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Varming et al. Microbiome Res Rep 2024;3:15 https://dx.doi.org/10.20517/mrr.2023.50 Page 13 of 16

φ 13, the corresponding residue to Phe75 is a Tyr; this could indicate that in φ13, the CI:MOR complex is
slightly weaker; however, the interacting residues on Mor are not conserved between TP901-1 and φ13. This
may have functional implications that are not yet understood, though as we demonstrate here, the φ13
CI:MOR complex can definitely form in vitro.

The refined crystal structure of the CI-NTD in the present study enabled us to see the changes in CI
conformation upon CI:Mor complex formation in greater detail. No large changes in the location of Phe75
were observed between the CI-NTD and CI-NTD:Mor structures, and neither in the following amino acids
Asp76 to Val80. Instead, the most notable rearrangement upon complex formation is Glu69, which forms
an unusual interaction with the aromatic ring of Phe67 in Mor through its aliphatic chain. In the solution
NMR structure ensemble of Mor alone, Phe67 assumes several conformations, about half very similar to the
one in the crystal complex of CI-NTD and Mor, but all clashing with CI-NTD Glu69 in its uncomplexed
form. The functional role of Glu69 residue was not previously investigated and would be an interesting
topic for future mutagenesis studies.

Recently, the lysogeny switch of the Sa phage φ13 - important for human colonization by S. aureus - has
[28]
been partly characterized . This switch contains elements similar to both the TP901-1 and λ lysogeny
switches. In particular, the sequence similarity of CI-NTD and Mor to the TP901-1 counterparts suggests
similar interaction mechanisms. It has therefore been important to establish that φ13 CI and Mor can also
form stable complexes, which we demonstrate here. Furthermore, through the appropriate use of AF2
modeling, we have been able to model and analyze the φ13 CI-NTD:Mor interface and suggest further areas
of investigation.

Recent work has shown that in addition to the interplay between CI, Mor, and RecA, host-specific factors
and additional phage regulatory genes are also involved in determining the mobility of φ13 and other
S. aureus phages . This opens new possibilities for discovering important regulatory mechanisms beyond
[37]
the CI:Mor switch. However, it is important to remember that even the basic switching mechanism and its
interplay with host factors is not yet understood in φ13, and there is a danger in assuming similarity to other
systems. The structural conservation certainly suggests strong functional conservation in the switch
mechanisms of TP901-1 and φ13. Thus, we could expect a similar mechanism for the antirepressor function
of MOR in φ13 as in TP901-1, implying that Mor binding to CI interferes with high affinity binding to O .
L
However, it is puzzling then that the φ13 switch should also require a λ-like CI protease domain for its
function, since the TP901-1 switch does not. Therefore, future work needs to ascertain experimentally if
either or both mechanisms (CI:Mor binding and/or CI autocleavage) are at play during the establishment of
the lytic state in φ13. Furthermore, the presumed additional function of CI and Mor as corepressors of P is
R
still poorly characterized. In TP901-1, Mor Arg31 has been implicated in the binding of the CI:Mor complex
to the composite operator site, such that its mutation to Ala resulted in 100% lysogeny. This residue is
structurally conserved in φ13, where it might have a similar role. However, the way in which the complexes
interact with the operator sites to repress transcription of the lysogenic genes is still a significant question
mark for both phages.

For φ13, determining the details of the switch will enable an understanding of which conditions promote
the excision of the phage and which cells it may successfully integrate into. As φ13 and Sa3int phages
promote human colonization, their establishment in S. aureus strains such as those of animal origin can
cause host jumps of strains, as has recently been observed for livestock strains infecting humans .
[10]
Knowledge of the molecular details of φ13 lysogeny and the mechanistic details behind phage excision may
be leveraged in the future to limit dissemination of the phage, thereby reducing the human risk of S. aureus