Figure 1.
The T. elongatus TeI3c group II intron RNA and TeI4c RT components of the thermotargetron.
(A) A secondary structure model of group II intron TeI3c showing modifications used for retrohoming assays and the construction of the thermotargetron. Nucleotide residues that differ from wild-type TeI3c are shown in lower case letters, exon sequences are boxed, and restriction sites used in plasmid constructions are in bold. The T7 promoter sequence inserted in intron domain IV for plasmid-based retrohoming assays in E. coli (Fig. 2) is in italics. Greek letters denote sequence elements involved in predicted tertiary structure interactions [16]. The loops of two stem-loop structures in subdomain DIVa (shaded boxes) can potentially base pair to form the pseudoknot shown. (B) Schematic representation of the TeI4c RT, which splices and mobilizes group II intron TeI3c. Conserved protein domains are: RT, containing conserved amino acid sequence blocks RT1–7 characteristic of the finger and palm regions of retroviral and other RTs; X/Thumb; D, DNA binding; and En, DNA endonuclease. RT-0 and -2a (hatched) are additional conserved sequence blocks found in the RT domains of non-LTR-retroelement RTs [16], [72], [73]. The RT and X/Thumb domains function together in reverse transcription and specific binding of the intron RNA, which stabilizes the catalytically active RNA structure for RNA splicing and reverse splicing of the intron into the DNA target site; domain D contributes to DNA target site recognition; and the En domain cleaves the opposite strand of the DNA target site to generate the primer for reverse transcription of the reverse-spliced intron RNA.
Figure 2.
Temperature profiles of retrohoming by thermophilic group II introns in E. coli.
(A) E. coli plasmid-based retrohoming assay [13], [14], [55]. The CapR intron-donor plasmid uses a T7lac promoter (PT7lac) to express a group II intron RNA with short flanking 5′ and 3′ exons (E1 and E2, respectively) and the group II RT cloned downstream of E2. The group II intron, which has a T7 promoter sequence (PT7) inserted near its 3′ end, integrates into a target site (the ligated E1–E2 sequence) cloned in a compatible AmpR recipient plasmid upstream of a promoterless tetR gene, thereby introducing the T7 promoter and activating that gene. The assays are done in E. coli HMS174(DE3), which contains an IPTG-inducible T7 RNA polymerase. Intron expression is induced with IPTG, and mobility efficiencies are calculated as the ratio of (TetR+AmpR)/AmpR colonies. (B) Temperature dependence of intron retrohoming. Retrohoming assays were done as described in panel (A) in E. coli HMS174(DE3), using intron-donor plasmids pACD2X-TeI4h*/4h*, pACD2X-TeI3c/4c, and a derivative of pACD2X-TeI3c/4c that has been retargeted to insert into a site in the E. coli lacZ gene (see Fig. 4). Targetron expression was induced with 500 µM IPTG for 1 h at different temperatures. Recipient plasmids contain the DNA target sites for each intron from positions −30 to +15 from the intron-insertion site. Target sites 1 and 2 for TeI3/4c are the native target site for the wild-type intron and the lacZ site target site for the retargeted intron, respectively. The figure shows data from a single experiment, which was repeated with similar results.
Figure 3.
DNA target site recognition by thermotargetron TeI3c/4c.
