterium known. This obligate anaerobe is capable of degrading lignocellulosic biomass including hardwood (e.g., poplar) and grasses with both low lignin (e.g., napier grass and bermuda grass) and high lignin (e.g., switchgrass) content without chemical pretreatment (Yang et al., 2009 Appl Environ Microbiol 75:4762-4769). When grown on crystalline cellulose, it produces lactate, ethanol, acetate, H2, and C02 (Svetlichnyi et al,
1990 Mikrobiologiya 59:598-604). Its genome includes sequences encoding cellulases, glycoside hydrolases, pectinases, pullulanases, and transporters that are important in biomass
deconstruction (Kataeva et al, 2009 J Bacteriol 191 :3760-3761). This variety of cellulolytic enzymes and end products, in combination with an optimal growth temperature near 80°C make C. bescii an important microorganism not only in the study of biomass deconstruction, but also in the industrial development of ethanol and other biofuels. This genus has many advantages for consolidated bioprocessing (CBP) and offers the possibility for production of bioenergy and bioproducts from lignocellulosic biomass by a single organism in a single step fermentation (Lynd et al, 2002 Microbiol Mol Biol Rev 66:506-577).
SUMMARY OF THE INVENTION
In one aspect, the invention provides an isolated polynucleotide comprising the coding of Cbes 2438. In some cases, the invention can provide a vector that includes such a polynucleotide operably linked to a promoter. In some cases, the invention can provide a cell that includes such a polynucleotide and/or such a vector.
In another aspect, the invention provides an isolated polynucleotide comprising the coding region of Cbes 2437. In some cases, the invention can provide a vector that includes such a polynucleotide operably linked to a promoter. In some cases, the invention can provide a cell that includes such a polynucleotide and/or such a vector.
In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence encoded by the coding region of Cbes 2438.
In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence encoded by the coding region of Cbes 2437.
In another aspect, the invention provides a method that generally includes incubating a DNA molecule comprising at least one 5'-GGCC-3' sequence with a Cbel polypeptide under conditions effective for the Cbel polypeptide to digest the DNA at the 5'-GG/CC-3' sequence.
In another aspect, the invention provides a method that generally includes treating a DNA molecule comprising at least one 5'-GGCC-3' sequence with a M.Cbel polypeptide under condition effective for the M.Cbel polypeptide to methylate at least one C residue of the 5'- GGCC-3' sequence.
In another aspect, the invention provides a method that generally includes introducing a polynucleotide into a microbial cell that comprises a thermophile or a hyperthermophile. In some cases, the method can include treating the DNA with a M.Cbel polypeptide under condition effective for the M.Cbel polypeptide to methylate at least one C residue of the DNA.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Detection of a Haelll-like restriction/modification system in C. bescii. (A) pDCW68 (B) pATHEOl (C) pATHE02 (D) pDCW68 isolated from E. coli DH5a incubated with CFE from C. bescii (E) pATHEOl and pATHE02 isolated from C. bescii incubated with CFE from C. bescii (F) pDCW68 isolated from E. coli treated with M.Haelll and incubated with CFE from C. bescii. Features originating from E. coli are shaded in black and from C. bescii are white; AprR, apramycin resistant gene cassette; Trp, Cbes 2105-trytophan synthetase a subunit; OriT, origin of transfer for conjugation; pSClOl, low copy replication origin in E. coli; Haelll restriction sites are indicated. All incubation times were as indicated.
Figure 2. Cloning, expression and purification of Cbel. (A) The region surrounding the location of Cbel in the C. bescii genome. (B) pDCW72; KanR, kanamycin resistance gene; bacteriophage T7 promoter and T7 terminator; lacl, the gene for the lactose repressor protein; ColEI, origin of replication derived from pBR322 (C) Lane 1 : protein molecular weight standards; lane 2: 15 ng of purified Cbel protein displayed on a 10-20% Tris-HCl gradient gel (CRITERION Precast Gel, Bio-Rad Laboratories; Hercules, CA).
Figure 3. Temperature profile of purified Cbel endonuclease activity. (A) The 2.558 kb DNA substrate synthesized as described in the Materials and Methods. Haelll cleavage sites are marked by vertical lines and predicted cleavage fragment sizes are indicated below the line. (B) pDCW72 used to his-tagged expression of Cbel. The substrate was incubated for 10 minutes with 5 ng of protein at the temperatures indicated and the cleavage products were separated on 1.2% agarose gel. The position of the full length undigested fragment and the three major cleavage products derived by digestion with Cbel are indicated by arrows.
Figure 4. Phylogram alignment of 46 Haelll-like restriction enzymes. The host organism for each restriction enzyme is indicated as well as the protein name, when available. Otherwise, the GenBank locus tag or accession number is given. The distance scale is indicated by a bar defining the distance for 0.1 amino acid substitution per site. The bracketed organisms represent those containing this new subfamily of Haelll-like enzymes that includes Cbel.
Figure 5. Amino acid sequence alignment of Cbel with the subgroup identified in
Figure 4. Amino acid identity is shown as shaded areas with the position of the motif within the protein sequence. Motif start site is indicated. Motif 1 : 7.0e-220, motif 2 1.9e-278, motif 3 1.6e- 185. Subgroup sequences shown are Cbel (SEQ ID NO:36); Bhall (SEQ ID NO:37); Haelll (SEQ ID NO:38); Hac_1214 (SEQ ID NO:39); Cthe_2319 (SEQ ID NO:40); HPSH 02550 (SEQ ID NO:41); HMPREF0105 0967 (SEQ ID NO:42); Smon_0161 (SEQ ID NO:43); PRU 0937 (SEQ ID NO:44); HMPREF0573 11018 (SEQ ID NO:45); CUY 2194 (SEQ ID NO:46); Bgr_19490 (SEQ ID NO:47); and GOS 4010239 (SEQ ID NO:48).
Figure 6. Distribution of Haelll-like restriction/modification systems in
Caldicellulosiruptor species. Total DNA isolated from 7 different species were incubated (-) without or (+) with commercially available Haelll endonuclease at 37°C for 1 hour according to the manufacturer's instructions (NEB). C. bescii; C. hydro, C. hydrothermalis; C. krist, C.
kristjansonii; C. sacc, C. saccharolyticus; C. obsid, C. obsidiansis; C. lacto, C. lactoaceticus; C. krono, C. kronotskyensis . Also visible in the C. bescii lanes are the undigested native plasmids from that strain, pATHEOl (8.3 kb) and pATHE02 (3.6 kb).
Figure 7. Plasmid DNA (pUC18) was isolated from E. coli and incubated in vitro with either M.Haelll methyltransferase (NEB) or M.Cbel methyltransferase. After digestion with either Haelll or Cbel (as indicated) fragments were displayed on a 1.2% TAE-agarose gel stained with ethidium bromide. Lanes 1) un-methylated pUC18 DNA; 2) pUC18 treated with M.Haelll; 3) pUC18 treated with M.Cbel are as indicated in each panel; Panel (A) no restriction enzyme added (B) with Haelll for 30 minutes at 37°C or (C) with Cbel for 30 minutes at 75°C. MW: 1 kb DNA ladder (NEB).
Figure 8. Expression, purification, and characterization of M.Cbel. (A) Physical map of surrounding region of M.Cbel in the C. bescii genome. (B) Schematic diagram of pDCW73; KanR, kanamycin resistance gene; bacteriophage T7 promoter and T7 terminator; lacl, the gene for the lactose repressor protein; ColEI, origin of replication derived from pBR322 (C)
Coomassie blue stained SDS-PAGE gel. Lane 1 : protein molecular weight standards; lane 2: 15 ng of purified M.Cbel protein displayed on a 10-20% Tris-HCl gradient gel (CriterionTM Precast Gel, Bio-Rad Laboratories, Hercules, CA). The molecular weight of the purified His- tagged M.Cbel proteins is indicated by an arrow on the right. (D) M.Cbel methylation sensitivity of Cbel. Lane 1 : Undigestged unmethylated pDCW 70; Lane 2: Undigested M.Cbel methylated pDCW 70; Lane 3: Digested with purified Cbel of unmethylated pDCW 70; Lane 4: Digested with purified Cbel of M.Cbel methylated pDCW 70; M: 1 kb DNA ladder (NEB). (E)
Differences between G signals to M.Haelll (top panel; SEQ ID NO:49) and M.Cbel (bottom panel; SEQ ID NO:50) methylated innercytosine residue in 5'-GGCC-3' sequence. Trace differences in G residue between M.Haelll and M.Cbel methylated pUC18 is shown in middle panel. Figure 9. Confirmation of transformation and marker replacement in ApyrBCF strain. (A) A schematic diagrams of the pyrBCF locus wild type (Ura /5FOA ), ApyrBCF (Ura~ /5FOAR), pDCW 70, and pyrBCF locus in result transformant. pDCW 70 having 0.892 kb region of pyrF and 0.662 kb region of pyrB for homologous recombination. Marker replacement by homologous recombination can occur in the chromosome in the pyrBCF region. Engineered Kpnl site is indicated, and bent arrows depict primers used for verification of transformation. (B) Electrphoration performance of ApyrBCF strain with unmethylated and M.Cbel methylated pDCW 70. Top plate (Defined + Uracil plates), competent cell after electro pulsing; Middle plate (w/o Uracil plate), transformed with unmethylated pDCW 70; Bottom plate (w/o Uracil plate), transformed with M.Cbel methylated pDCW 70. (C) Gel depicting PCR products (amplified by DC 163 and DC 188), and its cleavage products by Kpnl (M: lkb DNA Ladder (NEB); Lane 1 : Wild type, 3.2 kb; Lane 2: ApyrBCF, 1.63 kb; Lane 3: Transformat, 3.2 kb; Lane 4: Wild type cleaved by Kpnl, No cleavage; Lane 5: ApyrBCF cleaved by Kpnl, No cleavage; Lane 6:
Transformant cleaved by Kpnl, 1.9 and 1.3 kb cleavage products by Kpnl ).
