Figure 1.
Key features of integration and self-replicating constructs.
Panel A, OligoIR and OligoDR constructs. The hph (HygR, dark blue) gene was interrupted by two pairs of IRs (top) or one pair of DRs (bottom; black and maroon arrows over blue/red) flanking the aacC1 (GmR, green) gene in both orientations (A and B). These cassettes were further flanked by the aph (KmR, orange) gene on the left and the lacZ (blue color, light blue) gene on the right. Panel B, detailed DNA sequence of the IRs and DRs and the relevant restriction sites (EcoRI and ScaI) used during cloning. Note that because the sequence is given in the 5′→3′ direction with respect to the hph gene, the map is opposite to that shown in Panel A.
Figure 2.
Direct and inverted repeats are overrepresented in mycobacterial and E. coli genomes.
Genome-wide distributions of DRs and IRs. Panels A and B, E. coli K12; panels C and D, M. smegmatis; panels E and F, M. tuberculosis; left panels, direct repeats (DR); right panels, inverted repeats (IR); x-axes, length of repeats in nucleotides; y-axes, number of repeats; open symbols, predicted random distributions; closed symbols, observed distributions.
Figure 3.
OligoDR and OligoIR induce genetic instabilities in M. smegmatis and E. coli.
Panel A, reconstitution of hph (HygR) gene through the precise deletion of the aacC1 (GmR) is only detected with the integrated OligoDR, but not OligoIR, in M. smegmatis. x-axis, M. smegmatis mc2155 strains: pCI, pMV306KmHyg::Gm (control); pIRIA, pMV306KmHyg::OligoIRGmAlacZ; pIRIB, pMV306KmHyg::OligoIRGmBlacZ; pDRIA, pMV306KmHyg::OligoDRGmAlacZ; pDRIB, pMV306KmHyg::OligoDRGmBlacZ; y-axis, frequencies of HygR CFUs: asterisks, p<0.05 (pair-wise Holm-Sidak test for pIRIA, pIRIB, pDRIA, pDRIB against pCI). Panel B, large deletions occur in both the integrated OligoDR and OligoIR constructs in M. smegmatis. x-axis, as in Panel A; y-axis, percent GmS (black bars), KmS (light grey bars), and white (dark grey bars) CFUs; IR, OligoIR; DR, OligoDR.; asterisks, p<0.05 (χ2 test of pIRIA, pIRIB, pDRIA, pDRIB against pCI, z-test of pIRIA against pIRIB and pDRIA against pDRIB). Panel C, OligoIR induces deletions in a supercoiling-dependent manner in E. coli. Percent GmS and KmS CFUs in E. coli strains containing various steady-state levels of negative supercoil density. x-axis, E. coli strains carrying the self-replicating OligoIR (pIREA) or OligoDR (pDREA) vectors: JTT1, wild-type; RS2, topA mutant, higher negative supercoil density than JTT1; SD2, topA and gyrB mutant, lower negative supercoil density than JTT1; y-axis, percent GmS and KmS CFUs, geometric mean (+/−SD) from 100 CFUs obtained from 3 independent experiments; black bars, OligoIR (pIREA); grey bars, OligoDR (pDREA); asterisks, p<0.05 (χ2 test of SD7 against RS2 and RS2 against JTT1).
Figure 4.
Deletions occur independently of NHEJ and HR in M. smegmatis.
Panel A, precise deletions stimulated by DRs occur independently of NHEJ and are increased in the absence of RecA. x-axis, M. smegmatis strains: wt/pDRIA, wild-type/pMV306KmHyg::OligoDRGmAlacZ; Δku, Δku/pDRIA; Δ(ku ligD), ΔkuΔligD/pDRIA; ΔrecA, ΔrecA/pDRIA; Δ(recA ku ligD), ΔrecAΔkuΔligD/pDRIA; y-axis, frequencies of HygR CFUs; asterisks, p<0.05 (pair-wise Holm-Sidak test for mutant strains against wild-type). Panel B, large deletions are stimulated by HR (RecA) in E. coli. x-axis, E. coli strains carrying the self-replicating OligoIR vector pIREA; wt, KMBL1001; ΔrecA, Top10 mutant; asterisks, p<0.05 (χ2 test of mutant strains against wild-type). Panels C and D, large deletions occur independently of NHEJ and HR in M. smegmatis. x axis, M. smegmatis strains; y-axis, percent KmS (black bars) or GmS (grey bars) CFUs. Panel C, M. smegmatis strains: wt/pCE, wild-type/pMV206KmHyg::Gm (control); wt/pIREA, wild-type/pMV206KmHyg::OligoIRGmAlacZ; Δ(ku ligD), ΔkuΔligD/pIREA; ΔrecA, ΔrecA/pIREA. Panel D, M. smegmatis strains: wt/pCE, as in Panel D; wt/pDREA, wild-type/pMV306KmHyg::OligoDRGmAlacZ; Δ(ku ligD), ΔkuΔligD/pDREA; ΔrecA, ΔrecA/pDREA (asterisks, p<0.05 (χ2 test of mutant strains against wild-type).
Figure 5.
RecBCD deficiency increases the frequency of precise deletions between IRs. Panels A and B, x-axis, M. smegmatis strains; y-axis, frequencies of HygR CFUs; asterisks, p<0.05 (pair-wise Holm-Sidak test for ΔrecBCD M. smegmatis against wild-type). Panel A, wt/pCE, wild-type/pMV206KmHyg::Gm (control); wt/pIREA, wild-type/pMV206KmHyg::OligoIRGmAlacZ; ΔrecBCD/pIREA, ΔrecBCD/pMV206KmHyg::OligoIRGmAlacZ. Panel B, wt/pCE, as in Panel A; wt/pIREB, wild-type/pMV206KmHyg::OligoIRGmB; ΔrecBCD/pIREB, ΔrecBCD/pMV206KmHyg::OligoIRGmB.