Resistance to oxidative stress by inner membrane protein ElaB is regulated by OxyR and RpoS

Summary C‐tail anchored inner membrane proteins are a family of proteins that contain a C‐terminal transmembrane domain but lack an N‐terminal signal sequence for membrane targeting. They are widespread in eukaryotes and prokaryotes and play critical roles in membrane traffic, apoptosis and protein translocation in eukaryotes. Recently, we identified and characterized in Escherichia coli a new C‐tail anchored inner membrane, ElaB, which is regulated by the stationary phase sigma factor RpoS. ElaB is important for resistance to oxidative stress but the exact mechanism is unclear. Here, we show that ElaB functions as part of the adaptive oxidative stress response by maintaining membrane integrity. Production of ElaB is induced by oxidative stress at the transcriptional level. Moreover, elaB expression is also regulated by the key regulator OxyR via an OxyR binding site in the promoter of elaB. OxyR induces the expression of elaB in the exponential growth phase, while excess OxyR reduces elaB expression in an RpoS‐dependent way in the stationary phase. In addition, deletion of elaB reduced fitness compared to wild‐type cells after prolonged incubation. Therefore, we determined how ElaB is regulated under oxidative stress: RpoS and OxyR coordinately control the expression of inner membrane protein ElaB.

. Oligonucleotides used for cloning, qRT-PCR, flag insertion via the chromosomal copy of elaB, and probe amplification. f indicates the forward primer and r indicates the reverse primer. KM indicates kanamycin resistance gene.

Purpose/Name
Sequence (5'-3') Figure S1. Production of the ElaB-Flag fused protein complemented the oxidative stress sensitivity of the elaB mutant strain. Overnight cultures were diluted, cultured to a turbidity of 600 nm of 1.0, treated with 20 mM H2O2 for 10 minutes, and then cell survival (%) was assayed. Three independent cultures were used.

Figure S2
ElaB mutation weakens cell membrane. Cells were treated with H2O2 as indicated in Figure 1A and stained with plasma membrane specific red-fluorescent dye FM® 4-64. WT cells before (A, B) and after (C, D) H2O2 treatment were observed with a fluorescent microscope. The percentage of cells with weakened membranes are marked with white arrows. Three independent cultures for each strain were used and about 1000 cells in each culture were observed, and only one representative image for each strain was shown here. The percentages of cells with weak cell membrane were calculated. Figure S3. ElaB protects the cell membrane against exogenously added H2O2. TEM micrographs of wild-type cells before (A) and after (C) 50 mM H2O2 treatment for 30 min. The ultrastructure of the △elaB strain treated without (B) and with (D) 50 mM H2O2 for 30 min are also shown. Cells were cultured and collected as mentioned in the Material and Methods section. The ultrastructure of the cell membrane is indicated with a red arrow for a representative cell in each panel.

Figure S4
OxyR and RpoS were expressed in the oxyR and rpoS double mutant (ΔΔ) ΔΔ/pHGR01-PelaB-L, and β-galactosidase activities were determined as in Figure 4D. Three independent cultures for each strain were used. For statistical analysis, p< 0.01 is marked as **. Figure S5. The expression plasmids pCA24N-oxyR and pCA24N-rpoS were transferred into the ΔrpoS and ΔΔ cells. Production of OxyR (red arrows) and RpoS (green triangles) was induced by 0.5 mM IPTG at OD600 0.1 for 2 h and 6 h. Cm indicates the chloramphenicol resistance protein. The levels of OxyR, RpoS and ElaB were determined in ΔrpoS (A) and ΔΔ (B). The same amount of total protein was loaded as controls. Figure S6. Competition of WT, ΔelaB, ΔoxyR and ΔrpoS was tested, and all the three mutant strains without kanamycin (Km) resistance. (A) Overnight cultures of WT and ΔelaB were diluted to OD600 0.1 and were cultured till OD600 1.0. Then different ratios of ΔelaB and WT were mixed, and the percentages of ΔelaB in total cells were determined at different time points by PCR amplification of 96 randomly selected colonies using primers flanking the elaB gene region. (B) The ΔrpoS cells were mixed with WT and ΔelaB at the ratio of 1:1, and the percentages of ΔrpoS in total cells were determined at different time points. (C) The ΔrpoS cells in (B) were replaced by ΔoxyR and the percentages of ΔoxyR in total cells were determined at different time points. Triplicates were used for each strain, and data were shown as mean± SD.

Figure S7. ElaB increases cell fitness under microaerobic and oxidative stress
conditions. Overnight cultures of BW25113 wild type (WT) and ΔelaB::km were diluted to OD600 0.1 and were cultured till OD600 1.0. Then the same number of ΔelaB::km and WT cells were mixed and cultured under two conditions: (i) The mixed cells are treated with 1 mM H2O2 and the H2O2 was added again every day when the cells are re-cultured; (ii) The mixed cells are cultured in BACTROX-2 microaerobic chamber (SHELLAB, USA) equilibrated to a 5% O2 and 10% CO2 atmosphere. The percentages of ΔelaB::km in total cells were determined at different time points. Triplicates of each strain were used and error bar indicates standard error of mean (n = 3).  Figure S8. Growth of BW25113 wild type and ∆elaB strains under microaerobic condition. Cells were cultured in LB medium overnight. Then they were diluted to a turbidity at 600 nm 0.1, and cells were incubated in BACTROX-2 microaerobic chamber (SHELLAB, USA) equilibrated to a 5% O2 and 10% CO2 atmosphere, and turbidity (A) and CFU (B) were determined at indicated time points. Triplicates of each strain were used and error bar indicates standard error of mean (n = 3).