Figure 10. Linear order of the three functional groups of M.Cbel. Sequence alignment of three members of Caldicellulosiruptor species and DmtB from Anabaena variabilis, which contain a M.Cbel homologue. Sequences are shown for C. bescii DSM 6725 (SEQ ID NO:51); C kristjanssonii 177R1B (SEQ ID NO:52); C hydrothermalis 108 (SEQ ID NO:53); and Anabaena variabilis (SEQ ID NO:54).
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a Haelll-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.Cbel, that methylates at least one inner cytosine residue in the Cbel recognition sequence (5'-GGCC-3') to m4C. Thus, the invention provides, in various aspects, isolated Cbel or M.Cbel polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the Cbel or M.Cbel polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a Cbel polypeptide; methods of treating a DNA molecule using a M.Cbel polypeptide; and methods of transforming a Caldicellulosiruptor cell. Despite prior attempts to directly transform members of the Caldicellulosiruptor genus, this is the first report of success. Because members of the genus Caldicellulosiruptor possess certain biology properties of potential commercial value (e.g., biomass conversion), the ability to genetically manipulate these organisms can assist in metabolically engineering members of this genus for, for example, their use in consolidated bioprocessing that produces one or more biofuels and/or one or more bioproducts. Thus, certain aspects of the invention can be used to overcome restriction that may assist methods of DNA transformation of Caldicellulosiruptor species using DNA from, for example, homologous and/or heterologous sources. Moreover, these aspects may be generalized to permit transformation of other thermophilic and/or hyperthermophilic microbes.
As noted above, Caldicellulosiruptor bescii was formerly classified as Anaerocellum thermophilum. The genome of C. bescii was originally annotated when the organism was known as A. thermophilum. The annotations were modified upon reclassification of the organism to replace Athe annotations with Cbes annotations, reflecting the reclassification of the organism. Neither the substantive content of the annotation nor the numerical portion of the annotations changed. Thus, for example, the original annotation Athe 2438 is now referred to as Cbes 2438. Nevertheless, Athe annotations and Cbes annotations may be used interchangeably.
With the reclassification of A. thermophilum to C. bescii, the nomenclature used to refer to, for example, certain plasmids shown in Figure 1 also has changed. For example, pATHEOl is now known as pBAL. Similarly, pATHE02 is now known as pBAS2.
As used herein, the following terms shall have the indicated meanings.
"Cbel" refers to a polypeptide encoded by at least a portion of the coding region of Cbes 2438 and that cleaves DNA at a 5'-GG/CC-3'. Cbel can refer to a 38 kDa polypeptide encoded by a 981 bp coding sequence of Cbes 2438, or a biologically active fragment of such a polypeptide. Biological activity, in the context of Cbel, refers to the ability to digest DNA specifically at a 5'- GG/CC-3' recognition site at a temperature from 35°C to 85°C.
"M.Cbel" refers to a polypeptide encoded by at least a portion of the coding region of Cbes 2437 and that, when incubated with DNA at a temperature from 35°C to 85°C methylates a cytosine in the 5'-GG/CC-3' recognition site of Cbel.
"Methylase" and "methyltransferase" are synonymous as used herein and may be used interchangeably. The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.
The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.
Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
A potent Haelll-like DNA restriction activity was detected in cell- free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from E. coli as substrate.
Incubation of the plasmid DNA in vitro with Haelll methyltransferase partially protected it from cleavage by Haelll nuclease as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981 bp nucleic acid sequence, was optimally active at temperatures between 75°C and 85°C and was stable for more than a week when stored at 35°C. The cleavage sequence was determined to be 5'-GG/CC-3' indicating that Cbel is an isoschizomer of Haelll. A search of the C. bescii genome sequence revealed the presence of both a Haelll-like restriction endonuclease (Cbes 2438) and DNA methyltransferase (Cbes 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this
restriction/modification activity is widespread in this genus. A phylogenetic analysis based on sequence alignment and conserved motif searches identified features of Cbel distinct from other members of this group and classified Cbel as a member of a novel subfamily of Haelll-like enzymes.
While described below in terms of introducing heterologous polynucleotides into thermophilic and/or hyperthermophilic species, the methods described herein also may be used more generally to introduce polynucleotides - whether heterologous or homologous - into such species in order to, for example, achieve overexpression of the polynucleotide, overproduction of at least one polypeptide encoded by the introduced polynucleotide, and an increase in the cellular level of activity of the encoded polypeptide. Also, the methods described herein may be used generally to introduce polynucleotides into other thermophilic species such as, for example, certain Clostridium spp. and/or
hyperthermophilic species such as, for example, Thermo anaerobacter spp.
The ability to genetically manipulate Caldicellulosiruptor species - and other
thermophilic or hyperthermophilic species - is a prerequisite for their use in consolidated bioprocessing. In our efforts to develop a method of DNA transformation, we discovered several potent restriction activities, one of which we describe in this report. The activity of host restriction enzymes is a major barrier to the introduction of DNA into cells. Identifying and overcoming restriction systems has allowed the development of genetic systems in previously non-transformable bacteria. Often this is accomplished by in vitro methylation or by in vivo methylation systems in, for example, E. coli. For example, in the thermophile Bacillus methanolicus, plasmids are engineered with fewer Bmel recognition sites and prepared in a dam+ E. coli strain in order for transformation to occur (Cue et al., 1997 Appl Environ Microbiol 63: 1406-1420). In Prevotella species and Helicobacter pylori, plasmid DNA is methylated by cell-free extracts to achieve transformation by electroporation (Accetto et al., 2005 FEMS
Microbiol Lett 247:177-183; Donahue et al, 2000 Mol Microbiol 37: 1066-1074). In Clostridium difficile, plasmids are constructed that lack Cdil and Sau96I recognition sequences (Purdy et al, 2002 Mol Microbiol 46:439-452). Clostridium perfringens type B transformation can occur only when the transforming plasmid DNA is isolated from a dam+dcm+ strain of E. coli (Chen et al., 1996 FEMS Microbiol Lett 140: 185-191). Clostridium cellulolyticum can be transformed only if the plasmid DNA is protected from Ccel cleavage using either in vitro or in vivo methylation (Jennert et al, 2000 Microbiology 146(Ptl2):3071-3080).
In our efforts to develop efficient methods for DNA transformation of C. bescii, we identified a potent thermostable Type II restriction endonuclease that is an isoschizomer of Haelll (Middleton et al., 1972 J Virol 10:42-50). Haelll-like enzymes are a diverse group of proteins with distinct catalytic domains that have in common the ability to cleave the same DNA sequence. The prototype of this group, Haelll, was first identified in Haemophilus aegyptius in 1972 (Middleton et al, 1972 J Virol 10:42-50). This enzyme recognizes 5*-GGCC-3* and cleaves the DNA between the second G (in the second position) and first C (in the third position) leaving a blunt end. Haelll-like restriction activity is widespread in bacteria and archaea (Roberts et al., 2010 Nucleic Acids Res 38:D234-D236) allowing efficient restriction of foreign DNA. Four- base cutters like Haelll can present a challenge for DNA transfer since the expected frequency of the cleavage sites sequence is greater than the expected frequency of longer recognition sequences. The enzyme identified from C. bescii was named Cbel and its cleavage activity, temperature profile, and thermostability are described.
This is the first investigation of a restriction-modification system in any species of
Caldicellulosiruptor. Bioinformatic analysis of Cbel and other Haelll isoschizomers reveals a previously unidentified subfamily of this group of restriction endonucleases. The work described herein advances the study of the nature of restriction-modification systems and advances efforts to establish genetic methods for this important group of organisms.
Identification of a Haelll-like restriction activity in C. bescii.
A cell-free extract from C. bescii was prepared (Jennert et al., 2000 Microbiology 146(Ptl2):3071-3080) and incubated with pDCW68 DNA (FigurelA), a vector constructed for use in transformation experiments and that had been isolated from E. coli (DH5a dam+ dcm+). The plasmid DNA, when incubated with the C. bescii cell-free extract, was completely digested within 10 minutes and had a similar restriction cleavage profile to that of a digest with commercially available Haelll (Figure ID). DNA of the two native C. bescii plasmids, pATHEOl and pATHE02 (Figure IB and 1C), were not digested when incubated with the same cell-free extract nor were they digested by commercially available Haelll endonuclease (Figure IE) suggesting the presence of Haelll methyltransferase-like activity in C. bescii. In addition, pDCW68 DNA that had been methylated in vitro by commercially available Haelll
methyltransferase was protected from cleavage from either the commercially available Haelll restriction enzyme or cell- free extracts from C. bescii (Figure IF), suggesting the presence of a cognate methyltransferase activity in C. bescii. In support of the notion that C. bescii contains a Haelll-like enzyme is the observation that Haelll recognition sites (GGCC) are much less abundant in the C. bescii genome sequence than would be expected based on random nucleotide composition (0.468 observed/expected). Organisms that produce restriction enzymes often have a bias against the presence of the recognition sequences of those enzymes in their genomes (Nobusato et al, 2000 Gene 259:89-98; Rocha et al, 2001 Genome Res 11 :946-958). We have named this Haelll-like restriction endonuclease from C. bescii, Cbel. Cloning, expression, and purification of Cbel from Caldicellulosiruptor bescii.
A query of the C. bescii genome using the GenBank (Benson et al, 2009 Nucleic Acids Res 37:D26-31) database as well as REBASE (Roberts et al, 2010 Nucleic Acids Res 38:D234- D236) identified a methyltransferase (Cbes 2437) adjacent to a candidate gene for a Haelll-like restriction endonuclease (Cbes 2438) (Figure 2A), which is a common feature of Type II restriction/modification gene arrangements (Kong et al., 2000 Nucleic Acids Res 28:3216-3223).
The open reading frame encoding Cbel (Cbes 2438) was cloned into an E. coli expression vector, pDCW72 (Figure 2B) under the transcriptional control of the T7 promoter to allow regulated expression of the restriction endonuclease as it would be expected to be toxic to cells that did not contain the corresponding methyltransferase. In fact, initial attempts to clone the gene encoding a His-tagged version, Cbel- His6, at 37°C, even without induction of the T7 promoter, failed. Because C. bescii grows optimally near 80°C, we investigated the possibility that the cloning and expression of Cbel at lower temperatures would be more efficient because at lower temperatures the enzyme might be inactive or non-functional. When experiments were done at 23°C (the equivalent of expressing E. coli enzymes at -6°C) both cloning and expression were successful. Since there are also significant differences in codon usage between C. bescii (35.2% GC content) and E. coli (50.5% GC content), BL21-CodonPlus(DE3)-RIPL cells which contain rare tRNAs were used for expression. Preparations of purified C-terminal His-tagged Cbel contained a single band on an SDS-PAGE gel (Figure 2C) with a molecular mass of approximately 38 kDa which is consistent with the calculated value of 37.9 kDa determined using the Compute pI/Mw analysis tool.
