Fig 2. Rv1460 represses its own expression.

(A) Schematic representation of the β-galactosidase assay procedure. Each reporter vector (pJEM15, pJEM139pro or pJEM314pro) (containing the 139 bp or 314 bp regions upstream of Rv1460 fused to a lacZ reporter gene) was co-transformed into M. smegmatis with a protein expression vector either encoding no protein (pSE100) or encoding Rv1460 (indicated by the grey oval). (B) β-galactosidase activity from the 139 bp and 314 bp promoter fragments with (pSE1460) and without (pSE100) co-expression of Rv1460 in M. smegmatis. Transcriptional repression by wild-type or variants of Rv1460 results in a decrease in β-galactosidase activity, which is expressed in Miller units calculated as follows: 200 × (change in OD_450nm) per mg protein per min. The results shown are the mean and standard deviation for three experiments. Statistical analysis compared the mean activity for each plasmid with or without co-expression of Rv1460 e.g. pJEM15 (pSE100) vs. pJEM15 (pSE1460) using an unpaired t-test (*p ≤0.05). (C) β-galactosidase activity from the 314 bp promoter (pJEM314pro) when wild-type (pSE1460) or mutated forms of Rv1460 (pSE_C203S or pSE_C216S or pSE_C242S or pSE_C244S or pSE_C203S/C216S/C244S) are expressed in M. smegmatis. The results shown are the mean and standard deviation for three experiments. Statistical analysis compared the mean activity for wild-type with each variant using an unpaired t-test.