Figure 2

Monitoring expression of the gene ribM from Streptomyces davawensis in recombinant Bacillus subtilis strains containing the plasmid pHT01ribM _opt . (A) Cell samples (normalized to an OD₆₀₀ of 1) were taken 3, 6 and 9 h after treatment with IPTG and total RNA was prepared. Control cells were not treated with the inducer. The level of ribM _opt -transcript was assessed by Northern blot analysis using a ribM _opt -specific digoxigenin labelled DNA probe. The predicted size of the ribM _opt transcript is 0.8 kb (see arrow). Lanes 1, 6 and 10: B. subtilis 168 < pHT01 >; Lanes 2, 7 and 11: B. subtilis 168 < pHT01 > induced with 1 mM IPTG; Lanes 3, 8, and 12: B. subtilis 168 < pHT01ribM_opt >; Lanes 4, 9 and 13: B. subtilis 168 < pHT01ribM_opt > induced with 1 mM IPTG; Lane 5, no sample. (B) After growth in LB and treatment with IPTG B. subtilis < pHT01ribM_opt > cells were collected and cell free extracts were prepared. Cytoplasmic fractions and membrane fractions were subjected to SDS-PAGE, followed by transfer to a nitrocellulose membrane and analysis with anti-penta-his mouse monoclonal antibodies/goat anti-mouse IgG alkaline phosphatase (AP)-coupled secondary antibodies. Lane M, protein marker (in kDa); lane 1, B. subtilis < pHT01 > cytoplasmic fraction; lane 2, B. subtilis 168 < pHT01 > membrane fraction; lane 3, B. subtilis 168 < pHT01ribM_opt > cytoplasmic fraction; lane 4, B. subtilis 168 < pHT01ribM_opt > membrane fraction; (C-terminally his₆-tagged RibM from Streptomyces davawensis, calculated molecular mass: 24.7 kDa).