FIG. 4.

Conditionally expressed I-SceI efficiently cleaves the M. smegmatis chromosome at a supplied I-SceI site. (A) Plasmid map of pMSG375 which contains the I-SceI homing endonuclease with an N-terminal HA epitope tag under the control of Pmyc1 TetO, which confers tetracycline-inducible expression when tetR is coexpressed. The plasmid also contains a single I-SceI site and an integrase which catalyzes site-specific integration at the attB site in the mycobacterial chromosome. When integrated at attB, all plasmid sequences are contained on the chromosome, thereby creating a single unique I-SceI cleavage site. (B) AHT-dependent expression of HA-I-SceI. The indicated strains transformed with the tetracycline-inducible HA-I-SceI construct were cultured with or without 50 ng/ml AHT. Whole-cell extracts were analyzed by Western blotting with an anti-HA monoclonal antibody. The predicted size of the HA-I-SceI protein is 29.4 kDa. The anti-DlaT antibody serves as a loading control. (C) Map of the I-SceI expression cassette when integrated at the attB locus with probe location and predicted sizes of bands by Southern blotting. (D) I-SceI cleaves the mycobacterial chromosome. Southern analysis of genomic DNA prepared from M. smegmatis transformed with pMSG375 without (−) and with (+) AHT for 0.5, 1, and 2 h after AHT addition. The probe spans the I-SceI site at attB, yielding a band of 800 bp with I-SceI cleavage (Cut). wt, wild type.