(A) Analysis by Western blotting of two T-POP mutants (M-1, lane 1; M-2, lane 2) of serotype Typhimurium strain MA1 (LT2 misL::lacZYA) that exhibited a “blue” colony phenotype on LB-tetracycline-X-Gal agar plates for expression of MisL. Bacteria were grown in LB broth in the presence (Tc+) or the absence (Tc−) of tetracycline. The presence of an approximately 100-kDa protein band (MisL) in the mutants is indicated by an arrow. (B) Quantification of β-galactosidase activity in T-POP mutants (MA1-1 and MA1-2) of serotype Typhimurium strain MA1 (LT2 misL::lacZYA) grown in LB broth in the presence (open bars) or the absence (closed bars) of tetracycline. The bars represent averages from three independent measurements ± standard deviations.

Expression of misL in serotype Typhimurium strain MA1 (LT2 misL::lacZYA) detected by Western blotting (right) or by measuring β-galactosidase activity (left). Strain MA1 was transformed with a plasmid carrying the promoterless marT gene cloned behind the arabinose promoter (pCT6), and the bacteria were grown in the presence of the l-arabinose concentrations indicated on the left. The presence of an approximately 100-kDa protein band in the Western blot, which corresponds to MisL, is indicated by an arrow.

(A) EMSA for binding of GST or GST-MarT to the misL promoter region. Purified GST (Lane 1; 5 μg/lane) or purified GST-MarT fusion protein (lane 2; 5 μg/lane) incubated with a biotin-labeled DNA fragment containing the misL promoter region. (B) EMSA for binding of GST-MarT to the misL promoter region in the absence of unlabeled DNA (lane 1) or in competition with 10-fold excess of the unlabeled DNA fragment containing the misL promoter region (lane 2) or 10-fold excess of an unrelated promoter region (lane 3).

Detection of MisL expression in serotype Typhimurium strain LT2 by Western blotting. A plasmid in which the arabinose promoter drives the expression of a promoterless marT gene (pCT6) was introduced into strain LT2, and the bacteria were grown in the presence of the l-arabinose concentrations indicated on the right. The presence of an approximately 100-kDa protein band corresponding to MisL is indicated by an arrow.

Induction of MisL expression (A) and binding of the misL promoter region (B) by a GST-MarT fusion protein. (A) Western blot of serotype Typhimurium strain LT2 containing plasmid pGEX-4T-2 (lane 1) or a plasmid encoding a GST-MarT fusion protein (pCT17) (lane 2) detected with anti-MisL serum. The presence of an approximately 100-kDa protein band in the Western blot, which corresponds to MisL, is indicated by an arrow. (B) EMSA with a biotin-labeled DNA fragment containing the misL promoter region. Lanes 1 to 7 show biotin-labeled DNA in the absence of protein (lane 1) or in the presence of increasing concentrations (1, 2, 4, 6, 7, and 7.5 μg/lane) of purified GST-MarT fusion protein (lanes 2 to 7). Lane 8 contained GST-MarT in the absence of DNA.