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Page 12 of 17 Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28

Figure 5. Lesion formation in E. coli Lemo21 (DE3) cells synthesizing MS2-L. Toxin expression was induced at OD 600 = 0.27 with 1 mM
IPTG and samples were taken 60 min after induction and immediately vitrified. Electron microcopy images were acquired with a Titan
Krios and a K2 Summit direct detector. Different stages of lesion formation are shown in a proposed sequential order. Data were
acquired at a magnification of 81,000x at a defocus of -50 µm and with 30-40 e-/Å total dose (scale bar = 0.05 µM). OM (outer
membrane), IM (inner membrane) and PG (peptidoglycan) are indicated by arrows. The fission zone is indicated by brackets.
IPTG:Isopropyl-β-D-thiogalactopyranoside.

membrane integration [Figure 4A and B]. The clusters were detectable 10 minutes after induction and
became more prominent with extended induction times. Similar clusters were observed after the expression
of the constructs in T7 Express cells (data not shown). PI staining revealed that the cluster formation of
MS2-L in bacterial cells was accompanied by membrane permeabilization, probably due to pore formation.
The vast majority of control cells did not take up the dye [Figure 4C]. The pore formation could be verified
with electron microscope images of vitrified bacterial cells. Pores at different formation stages were detected
in most cells in samples taken 60 min after induction of the full-length toxin MS2-L [Figure 5]. Apparently,
the outer membrane is disrupted first, followed by disintegration of the underlying peptidoglycan layer. In
the next step, the inner membrane is disrupted, causing excessive leakage of cytoplasmic content. No fusion
of the outer and inner membranes at the periphery of the pores could be observed.

The fluorescent clusters could represent large assemblies of the toxins in the cell membranes and would
thus support our observation of oligomer formation in ND membranes in vitro. To provide further
evidence of MS2-L oligomerization in the bacterial membrane, we analyzed for co-localization of
MS2-L-GFP and mScarlet-MS2-L in Lemo21 (DE3) cells. Templates for MS2-L-GFP and mScarlet-MS2-L
were co-transformed into Lemo21 (DE3). The transformed cells were double-selected on ampicillin and
kanamycin and then inoculated in liquid medium containing both antibiotics. Samples were taken at