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Ambros et al. Microbiome Res Rep 2023;2:34 https://dx.doi.org/10.20517/mrr.2023.18 Page 15 of 19

present, but annotation failed due to poor matching with databases. To verify any of these options,
methylome data would be required, which is still an expensive venture, although high-throughput methods
[59]
are on the rise . From a host perspective, the presence of MTases is also very interesting, as methylation
has been shown to play an important role in the DNA repair of E. coli [60,61] . A previous study about
Burkholderia mallei phage ΦE125, whose genome encodes two DNA MTase genes, demonstrates the
specific methylation of its phage episome, neither host chromosome nor prophage. The authors conclude
that methylation of extrachromosomal phages must play an important role in their life cycle and is likely
[62]
connected to their replication rate . It is still unknown whether the methyltransferases of phages in this
study can interact with the host chromosome, regardless of prior induction.

We could not identify antibiotic resistance genes (ARG) transduced by the intact predicted prophages.
Wendling et al. have shown that prophages can benefit the fitness of their host independent of the
presence of phage-provided ARGs . The authors hypothesised that this may be caused by the release of
[9]
virions capable of lysing susceptible competitors, and that the competitiveness of lysogens, in turn, could
[9]
depend on and vary with the inducibility of prophages in their specific environment . This inducibility
was proven for some of the L. curvatus prophages in this study.

Prophage inducibility
We supported our predictive approaches with induction experiments, which demonstrated the manifold
lysis responses of different putative lysogens ranging from only a small step in the post-induction growth
curve (L. curvatus strain TMW 1.1447) to nearly complete lysis (L. curvatus strain TMW 1.2272). We used
the two common phage inducers, UV light and mitomycin C, to induce prophages within six L. curvatus
lysogens. Both inducers cause DNA damage to the host, activating its SOS response system and resulting in
conversion from lysogenic to lytic life cycle of phages, as shown in E. coli .
[63]
Notably, we wanted to show that induction is possible with both induction treatments, as each method has
its advantages. Mitomycin C might be the easier method to transfer and reproduce induction between
laboratories, yet UV light can be a powerful, cheap, and accessible alternative to antibiotics such as
mitomycin C. We achieved the best results using a four-min UV light treatment for induction; this protocol
was also used for the post-induction growth experiment depicted in Figure 3 and previously for phage
[21]
induction in L. sakei . When applied for the first time, various UV irradiation durations and/or intensities
should be tested. We found that UV light was a preferable induction method over mitomycin C. Following
induction treatment, the growth curves of all tested lysogenic strains displayed a halt or decrease in turbidity
measured at an optical density at 600 nm (OD ), indicating lysis and suggesting successful prophage
600
induction. Lysis differences also occurred between different strains with only one intact prophage (e.g.,
L. curvatus TMW 1.591 and L. curvatus TMW 1.1447). We conclude that the lysis response is strongly
phage/host-dependent and not necessarily reliant on the number of intact prophages in a specific strain.

Only virions with the siphovirus morphotype (icosahedral capsids and long non-contractile tails) were
found in the micrographs of the purified, post-induction lysates. Notably, only one phage morphotype was
found per lysate of each induced lysogenic L. curvatus strain, although multiple intact prophages were
predicted for some of them (L. curvatus strains TMW 1.706 and TMW 1.1365), similar to our previous
[21]
study describing prophages in L. sakei . There are multiple possible explanations for this. It is possible that
(i) only some of the intact predicted prophages have been extensively induced; or (ii) induction happened
for all prophages, but only one phage particle morphotype was assembled. The sequencing of viral DNA
extracted from post-induction analyses verified that multiple prophages were indeed induced
simultaneously, although at least one phage genome of multi-prophage harbouring strains was fragmentized