Functional analysis and temperature optimum of the purified Cbel endonuclease activity.
A DNA substrate containing three Haelll restriction sites (Figure 3A), which should generate fragments of 1393 bp, 772 bp, 293 bp, and 106 bp when digested, was used in restriction digestion assays. Purified Cbel protein (12-48 ng) was incubated with 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol at pH 7.9, and 100 ng of DNA substrate in 10 μΐ (Figure 3 A), and incubated for 10-20 minutes at 75°C. These conditions were chosen based on the reaction conditions of other isoschizomers of Haelll from thermophiles (Nolling and de Vos, 1992 J Bacteriol 174:5719-5726; Prangishvili et al., 1985 FEBS Lett 192:57-60) and on the composition of NEB Buffer 4 that is used with commercially available Haelll restriction endonuclease.
Experiments with cell free extracts indicated that Cbel cleavage activity occurred within 10 minutes even with low protein concentrations (4 μg total cell protein in 60 μΐ). Purified protein showed a similar time course and there was no difference in activity between 12 ng to 48 ng (in 10 μΐ) of purified protein in the enzyme assays. The experiment shown in Figure 3B used 24 ng (in 10 μΐ) of purified protein. In assays with less than 2.5 ng (in 10 μΐ) of protein, no activity was detected.
During the course of cloning and expressing Cbel in E. coli, the gene was not toxic to cells grown at 23°C but it was at 37°C, suggesting that the enzyme was less active at low temperature. To determine the optimal temperature for Cbel activity, purified protein was incubated with the DNA substrate at temperatures ranging from 23°C -100°C. As shown in Figure 3B, the enzyme is optimally active between 75°C and 85°C, exhibiting partial digestion activity at 45°C or below and non-specific activity above 85°C. No activity was detected at 23°C. The optimum temperature for commercially available Haelll activity is 37°C and the enzyme is inactivated by treatment at 80°C for 20 minutes. In contrast, Cbel is optimally active at 75°C - 85°C and is stable for more than 30 minutes at 75°C (Figure 3B). Cbel did not lose activity after storage for more than one week stored at up to 35°C and was heat-inactivated by incubation at 100°C for 5 minutes.
Cbel is an isoschizomer of Haelll.
Haelll recognizes a sequence that includes GGCC and cleaves between the G and C leaving a blunt ended fragment. To determine the cleavage site of Cbel, a DNA fragment containing three Haelll recognition/cleavage sites (Figure 3 A) was used as substrate with purified Cbel enzyme. The cleavage products were either ligated directly to pWSK29 that had been digested with EcoRV leaving a blunt end, or first treated with the Klenow fragment of DNA polymerase prior to ligation. The sequence of the region containing the cloning site revealed the dinucleotide 5'-CC-3' adjacent to the EcoRV site and the dinucleotide 5'-GG-3' adjacent to the EcoRV site, identifying 5'-GG/CC-3' as the Cbel cleavage site. The same result was obtained in eight independent cloning experiments, indicating that Cbel is an isoschizomer of Haelll. Cluster Analysis of Haelll-like proteins reveals that Cbel is a member of a new subfamily of Haelll-like enzymes.
A search of the REBASE and NCBI databases revealed 231 Haelll-like proteins. Of those, 183 had been fully or partially characterized and 48 were putative isoschizomers predicted from their DNA sequences. Of the 57 proteins for which there were sequence information available, subfamilies of the same genus and species were removed leaving 46 sequences used in this analysis. A phylogram based on protein sequence alignments is shown in Figure 4. Cbel falls into a distinct group of 13 proteins, five of which had previously been grouped (pfam09556) based on overall sequence similarity using the Conserved Domain Database (Marchler-Bauer et al, 2009 Nucleic Acids Res 37:D205-210). Our own motif-based sequence analysis (MEME Suite and GLAM2) of this group of 13 proteins with other Haelll-like proteins identified three conserved motifs shared by 11 of the 13 (Figure 5), but not present in other Haelll-like proteins. One protein (Bgr_19490) had motifs 1 and 2, and one (GOS 4010239) had only motif 1.
Evidence for Haelll-like restriction/modifications systems in other Caldicellulosiruptor species.
To investigate whether other Caldicellulosiruptor species contained Haelll-like restriction/modification activities, total DNA was isolated from a number of Caldicellulosiruptor species and incubated with commercially available Haelll restriction endonuclease. As shown in Figure 6, C saccharolyticus DSM 8903, C. hydrothermalis DSM 18901, C. kristjanssonii DSM 12137, and C. bescii DSM 6925 were resistant to Haelll nuclease while C. kronotskyensis DSM 18902, C. lactoaceticus DSM 9545, C. obsidiansis ATCC BAA-2073, were sensitive. These preliminary data suggest that this Haelll-like restriction/modification system may be widespread among members of this genus.
Identification of M.Cbel
As described above, Cbel from C. bescii DSM 6725 is an isoschizomer of Haelll (cuts at the same site) which is a Type II restriction enzyme. Type II restriction enzymes and their cognate methyltransferase genes are often adjacent to each other in the chromosome. The locations of the coding region for Cbel (Cbes 2438) and the coding region for the cognate methyltransferase (Cbes 2437) are indicated in FIG. 2A.
The coding region of M.Cbel was cloned and expressed shown in FIG. 8. Plasmid DNA (pUC18) was isolated from E. coli and incubated in vitro with either M.Haelll methyltransferase (NEB) or M.Cbel methyltransferase. FIG. 7 shows the result of those digestions. M.Cbel protected pUC18 DNA from digestion by Haelll for 30 minutes at 37°C (middle gel, lane 3) and from digestion by Cbel for 30 Minutes at 75°C (right panel, lane 3).
Cbes 2437 had been annotated in the GenBank database as a "D12 class N6 adenine- specific DNA methyltransferase," which would indicate that it is not a methyltransferase that would protect DNA from cleavage. Since Cbel cleaves the sequence 5'-GG/CC-3', a cytosine- specific methyltransferase would be necessary in order to protect the chromosomal DNA from Cbel restriction endonuclease activity. Thus, prior to our characterization of the expression product of M.Cbel, the expression product of Cbes 2437, Cbes 2437 was incorrectly identified to lead one away from the possibility that it could encode a cognate methyltransferase that would protect chromosomal DNA from digestion by Cbel.
Two other Caldicellulosiruptor species (C. hydrothermalis and C. kristjanssonii) may contain the same Haelll-like Cbel/M.Cbel restriction-modification system - i.e., homologues of Cbel and M.Cbel, suggesting that this may be a wide spread system in Caldicellulosiruptor species.
M.Cbel is a novel methyltransferase only present in Caldicellulosiruptor species (in C. hydrothermalis the gene locus tag is Calhy 0409 and in C kristjanssonii 177R1B the gene locus tag is Calkr 2088). M.Cbel is also distinctly different both in DNA sequence and protein sequence from M.Haelll and its isoschizomers, the models for 5'-GGCC-3' methylation.
After DNA replication in bacterial cells, methyltransferases can modify DNA in one of three ways to either methylate adenine to N6-methyladenine (m6A), cytosine to N4- methylcytosine (m4C), or cytosine to 5-methylcytosine (m5C). A study of methylation of nucleotide bases in thermophiles with optimal growth temperatures above 60°C revealed that the presence of m4C is favored over m5C, since m5C has a tendency to be deaminated to thymine by heat (e.g., temperatures greater than 70°C), thereby causing C-T transition mutations. Thus, the Cbel cognate methyltransferase (Cbes 2437) does not methylate cytosine to m5C as M.Haelll does, but instead methylates cytosine to the more heat-stable m4C. M.Cbel is the first example of an a-class (F G G - TRD - DPPY) N4-methylcytosine methyltransferase specific for 'GGCC. This may explain why commercially available 'GGCC methyltransferase (M.Haelll) treatment was not successful in protecting the DNA for
transformation into C. bescii.
Thus, in a screen of C. bescii cell-free extracts for restriction endonuclease activities we discovered a potent Haelll-like restriction activity with novel features. Plasmid DNA from E. coli, but not plasmid DNA from C. bescii, was digested within 10 minutes of incubation with these extracts and treatment with Haelll methyltransferase partially protected the DNA from cleavage suggesting the existence of a Haelll-like restriction-modification system in C. bescii. Bioinformatic analysis of the C. bescii genome identified a gene encoding a protein homologous to the Haelll endonuclease and an adjacent gene encoding a Type II DNA methyltransferase. The gene for the endonuclease was cloned and expressed in E. coli with a His-tag. Purified enzyme from E. coli was optimally active between 55°C and 85°C and was stable at 35°C for more than a week. The cleavage site of the enzyme was determined to be GG/CC suggesting that it is an isoschizomer of Haelll and we have named this enzyme Cbel. A phylogram of Cbel with other Haelll-like enzymes identified a new subfamily of these enzymes with unique features.
The cloning and expression of Cbel in E. coli presented some challenges, as does the expression of other toxic genes including other restriction endonuc leases (Kong et al, 2000 Nucleic Acids Res 28:3216-3223; Rasko et al, 2010 Nucleic Acids Res 38:7155-7166). In addition to using BL21-CodonPlus(DE3)-RIPL cells to compensate for differences in codon usage between E. coli and C. bescii, we took advantage of the fact that Cbel is from an extreme thermophile and would be expected to have minimal activity at temperatures significantly below the growth temperature of C. bescii (Topt ~ 80°C). This appeared to be the case since expression of Cbel was apparently toxic to E. coli cells grown at 37°C, but it was expressed efficiently at 23°C. This strategy may be useful for expressing toxic genes derived from thermophilic organisms in E. coli, eliminating the need for complicated highly-regulated expression systems and without the corresponding methyltransferase.