(A) DNA target site for group II intron TeI3c showing positions recognized by the IEP (blue) and intron RNA base pairing (red). IBS1, 2, and 3 denote intron-binding sites 1, 2, and 3 in the DNA target site, and EBS1, 2, and 3 denote exon-binding sites 1, 2, and 3 located in three different regions of the intron RNA. The arrowhead indicates the intron-insertion site (IS). (B) Target site positions recognized by the TeI4c RT. Nucleotide residues recognized by the TeI4c RT were identified in a selection experiment in E. coli HMS174(DE3) with IPTG induction at 48°C for 1 h using the donor plasmid pADC2X-TeI3c/4c and a recipient plasmid library with randomized nucleotide residues at positions −35 to −13 and +2 to +20. After plating on LB medium containing antibiotics, AmpR+TetR colonies were analyzed by colony PCR and sequencing of the 5′- and 3′-integration junctions to identify nucleotide residues in active target sites. The WebLogo representation [74] depicts nucleotide frequencies at each randomized position in 105 selected target sites, corrected for biases in the initial pool based on sequences of 100 randomly chosen recipient plasmids. The x-axis shows the sequence of the intron-insertion site in the T. elongatus genome, with blue residues highlighting the positions recognized by the IEP. The Figure was redrawn from [55]. (C) Retrohoming efficiency of the TeI3c/4c targetron with different EBS3/IBS3 pairings between the intron RNA and DNA target site. Retrohoming assays were done in E. coli HMS174(DE3) with IPTG induction for 1 h at 48°C with all possible combinations of donor plasmids pACD-TT1A, pACD-TT1C, pACD-TT1G, or pACD-TT1T [EBS3(RNA)] and recipient plasmids pBRR-3c (WT, IBS3A), pBRR-3cC, pBRR-3cG, or pBRR-3cT [IBS3(DNA)]. The grid shows mobility efficiencies for each combination of nucleotides at the EBS3 position in the intron RNA and the IBS3 position in the DNA target site. The wild-type U-A pairing is indicated in bold letters. The data are from a single experiment, which was repeated with similar results.
Figure 4.
Targeted disruption of the E. coli lacZ gene at 48°C.
(A) DNA target sequences and EBS/IBS interactions for thermotargetrons designed to insert into the E. coli lacZ gene. The wild-type target sequence and EBS/IBS interactions are shown above for comparison. The arrowhead indicates the intron-insertion site (IS), and gray shading highlights nucleotide residues in the lacZ target sites that match those in the wild-type target site. The schematic of the lacZ gene below shows the location of the targetron-insertion sites and the flanking ApaI and EcoRI sites used for Southern hybridizations. (B) Time course of lacZ targeting. After inducing thermotargetron expression in E. coli HMS174(DE3) with 500 µM IPTG at 48°C, lacZ targeting frequencies were determined by blue-white screening on LB+X-Gal agar plates. The Table shows the fraction of white colonies found by Southern hybridization to contain a single targetron insertion at the desired site. (C) PCR analysis. Eight colonies (two blue (B) and six white (W)) were picked for each targetron and compared to the parental E. coli HMS174(DE3) strain (P) in three PCRs with primers that flank the targetron-insertion site to detect the targetron insert or amplify the 5′- or 3′-integration junctions (Materials and Methods). (D) Southern hybridization analysis of two blue (B) and six white (W) colonies after induction of targetron expression for 15 or 30 min (LacZ60a and LacZ369a) or 1 h (LacZ2586a) at 48°C The blots show ApaI+EcoRI-digested chromosomal DNA hybridized with 32P-labeled probes for the TeI3c intron (nucleotides 1–342) or lacZ gene (nucleotides 30–1850). The lacZ probe hybridizes to a 3.7-kb band containing the wild-type lacZ gene in blue colonies and to a 4.5-kb band containing the lacZ gene with the inserted targetron in white colonies. The intron probe hybridizes to the same 4.5-kb band in the white colonies. Additional bands due to off-target integrations are observed in some white colonies.
Figure 5.
Map of plasmid pHK-TT1A used for thermotargtetron expression in C. thermocellum.
The plasmid uses a C. thermocellum groEL promoter to express a thermotargetron cassette consisting of the T. elongatus TeI3c group II intron and flanking exon sequences followed by an ORF encoding the TeI4c RT. The targetron expression cassette is cloned in the E. coli/C. thermocellum shuttle vector pHK, which was derived from pNW33N (BGSC) and contains replication origins from E. coli plasmid pUC19 (ColE1) and Geobacillus stearothermophilus plasmid pTHT15 (RepB), as well as a chloramphenicol acetyltransferase (cat) gene from Staphylococcus aureus plasmid pC194 that has been used for selections in thermophiles at temperatures of 50–55°C [59], [60].
Figure 6.
Validated thermotargetrons for C. thermocellum.
