B. sutilis 168 and mutant strains were pregrown overnight in liquid LB medium
at 37℃
to stationary phase (ca. 2-8 x 109 cells/ml). Aliquots of cultures
were serially diluted (1:100, 1:1,000, 1:10,000 and 1:100,00) and cell
suspensions were subsequently transferred to 96-well microtiter plate serving as
a master plate. Two microliters of cell suspensions were spotted on plates
containing inhibitors or antibiotics. Plates were then incubated at 37℃ for 36 hrs, and growth and β-galactosidase activity of the mutants was scored by comparison
with that in the control condition (LB) or with growth of the control strain
(168).
The
following compounds and X-gal (100μg/ml)
were added to LB media after autoclaving. LB (LB broth
LENNOX) and NB (nutrient broth) were obtained from Difco. Growth
conditions are as follows (final concentrations in LB media): H2O2, 0.5mM and
1.3 mM; Paraquat, 10μM
and 70μM;
EtOH, 2% and 8%; NaCl, 0.5M and 1M; Xylose, 0.5M and 0.8M; SDS, 0.001% and
0.007%; SDC (sodium deoxycholate), 0.001% and 0.007%; CCCP (carbonylcyanide-m-chlorophenylhydrazone),
2μM
and 7μM;
MC (Mitomycin C), 10 ng/ml and 15 ng/ml; PEN G (Penicillin G), 1 ng/ml and 7 ng/ml;
STR (Streptomycin), 2μg/ml
and 10μg/ml;
TRIM (Trimethoprim), 0.1μg/ml
and 1ug/ml.