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Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28 Page 15 of 17

The focus of this study was to further pave the way for directed pharmacological applications of MS2-L and
related toxins. It needs to be considered that the presented results are obtained either with purified protein
or by overexpressing toxin derivatives in cellular context. Whether similar mechanisms occur during
coordinated phage infection of bacterial cells still needs to be demonstrated. Despite the similar two-domain
structure, no explicit amino acid conservation within the two peptides MS2-L and ΦX174-E is detectable.
Furthermore, the inhibition of a distinct target such as MraY by ΦX174-E has not been detected for MS2-L
yet. Inhibition of cell-wall biosynthesis by MS2-L is supposed to be rather unlikely, as no accumulation of
[29]
cell-wall precursor has been observed . Accordingly, we could not detect any interaction of MS2-L in NDs
with the lipid I precursor forming enzymes MurA-F in pulldown assays (data not shown). However,
inhibition of later steps in cell wall formation and interaction of MS2-L with a yet non-identified target can
still not be ruled out. DnaJ might also play a role in keeping the MS2-L conformation competent for such an
[4]
interaction . Based on the demonstrated ability of MS2-L and ΦX174-E to oligomerize, a likely function
could be their participation in the formation of membrane disintegrating or penetrating pores as at least an
important asset of cell lysis. A pore-forming ability is common to a large variety of naturally occurring
peptide antibiotics and a variety of basic structures is possible [37-40] . The oligomeric interface of MS2-L and
ΦX174-E is located within their transmembrane domains. Both domains share a common hydrophobic
[41]
leucine-rich region essential for lytic activity . The high-order assembly of MS2-L and ΦX174-E in vitro
might be a paradigm for a large number of related small phage toxins and could become an important
feature in future applications.

DECLARATIONS
Acknowledgments
We are grateful to Roman Levin for helpful advice and discussions. We thank Birgit Schäfer for technical
assistance. We further thank Prof. Klaas Martinus Pos for providing strain BW25113ΔDnaJ.

Author contributions
Wrote the manuscript: Mezhyrova J, Bernhard F
Cell-free and in vivo studies: Mezhyrova J, Börnsen C
Performed LSM studies: Mezhyrova J, Frangakis AS
LILBID analysis: Martin J, Morgner N
Provided essential materials: Frangakis AS, Dötsch V
All authors contributed to the project design, data analysis, reading, and approving the final version of the
manuscript.

Availability of data and materials
Not applicable.

Financial support and sponsorship
This work was funded by the DFG projects BE1911/8-1 and FR1653/12, by the LOEWE project GLUE of the
state of Hessen, by the Center for Biomolecular Magnetic Resonance (BMRZ) and the BMBF EFFORT. We
also thankfully acknowledge funding by the DFG/ Heisenberg – Project-ID 426191805 and the graduate
school GRK 1986 (CLiC).

Conflicts of interest
All authors declared that there are no conflicts of interest.