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

membranes. In accordance with the in vitro data, the assembly of MS2-L derivatives into large membrane located
clusters was monitored by overexpression of corresponding fusions with fluorescent monitors in E. coli cells.
Analysis by cryo-electron microscopy indicates that lesion formation is initiated in the outer membrane, followed
by disruption of the peptidoglycan layer and disintegration of the inner membrane.

Conclusion: MS2-L forms oligomeric complexes similar to the related phage toxin ΦX174-E. The oligomeric
interface of both peptides is located within their transmembrane domains. We propose a potential function of the
higher-order assembly of small phage toxins in membrane disintegration and cell lysis.

Keywords: Phage toxins, cell-free expression, native mass spectrometry, molecular assemblies, nanodiscs

INTRODUCTION
The 75-amino acid protein MS2-L is a prototypical toxin of the ssRNA Leviviridae group of bacteriophages.
The toxin shows an amphiphilic topology consisting of a proposed N-terminal soluble domain followed by
a transmembrane domain. MS2-L is extremely toxic to bacteria and its recombinant expression rapidly kills
[1,2]
E. coli cells by forming lesions in the cell envelope and subsequent release of cytoplasmic content . The
essential lytic activity of MS2-L was confined to the C-terminal 35 amino acids containing its
[3]
transmembrane domain and deletion of the soluble domain did not affect its lytic function . The chaperone
DnaJ was identified as an interaction partner of MS2-L. Its interaction with the soluble domain of MS2-L
was shown in pulldown experiments. Deletion of this domain abolished DnaJ interaction, while lysis
function remained unaffected. Thus, the chaperone action seems not to be essential for cell envelope
disintegration . The presence and nature of further MS2-L targets in the bacterial membrane are still
[4]
unclear. Previous electron microscopy studies using immunogold staining localized MS2-L in bacterial cell
membranes and to a major part within membrane adhesion sites . An early postulation was the
[5]
oligomerization of the MS2-L transmembrane domain . Furthermore, lysis as a result of inducing the
[6]
bacterial autolytic system was discussed .
[1,7]
MS2-L-like toxins are related to the ΦX174-E toxin family of the ssDNA Microviridae bacteriophages. They
are designated as “amurins” or “single gene lysis (Sgl)” proteins as they are proposed to inhibit specific steps
[7-9]
in bacterial peptidoglycan synthesis . Both toxins may play major roles in the future development of novel
antibiotics and they are of increasing commercial interest due to their ability to produce bacterial ghost cells
suitable for vaccination. Additionally, they find applications in structural biology, e.g., as a tool for electron
tomography [10,11] . ΦX174-E membrane insertion strongly depends on its interaction with the chaperone SlyD
and the lipid-I precursor forming enzyme MraY has been identified as one target within the bacterial
membrane [12-17] . Similar to MS2-L, ΦX174-E consists of one proposed transmembrane domain connected to
a short soluble domain. However, the organization of the domains is reversed and the transmembrane
domain of ΦX174-E is N-terminally located .
[8]
Successful phage infection is a complex mechanism depending on physiological conditions, growth phase
characteristics and gene expression regulation by the bacterial host cells. Delicate stoichiometry and
production timing of individual process compounds can be pivotal for the formation of essential
interactions and for a coordinated cycle of phage replication and host lysis. However, the implementation of
phage toxins such as MS2-L as biochemical tools in antibiotic development or ghost technology usually
requires their overproduction and usage in non-physiological concentrations. The major goal of this study
was, therefore, to characterize the function of the MS2-L toxin in vitro in a defined cell-free (CF) system and
in overproducing bacterial cells in order to provide a basic platform for future applications. In previous
work, we demonstrated the formation of ΦX174-E oligomers and the toxin’s co-translational interaction