Fig. 4: NQNO triggers degradation of the Epsilon antitoxin.
a, Whole-cell lysates prepared from indicated N. gonorrhoeae strains were probed with polyclonal antibodies against ε1 (top) or against ζ1 (bottom). Strains resistant to NQNO are indicated with a blue dot; 2.5 ng of recombinant His-tagged ε1 was loaded as a positive control. The asterisk indicates a non-specific band reacting with the anti-ζ1 antiserum. b, Left: N. gonorrhoeae MS11 was treated with 50 µM NQNO or DMSO for the indicated time and lysates were probed as in a. Blot representative of 3 independent replicates. The asterisk indicates a non-specific band reacting with the polyclonal anti-ζ1 antiserum. Right: graph showing the ratio of ε1 vs ζ1 protein levels. Bars represent mean ± s.e.m. (n = 3 biological replicates); *P < 0.05, **P < 0.01; ordinary-pairing one-way ANOVA with Dunnett’s multiple comparisons test; 95% CI and exact two-tailed P values also shown. c, E. coli expressing ε1 was treated for 45 or 60 min with 1 mM or 5 mM H2O2 or left untreated. Lysates were probed with rabbit polyclonal antibodies raised against ε1 (top). E. coli lysates taken before (BI) and after (AI) induction of ε1 expression by IPTG were used as controls. The blots were reprobed with a monoclonal antibody against GAPDH (bottom). Blot representative of 3 independent replicates. Graph at the bottom shows the ratio of ε1 protein levels vs GAPDH. Bars represent mean ± s.e.m. (n = 3 biological replicates). Statistics as in b. d, E. coli expressing ε1 was treated for 45 or 60 min with 25 µM or 50 µM NQNO, 1% DMSO or left untreated. Lysates were probed as in c. Bars represent means ± s.e.m. (n = 4 or 3 biological replicates); statistics as in b. e, Growth curves of E.coli incubated with 25 or 50 µM NQNO; control cultures were grown in the presence of 1% DMSO. Growth was monitored using OD600 readings every 30 min. Data representative for 3 independent replicates. f, Purified ε1 protein was incubated for the indicated times with solvent (1% DMSO), 50 µM NQNO or 1 mM H2O2 and samples were analysed for the presence of ε1 with an anti-His-tag antibody. Data representative of 3 independent replicates. g, N. gonorrhoeae strains WHO G, WHO N and MS11-R2 pTetM A were treated for 0 or 60 min with 50 µM NQNO (N) or with DMSO (D) and lysates were probed as in a. Graph at the bottom shows the ratio of ε1 vs ζ1 protein levels. Bars represent means ± s.e.m. (n = 5 biological replicates); statistics as in b. h, N. gonorrhoeae MS11 was treated with 50 µM NQNO, 36 µM Antimycin A (AA), 30 µM ciprofloxacin (Cipro) or with DMSO for 1 h and lysates were probed as in a. Bars represent means ± s.e.m. (n = 3 biological replicates); statistics as in b.
