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Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28 Page 9 of 17

Figure 2. Post-translational interaction of DnaJ with MS2-L derivatives in detergent and in NDs. Toxin expressions were carried out in
the BW25113ΔDnaJ lysate. MS2-L derivatives were expressed and solubilized co-translationally in either Brij78 (D-CF) or DMPG NDs
(L-CF). Purified toxins were then analyzed in pulldown experiments for interaction with DnaJ. The MS2-L derivatives were immobilized
via their C-terminal StrepII-tag and 50 µg purified DnaJ was added as prey. After washing, the formed complexes were eluted in 40 µL
of SDS-loading buffer and 15 µL samples were analyzed by SDS-PAGE. Bound DnaJ was detected by immunoblotting with α-His
antibodies and immobilized MS2-L derivatives were visualized with α-StrepII antibodies. (1) MS2-L; (2) MS2-Lp ; (3) MS2-Lp ; (4)
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MS2-Lp ; (5) MS2-Lp ; (6) MS2-Lp . (A) D-CF expression in Brij78; (B) L-CF expression in DMPG NDs. The smaller band detected in
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the DnaJ control is a putative degradation product. D-CF: Detergent-based cell-free expression; DMPG: 1,2-dimyristoyl-sn-glycero-3-
phospho-(1’-rac-glycerol); ND: nanodisc; P-CF: precipitate forming cell-free expression.
In contrast to the oligomerization in NDs, in detergent, only monomers of full-length MS2-L are detectable
[Figure 3, left panel, red trace]. However, lower oligomeric assemblies containing up to four monomers are
already formed in detergent by the truncated toxins MS2-Lp , MS2-Lp , MS2-Lp , and MS2-Lp . N-
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terminal deletion of further three amino acids in MS2-Lp completely restored the high oligomerization
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observed in NDs and assemblies containing up to 10 monomers could be detected.
As bacterial cell lysis by MS2-L may be modulated by DnaJ, we next analyzed whether the chaperone has
any effect on the oligomerization state of the toxin. MS2-L and the truncated MS2-Lp derivatives were CF
synthesized in BW25113ΔDnaJ lysate in the presence of NDs (DMPG) or Brij78 micelles, and the samples
prepared as above were analyzed by LILBID-MS [Figure 3, right panels]. The spectra of oligomeric
complexes of all samples prepared in BW25113ΔDnaJ lysate were similar to those prepared in the standard
S30 lysate. This indicates that interaction with DnaJ has most likely no direct impact on the homomeric
oligomerization of MS2-L, neither in detergent nor in ND (DMPG) membranes.

In vivo cluster and pore formation of MS2-L derivatives
Complementary to the in vitro studies, the in vivo effect of full-length MS2-L derivatives in the E. coli strain
Lemo21 (DE3) was analyzed. Plasmids encoding for the constructs MS2-L, MS2-L-GFP and
mScarlet-MS2-L were transformed into Lemo21 (DE3). Growth curves of the cells were recorded after
induction of toxin expression by IPTG to determine the cell lysis behavior [Supplementary Figure 4]. All
MS2-L constructs showed similar toxic effects and inhibited further growth of the cells shortly after
induction.

Samples of cells expressing the full-length derivatives MS2-L, MS2-L-GFP and mScarlet-MS2-L were taken
at several time points after induction and analyzed by fluorescence and electron microscopy [Figures 4 and
5]. The cells showed numerous large fluorescent clusters located in the periphery of the cells, indicating a