Figure 1.
SM10, a synthetic small molecule with antibacterial activity.
(A) Chemical structure of SM10. (B) SM10 causes a dose-dependent drop in E. coli MG1655 cell viability. MG1655 cells were incubated in the presence of SM10 in MHB at 37°C for 3 hr, then the cultures were diluted and plated on LB. (C) Cells were grown in MHB to an OD600 of 0.1, and then treated with different doses of SM10 or with DMSO, as one test for lysis. In panel C, the symbols denote the following treatments: x's, DMSO; triangles, 5 µg/ml SM10; circles, 10 µg/ml SM10; squares, 20 µg/ml SM10; and diamonds, 30 µg/ml SM10 final concentration.
Figure 2.
SM10 causes cell filamentation, membrane alteration and DNA condensation.
E. coli MG1655 was incubated in MHB for 1.5 hr in the presence of 10 µg/ml SM10 (C, D) or SMs solvent, DMSO (A, B). The membrane and the DNA were stained with FM4-64 (A, C) and DAPI (B, D). TEM of E. coli MG1655 incubated in MHB for 1.5 hr in the presence of DMSO (E, F) or 20 µg/ml SM10 (G–J). The samples were fixed in 2% glutaraldehyde and then with 1% osmium tetroxide. Samples were dehydrated with alcohol, embedded in epon and sliced. Staining was performed with uranyl acetate and lead citrate. The slices were viewed with a FEI TECNAI 12 TEM.
Figure 3.
Cerulenin inhibits membrane alteration but does not prevent the death caused by SM10.
E. coli MG1655 was incubated in MHB for 1.5 hr in the presence of (A) DMSO, (B) 10 µg/ml SM10, (C) 100 µg/ml cerulenin, or (D) 10 µg/ml SM10+100 µg/ml cerulenin. The membrane was stained with FM4-64. (E) Log sensitivity was calculated relative to DMSO treatment, by testing cell viability after 1 hr, 3 hr, and 20 hr exposure. The absence of a bar at 20 hr reflects cell recovery.
Table 1.
Effect of sublethal concentrations of SM10 on expression of 3 envelope stress responses (β–galactosidase activity expressed in Miller Units, ± SE).
Figure 4.
SM10 did not permit leakage of ONPG or β-galactosidase.
Log phase E. coli MG1655 cells were incubated in the presence of SM10 (10 µg/ml) or DMSO (1%). β-galactosidase activity was induced with IPTG and quantitated after 1 hour, using the substrate ONPG. β-galactosidase activity is expressed in Miller units. “Lysed” indicates cells that were permeabilized using a combination of 1 part 0.1% SDS and 2 parts CHCl3 as a positive control. A. ONPG to cross the cell membrane freely in cells that were not permeabilized with SDS and CHCl3. Cells were induced or not with IPTG, and ONPG was added to the lysed supernatant or to cells treated with DMSO or SM10 (amount). B. β-galactosidase did not leak out of SM10-treated cells. The supernatant of cells induced or not with IPTG was assayed for β-galactosidase activity, to test leakage of the enzyme out of the cytoplasm when cells were treated with DMSO or SM10. C. The membrane potential of cells treated with SM10 (or not) was tested using the bis-oxonol indicator molecule DiBAC4(3). The fraction of cells showing DiBAC4(3) fluorescence and the mean DiBAC fluorescence per cell (inset) are shown; cells were treated with the specified amount of SM10 or with the appropriate amount of DMSO solvent alone.
Figure 5.
SM10 induces DNA damage in MG1655.
For experiments in all panels, bacterial cells were treated with SM10 for 3 hr before the appropriate analysis. A. Expression of sulAp::mCherry. The sulAp::mCherry strain was incubated in MHB in the presence of DMSO, SM10, or MMC. The % of cells positive for mCherry fluorescence cells was determined by flow cytometry. B. Measuring DNA breaks induced by SM10 compared to DMSO, using the TUNEL assay. A representative histogram of MG1655 treated bacteria. C. DNA damage induced by SM10 treatment or overexpression of OmpC or OmpF includes double strand breaks, as shown by PFGE. Expression of OmpC or OmpF was induced with 0.1 mM IPTG for 3 hr before cells were embedded in agarose.