Since the first description of the Haelll restriction enzyme in 1972 (Middleton et al., 1972 J Virol 10:42-50), more than 200 isoschizomers have been reported or predicted (Roberts et al., 2010 Nucleic Acids Res 38:D234-D236). Of these, fewer than 40 are from thermophiles (organisms that have Topt> 50°C), and only three of these have been characterized: MthTI (Nolling and de Vos, 1992 J Bacteriol 174:5719-5726) from Methanobacterium
thermoformicicum THF (Topt 55°C), NspLKI (Zabaznaya et al, 1999 Biochemistry (Mosc) 64: 189-193) &om Nocardia species LK (Top, 50°C), and Sual (Prangishvili et al, 1985 FEBS Lett 192:57-60) from Sulfolobus acidocaldarius (Topt 82°C). A fourth, Phol from Pyrococcus horikoshii (Topt 98°C), is commercially available (New England Biolabs; Ipswich, MA) but there are no reports on this enzyme in the literature. Unlike Haelll itself, which is optimally active at 37°C and is inactivated by heating to 80°C, Cbel was optimally active in the range 75°C-85°C and required incubation at 100°C for 5 minutes for inactivation. The fact that Cbel isolated from E. coli is thermostable suggests that this feature is due to its conformation, hydrophobic, electrostatic, or other properties rather than by association with other proteins or co factors in C. bescii.
Haelll-like enzymes are widespread in both the archaea and bacteria. Genes encoding NgoPII from Neisseria gonorrhoeae, a bacterium, and MthTI from Methanobacterium thermoformicicum, an archaeon, have unexpectedly high similarity (54.5% nucleotide identity) suggesting horizontal gene transfer (Nolling and de Vos, 1992 J Bacteriol 174:5719-5726). In fact, a phylogenetic tree based on protein sequence similarity of Haelll-like proteins (Figure 4) identified a subgroup that includes four proteins from archaea {Pyrococcus horikoshii OT3, Sulfolobus islandicus, Sulfolobus acidocaldarius, and Methanothermobacter
thermautotrophicum) and nine from bacteria, suggesting that there may have been cross-domain horizontal gene transfer for these proteins. In support of this notion is the fact that the GC- content of some of the genes encoding Haelll-like proteins is significantly different from that of their host organism chromosomes: Mobiluncus curtisii (55% genome, 46%> HMPREF0573), Prevotella ruminicola (47% genome, 32% PRU_0939), and Roseiflexus castenholzii (60% genome, 52% Rcas_2133). Haelll-like enzymes are also widespread in both archeael and bacterial thermophiles, such as Clostridium thermocellum ATCC 27405, Methanothermobacter thermautotrophicum THF (Nolling and de Vos, 1992 J Bacteriol 174:5719-5726), Nocardia species LK (Zabaznaya et al, 1999 Biochemistry (Mosc) 64: 189-193), Roseiflexus castenholzii DSM 13941, Sulfolobus islandicus, Sulfolobus acidocaldarius DSM 639 (Prangishvili et al., 1985 FEBS Lett 192:57-60), Pyrococcus horikoshii OT3, and Thermodesulfovibrio yellowstonii DSM 11347. In an analysis of 46 Haelll-like proteins, those most similar to Cbel were found in other bacteria, both Gram-positive and Gram-negative. Cbel exhibits the highest amino acid sequence similarity (-60%) with the Haelll-like proteins from Bacillus halodurans (Bhall) and
Clostridium thermocellum (Cthe_2319). Examination of genomic DNA isolated from seven different Caldicellulosiruptor species showed that four of the seven were resistant to Haelll cleavage indicating that Haelll-like restriction-modification systems may be widespread in members of this genus (Figure 6).
An amino acid sequence alignment of Cbel and the 12 closely-related Haelll-like proteins (bracketed in Figure 4) revealed highly conserved residues that define three previously unrecognized motifs that may play a role in their structure or catalytic. Although these thirteen Haelll-like proteins could not be reliably matched to any other known protein structure or to the five known type II restriction endonuclease superfamilies (PD-(D/E)XK, HNH, PLD, GIY YIG, and HALFPIPE) (Orlowski and Bujnicki, 2008 Nucleic Acids Res 36:3552-3569), these observations make Cbel an interesting candidate for structural analyses since it may possess a novel tertiary structure. This new subgroup identified in our analysis that includes Cbel defines a new subfamily of structurally or functionally related proteins in this diverse group of enzymes. The results presented here also have important implications in the development of methods of genetic transformation for this interesting and biotechnologically-important group of relatively uncharacterized organisms.
Type II restriction endonucleases like Cbel can be a barrier to DNA transformation of several bacterial strains. Thus, successful transformation of such bacterial strains can involve overcoming restriction by the hosts. Approaches include engineering the transforming DNA to contain fewer restriction sites (Gallagher et al, 2008 J Bacteriol. 190(23):7830-7; Cue et al, 1997 Appl Environ Microbiol. 63(4): 1406-20; Purdy et al, 2002 Mol Microbiol. 46(2):439-52), in vitro methylation by purified methyltransferases (Jennert et al., 2000 Microbiology
146(Ptl2):3071-80) or cell extracts (Accetto et al, 2005 FEMS Microbiol Lett. 247(2): 177-83; Donahue et al., 2000 Mol Microbiol. 37(5): 1066-74), or in vivo methylation by E. coli (Cue et al, 1997 Appl Environ Microbiol. 63(4): 1406-20; Chen et al, 1996 FEMS Microbiol Lett.
140(2-3): 185-91). We were unable to transform C. bescii in many attempts using a variety of transformation procedures. Plasmid DNA treated with purified M.Haelll, in vitro, was partially protected from cleavage by both Haelll and Cbel in vitro (Figure 7), but no transformants were detected when this DNA was used in electroporation experiments or added to cells that had been subjected to a procedure to induce natural competence of C. bescii. In addition, various strains of E. coli containing combinations of methyltransferases that facilitated transformation of the thermophiles Bacillus methanolicus and Clostridium thermocellum were used to prepare DNA from E. coli for transformation but no C bescii transformants were detected using DNA from these strains (Table 2). A gene for an apparent cognate methyltransferase, M.Cbel (Cbes 2437) is present adjacent to Cbel in the C bescii genome as well as the genomes of C hydrothermalis 108 (Calhy 0409) and C. kristjanssonii 177R1B (Calkr 2088). Cytosine metyltransferases methylate cytosine to either 5-methylcytosine (m5C), as for M.Haelll, or more rarely to N4-methylcytosine (m4C).
Methylation to m4C may be more common than methylation to m5C, perhaps because m5C may be more readily deaminated to thymine by heat.
Here we show that restriction can be a barrier to transformation of Caldicellulosiruptor by DNA from E. coli and that methylation of a novel aclass Type II cytosine methyltransferase can overcome this barrier. While the apparent transformation frequency may be low, the combined frequencies of transformation and recombination allow maker replacement of chromosomal genes with non-replicating vectors providing an essential tool to generate deletions, gene substitutions, His-tags for protein purification and expression of heterologous proteins to identify genes important for biomass utilization as well as extend substrate utilization and biomass conversion in these organisms.
A spontaneous deletion of the C. bescii pyrBCF locus allows nutritional selection of transformants
Attempts to use a thermostable kanamycin resistance gene previously used for selection of transformants in Thermoanaerobacteria species at 60°C to select transformants in C bescii was complicated by the fact that C bescii, which that grows optimally at 75°C, grows very poorly at or below 70°C. In fact, growth at 60°C increased the spontaneous mutation frequency significantly, from 10"7 to 10"5, making the detection of transformants over this background of spontaneous drug resistance problematic. Attempts to use a hygromycin phosphotransferase (hph) gene from E. coli that had been selected for function at 85°C in Sulfolobus solfataricus were compromised by the level of natural resistance to hygromycin in C. bescii. To generate a mutant strain for nutritional selection of transformants, C. bescii cells were plated on 5- fluoroorotic acid (5-FOA). OMP decarboxylase, encoded by the pyrF gene in bacteria (ura3 in yeast), converts the pyrimidine analog 5-fluoroorotic acid (5-FOA) to 5-fluorouridine
monophosphate which is ultimately converted to fluorodeoxyuridine by the uracil biosynthetic pathway, a toxic product that kills growing cells that are synthesizing uracil. Mutants of pyrF are, therefore, uracil auxotrophs resistant to 5-FOA. Spontaneous resistance to 5-FOA (8 mM) was observed at a frequency of approximately 10~5 at 60°C. One such mutant contained a deletion the included part of the carboxy terminus of the pyrF (Cbesl377) open reading frame, the entire pyrC (Cbesl376) open reading frame and the amino terminus of pyrB (Cbesl375) open reading frame diagrammed in Figure 9 A, and was used for further analysis.
The extent of the deletion was defined by PCR amplification of the pyrBCF region in the mutant (Figure 9C) and subsequent sequencing of the PCR product. Since mutations in pyrE also lead to uracil auxotrophy and 5FOA resistance, the region around the pyrE locus was amplified from this strain and sequenced to ensure that it was wild type. While the deletion would be expected to affect only the pyrBCF genes, qPCR analysis was performed to monitor expression of the pyrA gene as well as the Cbesl374 open reading frame predicted to encode a uracil xanthine permease. Expression of pyrA and Cbesl374 in the deletion mutant was
indistinguishable from the wild type, suggesting that the deletion within the pyrBCF locus did not affect expression of surrounding genes.
The ApyrBCF strain was a tight uracil auxotroph and because it contained a deletion, reversion to uracil prototrophy was not a concern making prototrophic selection possible no matter how low the frequency of transformation. Growth of this mutant supplemented with uracil (20 μΜ) was indistinguishable from that of the wild type, reaching a cell density of 1.5 x 108 in 20 hours. To assay transformation, a non-replicating plasmid was constructed with the wild type copy of the pyrBCF locus but containing an engineered restriction site within the cassette to distinguish it from the chromosomal wild type allele. This plasmid was used to transform the pyrBCF deletion strain selecting marker replacement events that repaired the deletion (strategy diagrammed in Figure 9A).
