Supplementary Data for Figure 3 Aldridge et al.

Average OD600 values and Calculation of 28 and FlgM half-lives

Here we present supplementary data with respect to the protein stability assays performed in figure 3. There is an increase in 28 protein levels within the first 5 mins of these assays. Stopping protein synthesis by the addition of antibiotic is a stress situation for the bacteria. In Supp_Figure 1 we include the average OD600’s for the cultures used to obtain protein samples for the protein stability experiments along with the data shown in Figure 3. As can be seen the OD600’s do not increase significantly after the addition of spectinomycin suggesting that the increase in the first five minutes is not due to further growth as could be first assumed. Other assays we have previously reported for FlgM (Aldridge et al. 2006 Mol. Micro 60:630-43) and FlgK (Aldridge et al. 2003 Mol Micro 49:1333-45) do not show a similar trend suggesting this may even be a specific effect with respect to 28.

Supp_Figure 1.The stabilities of FlgM and 28 are interdependent. Stability assays were performed for three independent repeats using the antibiotic spectinomycin to inhibit protein synthesis. Solid lines = 28 protein levels where red = wild type and green = flgM. Dashed lines = FlgM protein levels where red = wild type and blue = 28.The stability data presented here in colour is the same as found in figure 3. The average OD600 for the three cultures used as also shown in colour: red = wild type; green = flgM and blue = 28.

To calculate the half lives of 28 and FlgM from Supp_figure 1 the trend-line function of excel was used. Supp_Figure 2 shows the corresponding trend-lines obtained during this analysis. Supp_table 1 includes the corresponding slope for the calculated trend-lines that were subsequently used to calculate the protein half-lives. As 28 protein levels fluctuate the half-lives for all proteins were calculated using only the data for T5, T10, T20 and T30.

Supp_Figure 2: Calculated Trend-lines using the Program MS-Excel. The colours used correspond to the same lines as shown in Supp-Fig. 1. Only the T5 to T30 time points are used to obtain an accurate T1/2 for all proteins. Solid lines = 28 protein levels where red = wild type and green = flgM. Dashed lines = FlgM protein levels where red = wild type and blue = 28.

Supp_table 1: Calculated slopes and the half-lives of 28 and FlgM from data shown in Supp_fig. 2.

Strain / 28 / FlgM
Slope / R2a / T1/2 (mins.) / Slope / R2a / T1/2 (mins.)
Wild type / -2.7921x + 133.46 / 0.968 / 30 / -1.4942x + 77.5 / 0.843 / 18
28 / - / - / - / -1.5728x + 100.14 / 0.887 / 32
flgM / -4.4656x + 157.72 / 0.990 / 24 / - / - / -

a: R2 values represent how well the trend-lines fits the actual data. An R2 value = 1 is a perfect fit.