Fig. 3: Growth inhibition by NQNO requires the presence of the zeta toxin.
a, Presence of the epsilon1/zeta1 operon in N. gonorrhoeae strains was determined by PCR. As control, PCR for opa genes was conducted with the same DNA samples. Blue font marks strains harbouring the epsilon1/zeta1 locus, blue dots mark high and intermediate NQNO-resistant strains as indicated. b, N. gonorrhoeae MS11 and N. cinerea were treated with 50 µM NQNO or DMSO for 30 min and stained with SYTO9 (all bacteria) and propidium iodide (bacteria with disintegrated cell wall). c, Quantification of b showing the percentage of propidium iodide-positive bacteria (% PI-positive bacteria) of the total SYTO9-stained bacterial cells. Mean ± s.e.m. (n = 6 microscope fields). Significance was analysed using ordinary-pairing one-way ANOVA with Tukey’s multiple comparisons test of the mean; NSP > 0.10, ***P < 0.001 ****P < 0.0001; 95% CI and exact two-tailed P values also shown. d, pTetM plasmid was conjugated in MS11-R2 and two independent conjugants were selected. Successful conjugation of pTetM was verified by PCR of the epsilon1/zeta1 operon or opa genes as in a. e, Growth of strains MS11, MS11-R2 (NQNO-resistant) and pTetM-positive conjugants MS11-R2 pTetM A and MS11-R2 pTetM B in the presence of 5, 10, 25 or 50 µM NQNO. Control cultures were grown in 1% DMSO. Growth was monitored using OD550 readings every 30 min. Data represent 3 independent replicates. f, Strains as in e were treated with 50 µM NQNO or solvent control (DMSO) for 30 min and stained, enumerated and statistically analysed as in c. g, The pTetM plasmid was conjugated into WHO P and two independent clones (WHO P pTetM A and WHO P pTetM B) were selected. WHO P was also transformed with a construct encoding epsilon1/zeta1 and the ErmC resistance (WHO P ε1ζ1). Expression of ε1 and ζ1 in MS11, WHO P, WHO P ε1ζ1 and WHO P pTetM conjugants was verified by western blotting of whole-cell lysates (WCL) with antibodies against ε1 (top panel) or ζ1 (second panel). The asterisk indicates a non-specific band reacting with the polyclonal anti-ζ1 antiserum. Western blotting of the same lysates with a monoclonal antibody against Opa protein (third panel) and Coomassie staining of the membrane (bottom panel) served as loading controls. Blue dot indicates NQNO-resistant strains. h, Growth of strains from g in the presence of 5, 10, 25 or 50 µM NQNO. Control cultures were grown in 1% DMSO. OD550 was monitored every 30 min. Data represent 3 independent replicates. i, Strains as in g were treated with 50 µM NQNO or solvent control (DMSO) for 30 min and stained, enumerated and statistically analysed as in c.