Table 2.
SM10 treatment increases the fraction of cells with DNA breaks and the amount of DNA breaks per cells, as measured by TUNEL assay.
Figure 6.
Overexpression of OmpC or OmpF reduces cell viability and perturbs the membrane.
A. Viability assay of cells overexpressing OmpC or OmpF. Cultures were grown in MHB at 37°C, and overexpression of OmpC or OmpF was induced with 0.1 mM IPTG in log phase and incubated for 3 hr before viable counts were measured. B. Overexpression of porins induces membrane alteration. MG1655 cells carrying a high-copy plasmid encoding OmpC (v, vi, vii, viii) or PurE (i, ii, iii, iv) were incubated 3 hr in the absence (i, ii, v, vi) or presence of 0.1 mM IPTG (iii, iv, vii, viii). The membrane and the DNA were stained with FM4-64 (i, iii, v, vii) and DAPI (ii, iv, vi, viii), respectively.
Table 3.
Overexpression of OmpC or OmpF, but not FolD or PurE, induces the SOS response (mCherry+) and increases the fraction of cells with DNA breaks (TUNEL+).
Figure 7.
SM10 induces the production of hydroxyl radicals.
E. coli MG1655 was incubated in MHB at 37°C for 1.5 hr in the presence of SM10 (A) alone or in combination with the iron chelator dipyridyl. The cells were washed and incubated with HPF, for quantification of hydroxyl radicals (A, B) or processed to quantify DNA breaks (C) with the TUNEL assay.
Table 4.
Effect of dipyridyl on the fraction of HPF+ cells in conjunction with SM10 treatment or overexpression of OmpC.
Table 5.
Effects of dipyridyl on viability of cells incubated for 3 hr with SM10 or cells over–expressing OmpC, given as the log decrease in cfu/ml.
Table 6.
Overexpression of OmpF and OmpC induces hydroxyl radical formation.
Figure 8.
Comparing the effect of SM10 treatment or OmpC overexpression in aerobically- and anaerobically- growing E. coli.
MG1655 cells were grown for 3 hr at 37°C, with shaking. Anaerobic cultures were grown using an anaerobic hood and/or in a closed-vial-system, in both cases after sparging the O2 gas with argon. The legend for panels A, B and C is shown in panel B. The legend for panels D, E and F is shown in panel E; in addition, the presence of O2 is indicated both under the X axes as well as by the darker shading of the bars denoting aerobically-grown cells. (A) Lethality of SM10 treatments was not greatly affected by lack of oxygen. (B) SM10 caused about 40% fewer DNA breaks in MG1655 cells grown anaerobically, as quantified by TUNEL assay. (C) SM10-induced formation of ROS in MG1655 cells, visualized by the HPF assay, is negligible in anaerobically–grown MG1655 cells. (D) Overexpression of OmpC but not of PurE causes lethality in either aerobically- or anaerobically-growing cells. (E) Over-expression of OmpC anaerobically results in about half the DNA breaks observed compared to aerobically, as quantified by TUNEL assay. (F) Reactive oxygen species are produced in aerobically-growing cells over-expressing OmpC, but not PurE.
Table 7.
SOS induction measured using the sulA::mCherry reporter in MG1655 treated with SM10 or norfloxacin aerobically and anaerobically.
Figure 9.
Treatment with indole and ethanol induce DNA damage.
MG1655 cells were incubated in MHB at 37°C in the presence of the indicated concentration of indole (A) or ethanol (B) for 3 hr. The DNA damage was detected using the TUNEL assay.
Table 8.
DNA breaks present in ΔrpoE versus its isogenic parent strain, MC4100, grown at different temperatures, determined using TUNEL.
Table 9.
Strain list.