We were unable to transform C bescii in many attempts using this strategy with DNA isolated from E. coli. We used and modified methods known to work well for other Gram- positive bacteria including electroporation, artificially induced competence, natural competence, and methods that altered membrane permeability. Mating with E. coli, a method of DNA transfer that works well for similar bacteria, did not work for C. bescii or the other Caldicellulosiruptor species we tested using the same approach.
In vivo and/or in vitro methylation of DNA from E. coli partially protects DNA from cleavage but does not allow transformation of C. bescii
Cbel, a potent restriction endonuclease in C. bescii, recognizes and cleaves the same sequence as Haelll, unmethylated DNA at the sequence 5'-GG/CC-3'. Plasmid DNA treated with purified M.Haelll in vitro was partially protected from cleavage by both Haelll and Cbel in vitro (Figure 7), but no transformants were obtained when this DNA was used in electroporation experiments or added to cells that had been subjected to a procedure to induce natural competence in Mycobacterium and Thermoanaerobaterium species. In addition, various strains of E. coli containing combinations of methyltranferases were used to prepare DNA for transformation (Table 3, below in Example 3), a method that was successful for transforming Clostridium thermocellum using a dam+dcm E.coli strain (Lynd and Guss, personal
communication). No transformants of C. bescii were detected using DNA from these strains. In total we performed more than 1000 electroporation experiments varying conditions for cell growth, transformation conditions, and assay conditions as well as using DNA from different strains of E. coli.
M.Cbel is a novel a-class N4-Cytosine methyltransferase
As shown in Figure 8A, the region of the chromosome that contains Cbel also contains an open reading frame, Cbes 2437, predicted to encode an adenine specific methyltransferase. This open reading frame was cloned into an E. coli expression vector, pDCW73 (Figure 8B) that placed a His-tag at the carboxy terminus of the protein allowing purification on a Ni-NTA column. E. coli cells containing this plasmid were viable at 23°C but not 37°C suggesting that expression of M.Cbel was toxic to growing cells. Expression of this methyltransferase was, therefore, performed at 23°C to avoid problems related to toxicity and in E. coli BL21- CodonPlus(DE3)-RIPL to alleviate problems arising from the significant differences in codon usage between M.Cbel and E. coli proteins. Purified M.Cbel from E. coli was the size predicted from the open reading frame, 33 kDa (Fig 8C). No cleavage of DNA was detected by purified Cbel at 75°C when DNA from E. coli was methylated in vitro by the purified methyltransferase (Fig 8D) and we named this enzyme M.Cbel. To determine the optimal temperature for M.Cbel methyltransferase activity, we performed the in vitro methylation reactions with purified M.Cbel at temperatures ranging from 25°C to 100°C and tested the modified DNA for restriction by Cbel. Reactions performed between 65°C and 85°C, the growth temperature range of C. bescii, resulted in the best protection against cleavage by Cbel.
Even though Cbel is an isoschizomer of Haelll and M.Cbel would be expected to methylate the same sequence as M.Haelll, methyltransferase vary in the sites of methylation and specific or cognate methylation may be required for full protection. The pattern of DNA methylation by M.Cbel was compared to that by M.Haelll using a method (Rao and Buckler- White, 1998 Nucleic Acids Res 26(10):2505-7; Bart et al, 2005 Nucleic Acids Res 33(14):el24) that relies on the fact that the extent of incorporation of fluorescently labeled dideoxynucleotides during DNA sequencing is influenced by methylated bases in the template DNA. pUC18 DNA was methylated in vitro by either M.Cbel or M.Haelll and direct sequencing of the DNA revealed that DNA methylated with M.Cbel showed a higher degree of incorporation of dideoxyguanosine in the 5'-GGCC-3' recognition sequence than DNA methylated with
M.Haelll. N4-methylcytosine results in an increase in the complementary G (GGCC) signal and this signature (Figure 8E) indicates that M.Cbel methylated DNA contains N4-methylcytosine (m4C). M.Haelll methylates the C5 position of cytosine (m5C).
Methylation of E. coli DNA, in vitro, with purified M.Cbel protein allows transformation of C. bescii
Plasmid DNA from E. coli (dam+dcm+) methylated by M.Cbel in vitro readily transformed the C. bescii ApyrBCF strain resulting in marker replacement of the deletion with the wild type allele containing the engineered Kpnl site (Figure 9C). Amplification of the pyrBCF region from wild type C. bescii resulted in a 3.2 kb product while the product generated from the deletion strain was 1.63 kb. Amplification of this region in the transformant generated a wild type size product. Digestion with Kpnl resulted in no cleavage of the product generated from the wild type or the ApyrBCF mutant. The product generated from transformant was digested with Kpnl showing that the transformant contained the allele from the plasmid and its presence in the C. bescii chromosome resulted from marker replacement (Figure 9C). Transformation efficiencies were routinely on the order of 50 transformants per microgram of non-replicating plasmid DNA (Table 3, Fig 9B). This extremely low transformation efficiency may be an underestimate of the actual efficiency as the plating efficiency of C. bescii on selective solid medium is less than 10~4 (plating 106 cells as determined by cell count resulted in fewer than 100 colonies).
While there are many challenges in the development of transformation protocols, restriction of DNA from E. coli by host bacteria is often an issue. Restriction/modification of DNA, first recognized as a mechanism of protection against phage infection, varies in effectiveness depending on the activity of restriction endonuclease and the methylation state of the DNA substrate. Methylation of DNA may either facilitate or limit the activity of
endonucleases and plays a major role in transformation of heterologous DNA no matter what the source of the DNA or the host for transformation. Transformation of DNA from E. coli to Caldicellulosiruptor bescii is apparently especially sensitive to restriction/modification and here we show that the use of a novel endogenous methyltransferase provided specific modification of DNA from E. coli that allowed efficient transformation.
M.Cbel was annotated as a D12 class N6 adenine-specific DNA methyltransferase in GenBank, but our analysis clearly shows that it functions as a cytosine specific
methyltransferase. Like all known metyltransferases it contains a conserved F G G amino acid motif that facilitates interaction with S-adenosylmethionine, the source of the methyl group in these reactions. M.Cbel also contains a DPPY motif typical of N6-adenine metyltransferases, all of which contain a (D/N)PP(Y/F) motif (Malone et al, 1995 J Mol Biol 253(4):618-32). Its SPP(Y/F) motif is the hallmark of N4 cytosine metyltransferases active site (Klimasauskas et al., 1989 Nucleic Acids Res 17(23):9823-32), making M.Cbel unusual in that it contains a DPPY motif in the active site (Fig.10). Furthermore, the M.Cbel protein has no reported significant sequence or structural similarity to any characterized N4 cytosine methyltransferase. M.Cbel possesses some similarity to DmtB from Anabaena variabilis ATCC 29413 (Figure 10), which has been shown to have m4C methyltransferase activity specific to the inner cytosine in the 5'- GGCC-3' recognition sequence. These proteins, which show 57% amino acid identity, represent a new a-class methyltransferase specific for GGCC sequence, different from the previously characterized β-class of N4 metyltransferases in hyperthermophiles, M.Sual and M.PhoI, isolated from the archaea Sulfolobus acidocaldarius and Pyrococcus horikoshii OT3, respectively.
M.Cbel is the first characterized a-class m4C methyltransferase from a hyperthermophile.
Homologs exist in two other Caldicellulosiruptor species, Calhy 0409 (88% of protein sequence identity) from C. hydrothermalis 108 and Calkr 2088 (85% of protein sequence identity) from C. kristjanssonii 177R1B.
One reason that M.Haelll is not sufficient to allow transformation of C. bescii even though it partially protects DNA from cleavage by Cbel may be the activity of Cbel itself.
M.Haelll-modified DNA (m5C) was cleaved at reasonable efficiency by purified Sual, a GGCC specific restriction enzyme completely blocked by m4C methylation at the inner cytosine residue in high concentrations. M.Haelll is also known to have a significant level of promiscuous methylation activity at non-canonical sites and may actually increase restriction activity in vector
DNA by methyl-directed restriction enzymes.
Efforts to optimize the transformation procedure for C. bescii have included adding cell wall weakening agents (isoniacin or glycine) during cell growth, altering temperature during the preparation of electro-competent cells, changing the composition of the washing and
electroporation buffers, altering incubation times and temperatures of the cells with DNA prior to electric-pulse, varying the electrical settings during the electric pulse, and altering the composition of the recovery medium and incubation period before plating onto selective medium.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
EXAMPLES
Example 1
Strains and Growth Conditions.
