Fig. 1

The inserted mutation of the tdk gene by IS1447 and its sequence features. a Diagnostic PCR confirmation of the inserted mutation of the tdk gene. The transformant ΔpyrF::pHK-HR-CglT [26] was continuously cultured in the liquid MJ medium without or with FUDR through 1–3 (lane 1–3) or 1–8 (lane 4–11) subculturings, respectively. The 2-kb band referring to the mutated tdk gene became more dominant along with increased subculturings. M, DNA marker. b Schematic representation of the IS1447 insertion in the tdk sequence during the counterselection of C. thermocellum mutant with the presence of FUDR. Three types of IS1447 insertion were detected, and the insertion positions were indicated by the coordinates based on the available Tdk-encoding sequence (1–582 bp, Teth514_0091). The duplicated DR sequences were boxed, and the IRs of IS1447 were in purple. c Predicted domain structures of IS1447. The putative encoding regions of OrfA and OrfB are indicated by blue and yellow, respectively. The terminal IRs are shown as black boxes. IRL and IRR indicate the left and right IR sequences, respectively. The helix-turn-helix (HTH), leucine zipper (LZ), and DDE motifs are shown with the positions in amino acid residues. Sequence alignments were performed for the HTH and LZ motifs of IS1447 Tpases with representative members of the major subgroups of the IS3 family. For the HTH motif, consensus hydrophobic and hydrophilic residues are yellow and green highlighted, respectively. Residues that fit the consensus are in bold. The helix structures are indicated above the sequence. For the LZ motif, the four-component heptad repeats are indicated by the numbers above the sequence, and the leucine repeats are in bold. d IRs of IS1447, IS911 and defining members of IS3-family subgroups. The dinucleotides at the 5′ terminal are yellow-highlighted. The sequence lengths and the identities between IRL and IRR are indicated to the right of the figure