The Figure shows target sites, EBS/IBS base-pairing interactions, and targeting efficiencies for seven targetrons that gave site-specific gene disruptions in C. thermocellum. The targeted genes and their gene products were: cipA (Clo1313_0627), cellulosome scaffoldin protein; hfat (Clo1313_2343), hypothetical formate acetyltransferase; hyd (Clo1313_0554), hydrogenase; ldh (Clo1313_1160), lactate dehydrogenase; pta (Clo1313_1185), phosphotransacetylase; pyrF (Clo1313_1266), orotidine 5′-phosphate decarboxylase. Gray shading highlights nucleotide residues in the C. thermocellum target sites that match those in the wild-type target site, which is shown above for comparison. The arrowhead indicates the intron-insertion site (IS). The targeting efficiency was calculated as the percentage of thiamphenicol-resistant transformants in which the disruption of the target gene was detected by colony PCR and confirmed by sequencing across the 5′- or 3′-intron integration junction (see Fig. 7).
Figure 7.
PCR analysis of thermotargetron insertions in chromosomal genes of C. thermocellum DSM 1313.
(A) Schematic representation of the insertion of seven targetrons into chromosomal genes of C. thermocellum DSM 1313. Genomic DNA is indicated by a double line, and the ORF of the target gene is indicated by an open arrow, whose direction indicates whether the ORF is located on the positive (5′ to 3′) or negative (3′ to 5′) DNA strand. Inserted targetrons are indicated by black boxes, with the insertion junctions indicated by arrowheads with nucleotide position numbers in the target gene. PCR-primer binding sites and primer orientations are indicated by horizontal arrows. The binding sites for the external primer sets are located within the target genes upstream or downstream of the targetron-insertion site. The internal primer Te680rc base pairs to the sense strand of the intron (nucleotide positions 658–675; Table S3). The expected sizes (kb) of the PCR products obtained with the external primers for the wild-type (WT) and disrupted genes are indicated to the right. (B) Colony PCR analysis of seven targetron insertions in chromosomal genes. Three PCRs were performed for each targetron. Lane 1, using the external primers and wild-type DNA as the template; lane 2, using the external primers and the disruptant DNA as the template; lane 3, using the external forward or reverse primer and internal primer Te680rc with the mutant DNA as the template; M, DNA markers.
Figure 8.
Southern hybridization analysis of thermotargetron insertions in chromosomal genes of C. thermocellum DSM 1313.
After curing the targetron expression plasmid, genomic DNAs of wild-type or disruptant strains were digested with EcoRI and BamHI, run in a 0.8% agarose gel, and blotted to a Nylon membrane (Hybond-NX, GE Healthcare). The blots were hybridized with a DIG-labeled probe for the TeI3c intron (nucleotide positions 539–710) and visualized by immunological detection according to the manufactor’s protocol (DIG-High Prime DNA Labeling and Detection Starter Kit I, Roche). M, DNA markers.
Figure 9.
HPLC analysis of extracellular metabolites produced by C. thermocellum wild-type DSM 1313 and mutant strains with cellobiose as the sole carbon source.
The strains were: WT, C. thermocellum wild-type DSM 1313; DSM 1313 ldh::Ldh309s; DSM 1313 pta::Pta318a; and double mutant DSM 1313 ldh::Ldh309s, pta::Pta318a. The fermentation time was 110 h, and the values are the mean for three independent fermentations with the error bars indicating the standard deviation.
Figure 10.
H1NMR spectra of the extracellular metabolites of C. thermocellum DSM 1313 strains cultured with cellobiose as the sole carbon source.
The strains were WT, C. thermocellum wild-type DSM 1313 and the double mutant DMS 1313 ldh::Ldh309s, pta::Pta318a. Peaks for lactate, acetate, ethanol, and pyruvate are marked. The ratios of the integrals of representative metabolite peaks and internal reference (0.5 mM DSS) were used to calculate the metabolite concentrations against standard curves, as described in Materials and Methods. The concentrations of pyruvate produced by the wild-type and double mutant strains were calculated to be 0.73 and 4.12 mM, respectively.