All Caldicellulosiruptor species were grown in the DSMZ 516 medium (Svetlichnyi et al., 1990 Mikrobiologiya 59:598-604) with the following modifications. The mineral solution contained the following (per liter): NH4C1, 0.25 g; KH2P04, 0.33 g; KC1, 0.33 g; MgCl2-6H20, 0.33 g; CaCl2-2H20, 0.33 g; yeast extract, 0.5 g; casein hydrolysate (enzymatic; US
Biochemicals; Cleveland, OH), 5 g; cellobiose, 5 g; resazurin, 0.25 mg; vitamin solution, 2 ml; trace minerals solution, 1 ml; amino acid solution, 40 ml. The vitamin solution contained the following (per liter): biotin, 10 mg; folic acid, 10 mg; pyridoxine-HCl, 500 mg; thiamine-HCl, 25 mg; riboflavin, 25 mg; nicotinic acid, 25 mg; calcium pantothenate, 25 mg; vitamin Bi2, 500 mg; /?-aminobenzoic acid, 25 mg; lipoic acid, 25 mg. The trace element solution contained the following (per liter): HC1 (25%: 7.7M), 1.0 ml; FeCl3 -4H20, 2 g; ZnCl2, 50 mg; MnCl2-4H20, 50 mg; H3B03, 50 mg; CoCl2-6H20, 50 mg; CuCl2-2H20, 30 mg; NiCl2-6H20, 50 mg; Na4EDTA (tetrasodium salt), 50 mg; (NH4)2Mo04, 50 mg; A1K(S04)2- 12H20, 50 mg. The amino acid solution contained the following (per liter): L-alanine, 1.9 g; L-arginine, 3.1 g; L-asparagine, 2.5 g; L-aspartic acid, 1.2; L-glutamic acid, 5.0 g; L-glutamine, 1.2 g; glycine, 5.0 g; L-histidine, 2.5 g; L-isoleucine, 2.5 g; L-leucine, 2.5 g; L-lysine, 2.5 g; L-methionine, 1.9 g; L-phenylalanine, 1.9 g; L-proline, 3.1 g; L-serine, 1.9 g; L-threonine, 2.5 g; L-tryptophan, 1.9 g; L-tyrosine, 0.3 g; L-valine, 1.3 g. The medium was prepared anaerobically under an argon atmosphere, NaHC03 (2 g/1) was added, and the mixture was reduced using 3 g/1 cysteine and 1 g/1 Na2S. The final pH was 6.4. The medium was filtered sterilized using a 0.22^m-pore-size sterile filter (Millipore Filter Corp., Bedford, MA). Cultures were incubated anaerobically overnight at the optimal temperature for each: C. saccharolyticus DSM 8903, 70°C; C. hydrothermalis DSM 18901 , 65°C; C. kristjanssonii DSM 12137, 78°C; C. bescii DSM 6925, 78°C; C. kronotskyensis DSM 18902, 70°C; C. lactoaceticus DSM 9545, 68°C; C. obsidiansis ATCC BAA-2073, 78°C. E. coli strain JW 261 (pDCW 68, apramycin1) was grown in LB broth supplemented with apramycin (50 μg/ml) with shaking at 37°C overnight. Chromosomal DNA from C. bescii DSM 6725 was extracted using the DNeasy® Blood & Tissue Kit (Qiagen; Valencia, CA) according to the manufacturer's instructions. The two native plasmids (pATHEOl and pATHE02) in C. bescii were isolated using the method described by O' Sullivan and Klaenhammer (O'Sullivan and Klaenhammer, 1993 Appl Environ Microbiol 59:2730-2733) with the following modifications: 200 ml of mid-log phase C. bescii cultures were harvested by centrifugation at 3500 x g for 15 minutes and suspended in Lysis buffer (containing 25% sucrose and 25 mg/ml lysozyme to enhance the cell wall degradation). pDCW68 DNA was isolated from E. coli using a Qiagen Mini-prep Kit. Plasmid constructions.
All primers used in these constructions are listed in Table 1. pJHW006 was constructed from pSET152 (GenBank: AJ414670.1) to replace the ColEI origin of replication with the pSClOl origin. The pSClOl origin was amplified from pWSK29 (GenBank: AF016889.1) using primers JH012 with an Xbal site and primer JH013 with a Kpnl site. The pSClOl containing fragment and the pSET vector digested with Xbal and Kpnl were ligated to form pJHW006. Construction of pDCW68, designed for transformation of C. bescii, required three cloning steps as well as overlapping PCR reactions. All PCR amplifications were performed using Pfu Turbo DNA polymerase (Agilent Technologies; Santa Clara, CA Technologies). A 3.936 kb PCR product containing the pSClOl replication origin, the apramycin resistance gene and the oriT (origin of transfer) was amplified from pJHW006 using primers DC 176 and DC 165 which contains a BamHI site. A 3.121 kb PCR product containing the pyrBCF region of the C. bescii genome was amplified from chromosomal DNA using primers DC 188 and DC 156 which also contained a BamHI site. The two PCR products were digested with BamHI and ligated to generate a 7.057 kb product. A 0.205-kb PCR product containing the regulatory region of a ribosomal protein (Cbes 2105) was amplified using primers DC 175 containing an Nhel site, and DC 187 using chromosomal DNA as template. A 7.066-kb fragment was amplified from the 7.057 kb product using primers DC 188 and DC 176 and ligated to the 0.205-kb fragment that had been digested by Nhel generating a 7.262-kb product. A 2.067-kb PCR fragment containing the 3' flanking region of the tryptophan synthase, alpha subunit (Cbes 1690) was amplified from chromosomal DNA using primers JF283 and JF287 and joined to a 2.045-kb kb PCR product containing the 5' flanking region of tryptophan synthase, alpha subunit (Cbes 1690) amplified from chromosomal DNA using DC 182 which contained an Aatll site and an overlapping primer JF286 using the high fidelity Pfu DNA polymerase (Agilent Technologies; Santa Clara, CA Technologies). A 4.112- kb product was then generated by overlapping PCR using the two fragments and C. bescii genomic DNA as a template. The 7.262-kb product from the second cloning step was amplified by PCR using DC 180 which contains an Aatll and DC 100. The 7.262-kb product and the overlapping product were digested with Aatll and ligated to yield pDCW68 (11.368 kb). To construct pDCW72, the 0.981 kb Cbel (Cbes 2438) open reading frame was amplified by PCR using primers DC216 and DC217 using C. bescii genomic DNA as template. The PCR product was digested with Ncol and Xhol and ligated to pET24d (Hethke et al, 1996 Nucleic Acids Res 24:2369-2376), which had also been digested with Ncol and Xhol. This vector contains a his-tag sequence that is added to the C-terminus of the expressed protein. The final plasmid was sequenced to confirm that the cloned cbel gene was in frame with the C- terminal His-tag followed by a translation stop codon.
Table 1.
Figure imgf000026_0001
Preparation of cell extracts and DNA substrates.
A cell free extract of C. bescii was prepared from a 500 ml culture grown to mid- log phase, harvested by centrifugation at 6,000g at 4°C for 15 minutes and resuspended in CelLytic B Cell Lysis Reagent (Sigma- Aldrich; St. Louis, MO) containing a protease inhibitor cocktail (Complete, ED TA- free from Roche; Madison, WI). Extracts were sonicated on ice and then centrifuged at 13,000 rpm for 15 minutes at 4°C. Supernatants were removed and used immediately for enzyme activity assays. Protein concentrations were determined using the Bio- Rad protein assay kit with bovine serum albumin as the standard.
For in vitro methylation of DNA, pDCW68 DNA (20 μg), isolated from E. coli DH5a (dam+, dcm+), was treated with Haelll Methyltransferase (New England Biolabs; Ipswich, MA) according to the suppliers instructions. To allow complete methylation, an additional 10 units of M.Haelll and 80 μΜ S-adenosylmethionine (SAM) was added to the reaction every four hours of incubation at 37°C for a total of 12 hours. The methyltransferase was inactivated by incubation at 65°C for 15 minutes. Methylated DNA was purified and concentrated using the DNA Clean & Concentrator™-25 Kit (Zymo Research; Irvine, CA). The extent of protection was determined using Haelll (New England Biolabs; Ipswich, MA) according to the supplier's instructions.
The 2.56 kb fragment containing three Haelll (5'-GGCC-3') sites used in assays with purified Cbel was generated by PCR amplification using primers DC222 and DC224 (Table 1) from pDCW68 template. PCR products were purified and concentrated by Qiaquick PCR Purification Kit (Qiagen; Valencia, CA) prior to use in the restriction assays.
Endonuclease Assays.
Reactions were performed in 60 μΐ volumes at 75°C using 0.5 μg to 1.0 μg of the DNA substrate: pDCW68, methylated pDCW68, pATHE 01 , or pATHE 02. For cell free extracts, 4 μg of total cell protein was incubated at 75°C in reaction buffer (10 mM Tris-HCl, pH 6.7, buffer containing 50 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol, 0.01% BSA). Samples (10 μΐ) were withdrawn at various time points, mixed with 6 X DNA gel-loading buffer (0.25%
Bromophenol blue and Xylene Cyanol, 18mM EDTA, and 30% of Glycerol), and then chilled to -20°C to stop the reaction. The cleavage products were separated electrophoretically on a 1.2% agarose gel.
Preparation of assay of purified Cbel protein.
BL21-CodonPlus(DE3)-RILP cells (Agilent Technologies; Santa Clara, CA
Technologies) were used for recombinant protein expression. Cells were grown at 23°C in LB broth supplemented with kanamycin (25 μg/ml) and chloramphenicol (50 μg/ml) to O.D260 0.6 and induced by the addition of 0.5 mM IPTG at 23°C for overnight. Cells were harvested by centrifugation, resuspended in CelLytic B Cell Lysis Reagent (Sigma-Aldrich; St. Louis, MO) containing protease inhibitor (Complete, EDTA-free from Roche; Madison, WI) and lysed by sonication. All purification steps were done at 4°C using the Ni-NTA Spin Kit (Qiagen;
Valencia, CA) following manufacturer's instruction. Protein concentrations were determined by Bio-Rad protein assay kit as described above. SDS/PAGE and Coomassie brilliant blue G-250 staining were as described (Sedmak and Grossberg, 1977 Anal Biochem 79:544-552). Enzyme assays with purified Cbel were carried out in 10 μΐ reaction volumes with NEBuffer 4 (20 mM Tris-acetate pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol) and 200 ng of DNA substrate. The amount of purified Cbel protein used in each reaction varied depending on the experiment and is indicated.
Bioinformatic analysis.
To produce an alignment and phylogenetic tree of 46 amino acid sequences of Haelll-like proteins, we used ClustalW, version 2 (Larkin et al, 2007 Bioinformatics 23:2947-2948) which is based on the neighbor-joining (NJ) method used for phylogenetic calculations. The tree was visualized with TreeView (Page, 1996 Comput Appl Biosci 12:357-358). To discover conserved motifs of groups of Haelll-like protein sequences, we used MEME (Bailey and Elkan, 1994 Proc Int Conf Intell Syst Mol Biol 2:28-36) and GLAM2 (Frith et al, 2008 PLoS Comput Biol 4:el000071). Default parameters were used for all analyses.
Identification of the Cbel cleavage site.
DNA fragments resulting from digestion by either Haelll or Cbel were separated electrophoretically and extracted from the gel matrix using a QIAquick Gel Extraction Kit (Qiagen; Valencia, CA). The products were then cloned into pWSK29 (Wang and Kushner, 1991 Gene 100: 195-199), which had been digested with EcoRV, using the Fast-Link™ DNA ligation kit (Epicentre Biotechnologies) and sequenced.
Determination of the site of DNA methylation by M.Cbel.
