Extended Data Fig. 7: The presence of the ζ1 toxin correlates with NQNO-induced membrane permeability in gonococci.
(a) N. gonorrhoeae MS11 and N. cinerea treated for 4 h with 50 µM NQNO or solvent only (DMSO) were analyzed by scanning electron microscopy. Scale bar represents 1 µm. (b) N. gonorrhoeae MS11-R2 (NQNO-resistant) and its pTetM-positive conjugants MS11-R2 pTetM A and MS11-R2 pTetM B were grown for 30 min in the presence of 50 µM NQNO or 1% DMSO. Bacteria were stained with SYTO9 (all bacteria) and propidium iodide (bacteria with disintegrated cell wall) and membrane integrity was analyzed by confocal microscopy. Scale bar, 10 µm. (c) Presence of the epsilon1/zeta1 operon in WHO P conjugated with the pTetM plasmid or transformed with the ε1/ζ1 construct was verified by PCR of the epsilon1/zeta1 locus using total DNA. As a control, PCR for opa genes was conducted with the same DNA sample. Red dot marks NQNO resistant strains. (d) Successful chromosomal integration of the epsilon1/zeta1 operon in WHO P ε1/ζ1 was verified by PCR of genomic DNA isolated from N. gonorrhoeae MS11, the parent strain WHO P, or a WHO P ε1/ζ1 transformant. PCR was performed with primers LbpA_fw and Zeta1_rev indicated in (e) yielding a 2 kb amplicon. Control PCRs for the epsilon1/zeta1 operon and opa genes was conducted as in (c). (e) Schematic overview of the insertion of the opa promotor driven epsilon1zeta1 operon and the erythromycin resistance gene (ErmC) into the Neisseria gonorrhoeae chromosome at the LbpA locus. Arrows indicate the location of primers used for PCR verification. DUS, neisserial DNA uptake sequence. (f) N. gonorrhoeae WHO P, two independent pTetM conjugants (WHO P pTetM A and WHO P pTetM B), and WHO P ε1/ζ1 were grown for 30 min in the presence of 50 µM NQNO or 1 % DMSO. Membrane integrity was analyzed as in (b). Scale bar, 10 µm.
