Supplementary Material and Methods s3

Supplementary Material and Methods

Statistical analysis of data

b-galactosidase assays were performed in duplicate. Approximately 40,000 duplicate assays were performed for this and an accompanying paper (Martin & Rosner, in preparation). When each pair of assays was normalized to 100 and compared, the standard deviation was ± 3.178%. However, when the duplicates for a particular strain were compared (standard error of the mean), the deviation was considerably higher and dependent on the promoter being analyzed. We believe the major contribution to this increase is the fact that the basal activity of several of the promoters is dependent on growth phase. In the most extreme example, the basal b-galactosidase activity of the fumC::lacZ fusion varied by over 10-fold between very early log phase growth (OD600 =

0.05) and early stationary phase growth (OD600 = 0.9). And although great care was taken to try and achieve the same cell density at the end of the induction period for each pair of assays, A600 readings of 0.1 to 0.25 were accepted. The standard error of the mean for most of the IPTG-derepressed promoter fusions in the lon clpP strains were ~

± 12%, for example: fpr, ± 14.5%; fumC, ± 14.7%; mar, ± 10.9%; micF, ± 9.5%; sodA,

± 7.7%; ybjC, ± 6.8%; yhbW, ± 10.3%; and zwf, ± 9.8%.

The standard errors of the mean for the parameter presented in Tables 2 and 4 and in

Figure 1 are considerably larger since they are dependent both on the subtraction of the values for the induced minus the uninduced and on the division of those values for mutant and wild-type. In those cases where the derepression by IPTG was small, the variance of the parameter became particularly prominent. For the worst example, fpr, the bgalactosidase activity for the uninduced fusions were 80 ± 12.8 MU and the induced cultures were only 2.3-fold greater, 180 ± 26.1 MU. This meant that the results for each of the mutants had to be divided by 100 ± 29.1. Thus, even though the derepressed and repressed values for SoxS were 2380 ± 190.4 and 80 ± 12.8 giving a calculated difference of 2300 ± 190.8 (i.e. 2300 ± 8.3%), the parameter listed in Tables 2 and 4 has a value of (2300 ± 190.8)/(100 ± 29.1) or 23.0 ± 7.0 (i.e. 30 %).

When the data was not normalized (which was necessary for the comparison of different promoters) and the standard errors of the mean for each set of assays was

<±15% as in Fig 3, the differences were striking.