Using the method of Bart et al. (Bart et al, 2005 Nucleic Acids Res 33:el24) we determined the site of methylation by M.Cbel. This method allow the detection of DNA methylation by comparing sequencing traces in Sanger sequencing reactions between methylated and unmethyltaed DNA. The peak height of G bases incorporated to pair with N4m-Cytosine is higher than if the cytosine is unmethylated. UC18 plasmid DNA isolated from E. coli (DH5a: darn , dcrn ), untreated or methylated with M.Cbel or M.Haelll was used in the SeqDoC program (Crowe, 2005 BMC Bioinformatics 6: 133). The sequences were used to align traces and visually display the difference in corresponding peak heights between traces. There is a clear pattern that the second G in GGCC sequences is higher in M.Cbel treated plasmid, as compared to untreated. M.Haelll treated plasmid does not appear significantly different. This confirms that M.Cbel methylates the N4 position of cytosine.
Example 2
Preparation of C. bescii "competent" cells
2.5 ml of Caldicellulosiruptor species from an overnight culture are inoculated into bottles of 500 ml of appropriate medium (either Defined or Defined + Uracil). The cells are incubated at optimal temperature (78°C) up to mid- log phase (as determined by either measuring OD6oo to 0.1 or by cell counts 2 x 107 cells/ml). The cells are centrifuged at 4500 x g for 15 minutes at 25°C and the supernatant is decanted. The pelleted cells are resuspended in 50 ml of ice cold 10% sucrose. The cells are washed by twice re-centrifuging and resuspending in 10% as just described.
The washed cells pellets are resuspended in a total volume of 500 μΐ of ice cold 10%>
Sucrose after the final wash. Aliquots of 40-50 μΐ of the resuspended cells are placed into cold microfuge tubes and flash frozen by dipping them into a dry ice and ethanol mix.
Transformation Protocol
DNA (0.5-1.0 μg) is added to competent cells, mixed gently, and incubated for 10 minutes on ice. For natural transformation, the mixed competent cells are injected into 10 ml of pre-warmed complex medium (per 1 liter of medium: 20 ml of 5 Ox salts, 2 ml of 500x vitamin mix, 1 ml of lOOOx trace minerals, 40 ml of 25x amino acid solution, 50 μΐ of 5 mg/ml resazurin, 50 ml of 10% cellobiose, 2.4 ml of 1 M KH2P04, 5 ml of 10% yeast extract, and 50 ml of 10% casein hydrolysate) at 75°C, and then incubated at the optimal temperature for the given species overnight. For electrotransformation, the electroporation of mixed competent cells was performed via single electric pulse (1.0 kV, 600Ω, and 25 mF) in 1 mm cuvettes using a Bio-Rad gene Pulser, and then incubated in 10 ml of complex medium at optimal temperature overnight.
After overnight incubation, the 10 ml recovery culture is centrifuged at 3500 x g for 10 minutes and the cell pellet is washed twice with IX AT base salts. After washing, the cells are suspended in 0.28 ml of IX AT base salts. 100 μΐ of the cell suspension is placed into 4 ml of overlay solution (1.0% agar in water) and the overlay suspension is spread onto an appropriate selective medium. The plates are placed in ajar, degassed, and observed for growth after incubation for four days at 75°C. Example 3
Table 2. Strains/plasmids used and constructed.
Strains/
Plasmids Description Source
Strains
Re-classified as Caldicellulosiruptor bescii (Yang et al., 2010 Int J Syst Evol
DSM 6725a Microbiol. 60(Pt 9):201 1-5)/ Wild type (ura+/5-FOAs) DSMZC pyrABCF (partial deletion in pyrB and pyrF, and entire deletion of pyrC /(ura~
JWCB 002a /5-FOAR) This study
Recover to wild type by homologous recombination gene replacement
JWCB 005a between pDCW 70 and JWCB 002/ (ura+/5-FOAs) This study
Cbes_2437 (M.Cbel) expression strain / KanamycinR, SpectinomycinR,
JW 284b Chloramphenicol This study
Plasmids
Integrating vector to gene replacement in pyrBCF locus in
pDCW 70 iWCB002/ApramycinR This study pDCW 73 M.Cbel Expression vector / KanamycinR, SpectinomycinR, Chloramphenicol11 This study
Strains and growth conditions
Caldicellulosiruptor species were grown in modified DSMZ 516 medium (Chung et al, 2011 J Ind Microbiol Biotechnol 38: 1867-1877) at a final pH 6.8. Liquid cultures were inoculated with a 1-2% inoculum or with a single colony and then incubated at 75°C overnight in anaerobic culture bottles or Hungate tubes degassed with at least three cycles of vacuum and argon. A solid medium was prepared by mixing an equal volume of liquid medium at a 2X concentration with 1% (wt/vol) Phytagel (Sigma- Aldrich; St. Louis, MO) previously autoclaved to solubilize. Both solutions were maintained at 95°C and poured into petri dishes immediately after mixing. Initial plating of C. bescii in soft agar overlays allowed the cells to grow but they did not form discrete colonies because of the soft and liquid nature of the agar matrix. Increasing the agar concentration from 0.3% to 1.5% in the overlay allowed both abundant growth and the isolation of discrete colonies. Cells from overnight cultures were pelleted, washed in IX base salts (Chung et al, 2011 J Ind Microbiol Biotechnol 38: 1867-1877) three times and resuspended in 300 - 500 μΐ of lx base salts. 100 μΐ of the cell suspension was mixed with 4 ml of soft top agar (1.5%) and poured across the top of a solid medium Plates were incubated in anaerobic jars degassed with at least three cycles of vacuum and argon at 75°C for 3 to 5 days. E. coli strains, DH5a (dam+dcm+), BL21 (dam+dcm+), or ET12567 {dam dcm ) were used to prepare pDCW70 DNA. Cells were grown in LB broth supplemented with apramycin (50 μg/ml) and plasmid DNA was isolated using a Qiagen; Valencia, CA Mini-prep Kit. Chromosomal DNA from C. bescii DSM 6725 was extracted using the Quick-gDNA™ MiniPrep (Zymo) according to the manufacturer's instructions.
Isolation and characterization of 5-FOA resistant/uracil auxotrophic mutants
C. bescii DSM 6725 was inoculated into 10 ml of modified DSMZ 516 medium and grown anaerobically at 60°C for 24 hours. Cells were harvested at 18,000 x g for five minutes, washed twice with mineral solution (Chung et al., 2011 J Ind Microbiol Biotechnol 38: 1867- 1877), resuspended in 1 ml of mineral solution and plated by mixing 100 μΐ of cells with 4 ml of 0.3% agar and overlaying onto defined modified DSMZ 516 agar medium (no yeast extract or casein) supplemented with 20 μΜ uracil and 8 mM 5-FOA (US Biologicals; Swampscott, MA). The plates were incubated anaerobically at 60°C for three days and 5-FOA resistant colonies were transferred to 10 ml of defined modified DSMZ 516 medium with 20 μΜ uracil and 8 mM 5-FOA and incubated overnight at 75°C anaerobically. To test for uracil auxotrophy, cells were subcultured in defined modified DSMZ 516 medium with or without uracil (20 μΜ). Cell number was measured in a Petroff Houser counting chamber using a phase-contrast microscope with 40X magnification. Plasmid Construction and DNA manipulation
Primers used in these constructions are listed in Table 4. All PCR amplifications were performed using Pfu Turbo DNA polymerase (Agilent Technologies; Santa Clara, CA
Technologies). A 1.858 kb fragment containing the pSClOl replication origin was amplified from pDCW68 (Chung et al., 2011 J Ind Microbiol Biotechnol 38: 1867-1877) using primers DC081 and DC230, which contain Kpnl and Aatll sites, respectively. A 4.343 kb fragment containing the apramycin resistance and pyrBCF cassettes was amplified from pDCW68 using primers DC084 and DC232 to which an Aatll and Kpnl site had been added. An additional fragment (1.801 kb) containing DNA sequences not relevant to the experiments described here was amplified using primers DC212 and DC213. These three DNA fragments were cut by restriction enzymes, Kpnl and Aatll, and then ligated to yield pDCW69 (8.014 kb). pDCW70 was constructed by introducing a single nucleotide change (an A to C transversion) in the +978 amino acid of pyrC (Cbes 1376) ORF using "PCR based Site Directed Mutagenesis", using DC 214 and DC 215 primers, to create the Kpnl site (GGTAC/C), in pDCW 69. To construct pDCW73, the 0.837 kb M.Cbel (Cbes 2437) open reading frame was amplified by PCR using primers DC238 and DC239 using C. bescii genomic DNA as template. The PCR product was digested with BamHI and Xhol and ligated to pET24d (Hethke et al., 1996 Nucleic Acids Res 24(12):2369-76), which had also been digested with BamHI and Xhol. This vector contains a His-tag sequence that is added to the C-terminus of the expressed protein. All plasmids used in this study were sequenced to confirm their structure.
Purification of His-tagged M.Cbel and in vitro methylation of DNA
Purification of M.Cbel was similar to the method described by Chung et al. (Chung et al, 2011 J Ind Microbiol Biotechnol 38: 1867-1877). BL21-CodonPlus(DE3)-RILP cells (Agilent Technologies; Santa Clara, CA Technologies), containing pDCW73, was used for M.Cbel protein expression. Cells were grown at 23°C in LB broth supplemented with kanamycin (25 μ /ιη1) and chloramphenicol (50 μ^ιηΐ) to OD60o 0.7 and induced by addition of 0.5 mM isopropyl b-D-l-thiogalactopyranoside (IPTG) at 23°C overnight. His-tagged (carboxy terminus) M.Cbel was purified as described previously (Chung et al, 2011 J Ind Microbiol Biotechnol 38: 1867-1877) except for the use of a His-Spin Protein Miniprep™ (Zymo Research; Irvine, CA). Protein concentration was determined by the Bio-Rad protein assay using bovine serum albumin (BSA) as the standard. Purified protein was displayed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 1996 Comput Appl Biosci 12(4):357-8) and stained with Coomassie brilliant blue G-250 as described (Sedmak and Grossberg, 1977 Anal Biochem 79(l-2):544-52). Protein purity was determined to be >98%.
