Fig. 1.

Autogenous regulation of the Rv0081 promoter by Rv0081. (A) Genomic organization of the Rv0079-Rv0089 locus. Numbers below genes indicate the intergenic distance. (B) Rv0081 promoter-lacZ reporter assays were conducted in wild-type M. smegmatis carrying vector only (white bars) or vector expressing hsp60-Rv0081 (black bars). Cultures were grown with shaking or were grown statically, and β-galactosidase activity was quantified. Data are presented in Miller units. (C) His-Rv0081 was overproduced and purified using nickel nitrilotriacetic acid-agarose column chromatography. Enriched His-Rv0081 is depicted with an arrow. M, marker; L, lysate; FT, flowthrough; B, binding buffer; W, wash buffer; E, eluate; DE, dialyzed eluate. (D) Binding by His-Rv0081 was assessed using electrophoretic mobility shift assays to regions upstream of Rv0081, Rv0079, Rv0082, or hycD. Reaction mixtures contained 1.0 ng radiolabeled probe alone (lane 1) or probe with 10 ng (lane 2), 40 ng (lane 3), or 80 ng (lane 4) of His-Rv0081. (E) Binding by Rv0081 to its own upstream region is sequence specific. Radiolabeled probe (1.0 ng) from the Rv0081 upstream region was incubated alone (lane 1) or in the presence of 100 ng of His-Rv0081 (lanes 2 to 6). Reaction mixtures also contained 150-fold and 300-fold excess cold DNA from the Rv0081 upstream region (lanes 3 and 4) or nonspecific cold DNA from the hycD upstream region (lanes 5 and 6). B, bound; F, free.