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Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28 Page 13 of 17

different time points (20 and 40 min) after IPTG induction and analyzed for GFP and mScarlet fluorescence
[Figure 4D]. Approx 20% of the cells showed fluorescence of both monitors and the corresponding clusters
perfectly matched in overlays.

DISCUSSION
The role of MS2-L in bacterial cell lysis remains a matter of debate. Electron micrographs revealed large cell
[2]
wall lesions associated with the efflux of cytoplasmic content in MS2-L expressing E. coli cells . In
combination with the observed localization of MS2-L at membrane adhesion sites, lesion formation by
[5]
direct involvement of MS2-L was suspected . Expression of the C-terminal MS2-L transmembrane domain
is sufficient to induce bacterial lysis and leakage in liposomes as well as in E. coli membrane vesicles. This
[3,6]
was shown with synthetic peptides covering the C-terminal 25 amino acids of MS2-L . A second and
potentially simultaneous mechanism is the activation of the bacterial autolytic system by MS2-L [1,29] . Lesion
formation would then be supported and associated by the activity of murein hydrolases. By combining both
mechanisms, a two-step process was suggested where membrane interaction of MS2-L first results in
membrane depolarization. This triggers the activation of autolytic enzymes, which then locally generate
large holes in the murein sacculus . The biological relevance for such a dual action of MS2-L could be a
[6]
more efficient release of the approx. 28 nm MS2 phage through the relatively tight murein-sacculus into the
environment.

By our combined approach including CF expression, ND technology and native LILBID MS, we could show
for the first time the high-order self-assembly of MS2-L monomers triggered by its transmembrane domain.
In combination with the fact that the C-terminal domain is also essential for lysis, this could support the
possibility of the participation of MS2-L higher-order assemblies in the disintegration of the bacterial cell
envelope. The assembly of MS2-L resembles previous data obtained with the related lysis toxin ΦX174-E.
Similar to MS2-L, in ΦX174-E, the lysis activity is confined to the transmembrane domain, which
additionally promotes the oligomerization process [2,17,30-32] .

The MS2-L soluble domain is dispensable for cell lysis and no function could be attributed so far. It is
encoded by overlapping sequences with the phage coat protein, leading to speculations that it might be
rather a byproduct of the control mechanisms in the MS2 phage expression process [3,33,34] . As some first
effects of the MS2 soluble domain, we show that its presence in MS2-L impairs the oligomeric assembly in
detergent environments. Furthermore, the insertion kinetics of MS2-L into a ND membrane was different if
compared with the construct MS2-Lp truncated in the soluble domain. These effects hint at a potential
23
regulatory function of the MS2-L N-terminal domain, similar to that observed with the soluble domain of
the toxin ΦX174-E . Here, a conformational lock formed by the intramolecular interaction of soluble and
[17]
transmembrane domains prevents membrane insertion and oligomerization. This intramolecular lock is
released by interaction with the chaperone SlyD. The biological relevance of this mechanism could be a
quorum sensing mechanism that contributes to determining optimal time points for phage release .
[8]
However, despite these coincidences, the effect of the soluble domain in the MS2 system seems to be
different. Its inhibitory function on MS2-L oligomerization was not observed in membrane environments of
NDs and membrane insertion was retarded but not completely prevented as in the ΦX174-E system.
However, our results were obtained in vitro with plain lipid bilayers devoid of any other proteins. The
insertion and oligomerization of MS2-L in crowded native bacterial membranes will be a different situation
and modulatory effects of the soluble domain could then become more important.

A previously identified interaction partner for MS2-L is the chaperone DnaJ . We confirmed previous in
[4]
vivo studies by demonstrating strong binding of DnaJ to the MS2-L soluble domain in the combined CF/