Fig. 2. ccDNA reverse-transcribed by DRT2 can be transcribed into RNA.

(A) Conservation at each position for 44 homologs of the K. pneumoniae DRT2 ncRNA. Conservation was calculated as the difference in entropy from maximum entropy at each position. The tan shaded background represents the template region. The moving average was calculated with a window size of 10 bp. (B) Sequence logo derived from an alignment of 42 homologs of the DRT2 ncRNA template region (shown in reverse complement to the ncRNA sequence). (C) The same sequence logo rearranged to correspond to a ccDNA repeat junction. Putative conserved promoter elements are shaded. (D) ccDNA assay by PCR, and RT-PCR assay to test for transcription of the ccDNA. WT, wild-type DRT2 system; YCAA, reverse-transcriptase active site mutant of DRT2 system; –10, mutation of the putative –10 sequence from TATAAT to CGCGGC; –10*, the same mutation combined with mutation of ncRNA bases ₂₄AUU₂₆ to ₂₄GGC₂₆ to restore pairing with the mutant –10 sequence; –35, mutation of the putative –35 sequence from TTGACA to AAAGGC; EV, empty vector control. (E) T5 defense (fold reduction in PFUs) and T5-induced ccDNA production (qPCR normalized to WT) for each of the promoter mutants. (F) GFP reporter assay. GFP production was assessed as the background-corrected GFP fluorescence when cells reached OD₆₀₀=0.5. Reporter 1 contains the reverse-complement of the 120-bp ncRNA template region upstream of the GFP start codon. Reporter 2 has the same repeat permuted as found in ccDNA leading up to the start codon. Reporters 3 and 4 are identical to 2 but with the –10 or –35 mutants described above. Fluorescence was determined for three independent replicates.