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Page 12 of 16 Varming et al. Microbiome Res Rep 2024;3:15 https://dx.doi.org/10.20517/mrr.2023.50

Figure 4. Computational model of the φ13 CI-NTD:Mor complex. (A) Superposition of the TP901-1 experimental CI-NTD:Mor complex
(PDB code 6TRI, color scheme as in previous figures) with the φ13 CI-NTD:Mor complex obtained with AF2 (in grey). The extreme N-
and C-terminus of Mor are omitted for clarity. Helices are shown as cylinders. Side chains of interface residues mentioned in the text are
shown as sticks representation. Label numbering is for TP901-1 proteins; (B) Close-up of the φ13 CI-NTD:Mor complex highlighting the
Mor pocket in which Gln55 from CI fits. A semitransparent surface is shown for Mor. CI-NTD: N-terminal domain of Phage repressor.

Substitution of Tyr5 and Phe67 had large effects in TP901-1 in vivo, while the role of Tyr3 and Trp43 was
not investigated by mutagenesis. The model for the complex suggests several further studies of the φ13
[28]
switch, for example, using the switch plasmids in , to understand the importance of these residues.

DISCUSSION
The lactococcal TP901-1 lysogeny switch is one of the best studied at the molecular level from Gram-
positive bacteria phages. As stated above, the mechanism for maintaining the lysogenic state is well
understood and depends on the dimeric form of the CI repressor binding to its operator sites with high
affinity [21,24] . The Mor protein has the DNA binding HTH motif but is unable to bind to DNA by itself;
however, through complex formation with Mor in the lytic life cycle, CI becomes unable to bind to both of
the half-sites in O due to steric hindrance. Furthermore, the CI:Mor complex appears to be able to bind to
L
a composite binding site, thereby repressing the lysogenic promoter [21,24] .

Because of its dual involvement in the Mor function as antirepressor and corepressor, the CI:Mor complex
is central to the TP901-1-like phage switches. Previously, the kinetics of CI:Mor complex formation was
analyzed by isothermal titration calorimetry, showing that the dissociation constant was reasonably high,
around 650 nM . Mutant switches with altered amino acids in the CI:Mor interface were constructed,
[24]
based on the crystal structure of the CI-NTD:Mor complex. Using switching frequencies as an assay for
CI:Mor complex formation, the CI:Mor interface mutants showed both increased and reduced complex
formation. Though the effect was smaller than for other residues, increased complex formation (low lytic
switch frequency) was seen for CI-Phe75Ala and CI-Phe75Val substitutions, while decreased complex
formation was seen for CI-Phe75Ile and CI-Phe75Tyr substitutions (increased lytic switch frequency). As in