SUPPLEMENTARY MATERIAL
The role of the soxRS regulon in cadmium induced oxidative stress in E. coli
Biological Trace Element Research
Milini Thomas and Ludmil Benov
Department of Biochemistry, Faculty of Medicine, Kuwait University, P. O. Box 24923 Safat, 13110 Kuwait City, Kuwait,
Contents
Figure S1: Effect of Cd on SOD overproducing strains.
Figure S2: Effect of Cd on the growth of SOD-proficient strain AB1157 andsodA sodB mutant JI132
Figure S3: Effect of Cd on the growth of mid-log E. colicultures.
Figure S4: Lack of induction of members of the soxRS regulon by Cd.
Figure S5: Cd prevents the induction of fumarase C in a sodA sodB mutant by paraquat.
Figure S1: Effect of Cd on SOD overproducing strains. Overnight KK 196 (FeSOD overproducer, panel A) and KK198 (MnSOD overproducer, panel B) cultures in LB medium were diluted to OD600nm 0.05 in low phosphate MOPS medium and CdCl2 was added. Cultures were incubated on a shaking water bath at 37oC and 200 rpm. Growth was monitored turbidimetrically at 600 nm.
Strains overproducing either FeSOD or MnSOD were not more protected from Cd toxicity than the parental strain AB1157 (see Fig. S2, A). At the same time, introduction of either FeSOD or MnSOD into sodA sodB mutant abolished its hypersensitivity to Cd (compare Fig. S1 with Fig. S2 B). Both SOD overproducers grew worse that the parent in low phosphate MOPS medium. It suggests that in conditions where nutrients are limited, overexpression of a protein causes imbalance that disturbs cell metabolism and eventually ability to proliferate.
Figure S2: Effect of Cd on the growth of SOD-proficient strain AB1157 (Panel A) and sodAsodB mutant derived form it (Panel B). Stationary phase cultures were diluted to OD600nm 0.05 in low phosphate MOPS medium and CdCl2 was added to the indicated final concentrations.Cultures were grown on a shaking water bath at 37oC and 200 rpm and growth was monitored turbidimetricallyat 600 nm.
Figure S3: Effect of Cd on the growth of mid-log E. coli. Cultures were grown to mid-log phase in low phosphate MOPS medium and were diluted to OD600nm 0.05 in the same medium. CdCl2 was added to the indicated final concentrations. Growth was monitored turbidimetrically at 600 nm. Closed symbols, parental GC4468 strain; open symbols, sodA sodBQC1799.
Figure S4: Lack of induction of members of the soxRS regulon by Cd. Parental (GC4468, panels A – D) and sodAsodB (QC1799, panels C and D) cultures were grown to mid-log phase in LB medium. Cells were washed, resuspended in MOPS medium to the original volume and challenged with the indicated concentrations of CdCl2. After one hour of incubation at 37oC with vigorous shaking (200 rpm) cells were washed, resuspended in phosphate buffer and French-pressed. Cell-free extracts were assayed for fumarase C, superoxide dismutase (SOD), glucose 6-phosphate dehydrogenase (G6PD), and nitroreductase A activities. Paraquat (PQ++), 25 M was used as a positive control. Results are presented as mean ± S.E. (n=3).
Figure S5: Cd prevents the induction of fumarase C in a sodA sodB mutant by paraquat. The SOD-deficient QC1799 mutant was grown in LB medium to mid-log phase. The cells were washed and resuspended to the same volume with MOPS medium. Aliquots were preincubated for 15 min with CdCl2 at37oC on a shaking water bath (200 rpm). Paraquat was added to a final concentration of 25M, and cultures were left on the shaker for additional 60 min. At the end of the incubation time, the cultures were washed, resuspended in phosphate buffer, and disrupted with a French press. Fumarase C and protein were determined in the cell-free extracts. Data are presented as mean ± S.E. (n=3).
S-1