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Page 14 of 17 Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28

ND system. We further showed that DnaJ also binds post-translationally to MS2-L and binding extends at
least to amino acid position 35, being very close to the proposed transmembrane domain. This is in clear
[17]
contrast to the ΦX174-E interaction with the chaperone SlyD that only happens co-translationally .
Moreover, DnaJ is also not able to keep MS2-L in a soluble, membrane insertion competent complex as
observed with the ΦX174-E/SlyD interaction. The DnaJ binding site of MS2-L remains accessible after its
membrane insertion and strong binding was monitored in truncations of the soluble domain up to amino
acid position 32. The reduced but still detectable DnaJ binding after further deletions up to amino acid
position 35 indicates that the binding site extends close to the proposed transmembrane domain. Approx.
25 amino acids are sufficient to span a membrane and the 41 amino acid construct MS2-Lp could still
35
contain protruding parts sufficient to interact with DnaJ [3,35] . The post-translational interaction could
indicate a function of DnaJ in coordination with the soluble domain to increase the efficiency of MS2-L
membrane insertion and oligomerization in more complex cell membranes. This would be in agreement
[4]
with the observed faster cell lysis of N-terminal truncated MS2-L derivatives in vivo . However, these
effects appear to be rather modulatory in view of the observation that DnaJ is not essential for MS2-
mediated bacterial lysis . Despite its different action, DnaJ might thus have a similar but less restrictive
[4]
function as SlyD in the timing of bacterial lysis and MS2 phage release.

MS2-L localizes in the bacterial cytoplasmic membrane and, to a lesser extent, in the outer membrane .
[1,5]
Furthermore, a significant increase in adhesion sites upon MS2-L lysis was noticed and approx. 30% of
MS2-L finally localized in clusters associated with newly formed adhesion sites . MS2-L lysis further
[5]
requires a certain spacing of inner and outer membranes, indicating the formation of rather defined high-
order structures . By LSM microscopy, we visualized cluster formation of overexpressed MS2-L derivatives
[36]
in the periphery of bacterial cells. The cluster formation, in contrast to a random distribution, agrees with
the in vitro data obtained with NDs and supports an oligomeric assembly in vivo as well. Furthermore,
global cellular influx of PI was only detected in cells containing MS2-L clusters, indicating that the cells were
lysed and at least some of the clusters could be associated with lesions in the cell wall. In ND membranes,
we could detect up to dodecameric complexes of MS2-L. However, complex disintegration by the laser
power upon LILBID-MS analysis might restrict the detection of higher oligomers. Furthermore, it is not
clear yet whether a distinct MS2-L stoichiometry in complex assembly or simply large cluster formation is
necessary to cause membrane and cell wall disruption.

The cryo-electron microscopy studies provided initial insights into the likely first steps in lesion initiation.
Previous data obtained by transmission electron microscopy revealed the local disruption of large areas of
the bacterial cell envelope by expression of MS2-L derivatives . The high release of periplasmic marker
[2]
proteins further indicated that the inner and outer membranes did not fuse during lesion formation. Its
initiation was proposed by permeabilization of the inner membrane and followed by a burst of the outer
membrane due to over-expansion caused by leakage of cytoplasmic content . The analysis of MS2-L
[2]
directed cell wall disruption by high-resolution cryo-electron microscopy partially agrees with that finding
as, indeed, no connection between the inner and outer membranes can be observed. However, lesion
formation appears to be initiated by the disintegration of small areas of the outer membrane, while the inner
membrane in those areas remains structured. Nevertheless, molecular changes of the inner membrane in
the lesion areas resulting in increased permeability and release of cytoplasmic content can still not be ruled
out. Lesion formation proceeds by disintegration of larger areas of the outer membrane and the underlying
peptidoglycan layer followed by disintegration of the inner membrane. The detected lesions localize
predominantly in the close vicinity of cell fission zones. Whether MS2-L oligomers directly or indirectly
cause local permeabilization of the inner membrane or whether they are even localized in the outer
membrane can currently only be speculated.