For in vitro methylation, DNA isolated from E. coli DH5a (dam+dcm+) was treated with either M.Cbel or M.Haelll methyltransferase (NEB). 50 ng of purified M.Cbel was incubated with 50 mM Tris-HCl, 50 mM NaCl, 80 μΜ S-adenosylmethionine (SAM, Samuelson and Xu, 2002 J Mol Biol 319(3):673-83), 10 mM Dithiothreitol (DTT) at pH 8.5 and 20 μg of DNA substrate in 400 μΐ reaction, and incubate for two hours at 78°C. The M.Haelll methylation reaction was performed according supplier's instructions. To allow complete methylation, an additional 10 units of M.Haelll and 80 μΜ SAM was added to the reaction every four hours of incubation at 37°C for a total of 12 hours. Methylated DNAs were purified and concentrated by Phenol/Chloroform extraction and ethanol precipitation. The extent of protection was determined by cleavage using Haelll and Notl (NEB) restriction enzymes according to the supplier's instructions.
Analysis of methylation by M.Cbel
To identify the site of methylation by M.Cbel, pre-modified DNA was compared to that after methylation and the changes were determined by direct visualization in automated DNA sequencing chromatograms (Rao and Buckler- White, 1998 Nucleic Acids Res 26(10):2505-7; Bart et al, 2005 Nucleic Acids Res 33(14):el24). In vitro methylation of pUC18 DNA isolated from E. Coli DH5a (dam+dcm+) was carried out using M.Haelll (NEB) and purified M.Cbel. The efficiency of methylation was determined by cleavage of the methylated and unmethylated DNA with Haelll (NEB), purified Cbel, and C. bescii cell free extracts (CFE). Digested DNA was displayed by agarose gel electrophoresis and visualization using ethidium bromide staining. Automatic sequencing was performed using primers M13F(-20) and M13R(-20) in an ABI automated PRISM big-dye-terminator system (Macrogen, Inc.; Rockville, MD). Sequences were analyzed using the Chromas Lite v2.01 (Technelysium Pty Ltd.) and ABI chromatograms were compared by aligning the Sequencing traces and using SeqDoc (Crowe, 2005 BMC
Bioinformatics 6: 133). Transformation of C. bescii
To prepare cells for transformation, 2.5 milliliter of a freshly grown JWCB002
(ApyrBCF) culture was inoculated into 500 ml of fresh medium, and incubated at 78°C to mid- log phase (O.D6oo - 0.1 or 2 x 107 cells/ml). The cultures were cooled to room temperature for 1 hour, harvested by centrifugation (5000 x g, 15 minutes) at 25°C and washed twice with 250 ml of pre-chilled 10% sucrose. After the final wash, the cell pellets were resuspended in a total volume of 1 ml of pre-chilled 10% sucrose and aliquots of 50 μΐ were freeze-dried in
microcentrifuge tubes in a dry ice/ethanol bath. Plasmid DNA (0.5-1.0 μg) was added to cells, gently mixed and incubated in 10% sucrose for 15 minutes at room temperature.
Electrotrans formation of the cell/DNA mixture was performed via single electric pulse (1.8 kV, 600Ω, and 25 μΕ) in a pre-chilled 1 mm cuvette using a Bio-Rad gene Pulser. After pulsing, cells were incubated overnight at 75°C in 10 ml modified DSMZ 516 medium supplemented with 20μΜ of uracil, harvested by centrifugation (at 5000 x g for 20 minutes) and resuspended in 1 ml of lx base salt. A cell suspension (100 microliters) was plated onto defined medium without uracil. A solid defined modified DSMZ 516 medium (no yeast extract and casein) was prepared by mixing an equal volume of 2X liquid medium with 1% (wt/vol) previously autoclaved Phytagel (Sigma-Aldrich; St. Louis, MO). Both solutions were maintained at 95°C prior to mixing and immediately poured into petri dishes. Transformation mixtures were incubated overnight at 78°C in 10 ml modified DSMZ 516 medium supplemented with uracil. Cells were harvested by centrifugation, resuspended in 1 ml of lx base salt (Chung et al, 201 1 J Ind Microbiol Biotechnol 38: 1867-1877), (100 microliters) mixed with 4 ml of soft agar (0.3 % agar), that had been melted at 100°C and cooled in a 45°C heating block and plated onto defined medium without uracil. Plates were incubated in anaerobic jars at 75°C for three to four days. To confirm marker replacement of the pyrBCF region in the transformants, DNA from uracil prototrophic transformants was used to amplify the chromosomal region using primers DC 163 and DC 188 which anneal outside the regions of the pyrBCF fragment contained on pDCW70. PCR products of this locus amplified from the wild type, the deletion mutant and the
transformants were digested with Kpnl and sequenced. RNA extraction and RT-qPCR analyses Total RNA was extracted using an RNeasy Mini kit (Qiagen; Valencia, CA) and stored at -80°C. RNA was treated with RNase-free DNase (Qiagen; Valencia, CA) according to manufacturer's instructions. cDNA was then prepared using the Affinity Script quantitative PCR (qPCR) cDNA synthesis kit (Agilent Technologies; Santa Clara, CA Technologies). All quantitative reverse transcription-PCR (RT-qPCR) experiments were carried out with an
Mx3000P instrument (Stratagene; a part of Agilent Technologies; Santa Clara, CA) with the Brilliant SYBR green qPCR master mix (Agilent Technologies; Santa Clara, CA Technologies). The gene encoding pyruvate ferredoxin oxidoreductase (Cbes 0876) was used as an internal control for RNA. The primers used in RT-qPCR experiments are listed in Table 4.
Table 3. Influence of different methylation status on pDCW70 in transformation efficiency into JWCB002 (ApyrBCF) strain.
E. coli strain as source of pDCW70/ Transformation efficiency
Methylation status (Transformants^g of DNA)a
DH5a (dam+dcm+) NDb
BL21 (dam+dcm) NDb
ET 12567 {dam dan ) NDb
DH5a (dam+dcm+)MMae NDb
DH5a (dam+dcm+)/M.CbeI ~50c
aEach transformation experiment used approximately 109 cells and 600 ng of transforming DNA bND (Not detected): based on at least 30 independent transformation experiments.
cAverage of the results of five independent transformation experiments.
Table 4. Primers
Primers Sequences (5'→ 3") SEQ ID NO:
DC081 ACCAGCCTAACTTCGATCATTGGA 19
DC084 TCTGACGCTCAGTGGAACGAA 20
DC156 TTAAGAGATTGCTGCGTTGATA 21
DC163 TCCTGAACCAATAACCAAAACCT 22
DC188 TTGAAACATTTGCTTGGGCTAAGA 23
DC212 ACCCTCAAATATAACACAAAAATTGTCCAC 24
DC213 GTTATTATTCTCTGTGGATAAGTC 25 DC214 AGCGGTACCATTGGGTTTGAGAC 26
DC215 TGCAGCAAGGTTAAATTCGACATT 27
DC230 TCATCTGTGCATATGGACAG 28
DC232 TAAGAGATTGCTGCGTTGATA 29
DC238 AGAGGATCCATGCTCAAAAACGTTCTTCGATAC 30
DC239 TCTCCTCGAGCAGACCAAGTGCGTATTTTTC 31
DC326 TCAGGTCCTGCTATAAAGCCAA 32
DC329 AGGTGTTTGAGAGATTTCCAAGG 33
M13F(-20) GTAAAACGACGGCCAGT 34
M13R(-20) GCGGATAACAATTTCACACAGG 35
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:
1. An isolated polynucleotide comprising the coding region of Cbes 2438.
2. A vector comprising the polynucleotide of claim 1 operably linked to a promoter.
3. An isolated polynucleotide comprising the coding region of Cbes 2437.
4. A vector comprising the polynucleotide of claim 3 operably linked to a promoter.
5. A cell comprising the vector of claim 2.
6. A cell comprising the vector of claim 4.
7. An isolated polypeptide comprising an amino acid sequence encoded by the coding region of Cbes 2438.
8. An isolated polypeptide comprising an amino acid sequence encoded by the coding region of Cbes 2437.
9. A method comprising:
incubating a DNA molecule comprising at least one 5'-GGCC-3' sequence with a Cbel polypeptide under conditions effective for the Cbel polypeptide to digest the DNA at the 5'- GG/CC-3* sequence.
10. A method comprising :
treating a DNA molecule comprising at least one 5'-GGCC-3' sequence with a M.Cbel polypeptide under condition effective for the M.Cbel polypeptide to methylate at least one C residue of the 5'-GGCC-3' sequence.
11. A method comprising:
introducing a heterologous polynucleotide into a microbial cell that comprises a thermophile or a hyperthermophile.
12. The method of claim 11 wherein the heterologous polynucleotide comprises a DNA molecule treated according to the method of claim 10.
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WO2022229330A1 (en) 2021-04-28 2022-11-03 BluCon Biotech GmbH Enzyme composition with at least two different thermostable polypeptides having type ii dna methyltransferase activity

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US20110306089A1 (en) * 2010-02-12 2011-12-15 Sowers Kevin R Thermophilic methanogenic consortium for conversion of cellulosic biomass to bioenergy

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US20110306089A1 (en) * 2010-02-12 2011-12-15 Sowers Kevin R Thermophilic methanogenic consortium for conversion of cellulosic biomass to bioenergy

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BISTA, S.: "Cloning and Characterization of a Novel Thermostable Endoglucanase from Caldicellulosiruptor Bescii in Heterologous Host Escherichia Coli.", TAMPERE UNIVERSITY OF TECHNOLOGY, August 2011 (2011-08-01), pages 1 - 63 *
CHUNG, D. ET AL.: "Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes.", J. IND. MICROBIOL. BIOTECHNOL., vol. 38, no. LL, May 2011 (2011-05-01), pages 1867 - 1877 *
CHUNG, D. ET AL.: "Methylation by a Unique a-class N4-Cytosine Methyltransferase is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725: Use for Construction of Mutants That Affect Biomass Utilization.", GENOMIC SCIENCE AWARDEE MEETING X, February 2012 (2012-02-01), pages 1 - 45 *

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WO2022229330A1 (en) 2021-04-28 2022-11-03 BluCon Biotech GmbH Enzyme composition with at least two different thermostable polypeptides having type ii dna methyltransferase activity
WO2022228669A1 (en) 2021-04-28 2022-11-03 BluCon Biotech GmbH Restriction/modification system and uses therof

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