Fig 1.
Predicted replication intermediates and two-dimensional gel pattern of linearized pBR322.
The diagram on the left depicts plasmid pBR322 linearized by PstI at 3,607 bp; leftward arrows, replication start site (2,535 bp). Inverted triangles, EcoRII methylation sites (at positions 131, 1,060, 1,443, 2,501, 2,622, and 2,635 bp from left to right); diamond, tightly bound MTase at a blocked fork. The diagram on the right depicts B, bubble; DY, double Y; X, X structures; OC, open circle; LM, linear monomer; star, replication intermediate blocked at the 1,060 methylation site and the resulting accumulation on the two-dimensional gel.
Fig 2.
DNA replication is blocked in cells expressing mutant EcoRII C186A methyltransferase.
DNA was digested with Pst1-HF, run on two-dimensional gel electrophoresis and visualized with Southern hybridization. Closed arrows show accumulation of bubble molecules at locations consistent with the EcoRII methylation sites at 131, 1,060, and 1443 bp. Open arrow shows disappearance of the 1,060 bp spot with the mutated pBR322-C1060A plasmid.
Fig 3.
Sensitivity to M.EcoRII C186A.
Overnight cultures of BW27783 (wild-type) and the indicated BW27783 derivatives, containing plasmid pBAD-MEcoRII-C186A, were serial diluted five-fold and spotted onto LB plates containing chloramphenicol with either glucose (0.01%) or arabinose (0.0003%, 0.001%, 0.003%, or 0.01%) and incubated at 37° overnight. Those mutants deemed to be hypersensitive are indicated with an asterisk.
Table 1.
Summary of growth in the presence of arabinose-induced TBCs.
Fig 4.
Sensitivity to quinolone antibiotics.
Overnight cultures of BW27783 (wild-type) and the indicated BW27783 derivatives, were serial diluted five-fold and spotted onto LB plates containing either ciprofloxicin (5 or 7.5 ng/mL) or nalidixic acid (1.5 or 2.5 μg/mL) and incubated at 37° overnight. Those mutants deemed to be hypersensitive are indicated with an asterisk.
Table 2.
Summary of growth in the presence of quinolones.
Table 3.
Sensitivity to M.EcoRII-C186A induced TBCs, quinolones, and aza-C induced DPCs.