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Page 4 of 16 Varming et al. Microbiome Res Rep 2024;3:15 https://dx.doi.org/10.20517/mrr.2023.50

general repressor DNA-binding domain structure and some analogies to the λ system, there are remarkable
differences at the molecular level in lysogeny switches from different temperate phages.

Among bacteriophages of Gram-positive bacteria, one well-characterized example is the switch of TP901-1
infecting Lactococcus lactis, a commonly used bacterium in the dairy industry, which we have previously
studied in detail at the molecular level [18-24] . The TP901-1 switch is encoded by a < 1 kb fragment of the
phage genome [Figure 1B], including genes for the CI repressor and a Mor (modulator of repression)
antirepressor (transcribed from the P and P promotors, respectively) instead of Cro. As for phage λ, CI in
L
R
TP901-1 contains a DNA-binding, N-terminal domain (CI-NTD), which consists of a characteristic helix-
turn-helix (HTH) structure . Interaction between CI and DNA is at three palindromic operator sites
[19]
related in sequence (O , O , and O ). Cooperative binding of the TP901-1 CI dimer to O ensures
L
D
R
L
maintenance of the lysogenic, immune state, and inhibition of expression of lytic genes and mor. In contrast
to the predominantly β-sheet fold of the λ phage CI-CTD protease domain, the TP901-1 CI-NTD is
connected by a flexible linker to a characteristic all α-helical C-terminal domain , within which a helical
[20]
[22]
hook subdomain mediates dimerization (CTD ) . TP901-1 CI does not contain a typical auto-cleavage site
[23]
1
or a peptidase domain at its C-terminus. However, induction is still RecA dependent through unknown
[25]
mechanisms. Furthermore, TP901-1 does not have a close functional counterpart to Cro. Instead, the lytic
state depends on the Mor antirepressor. Mor also consists of a typical HTH DNA-binding domain , in a
[24]
similar way to Cro, but unlike Cro, Mor has no demonstrated high-affinity interaction with DNA on its
own. Mor has a dual function in establishing the lytic state, primarily as an antirepressor of CI. Structural
[24]
evidence from a CI-NTD:Mor complex and a CI-NTD:DNA complex [19,23] strongly suggests that it
functions as an antirepressor by steric hindrance, preventing binding of CI-NTD monomers in a dimer to
[24]
adjacent half-sites on O . While CI is sufficient to keep the phage in the lysogenic state, direct interaction
L
of Mor with CI is needed for the lytic state . Evidence based on the use of switch plasmids and NMR
[21]
[24]
[24]
titrations suggests that in the lytic state, the CI:Mor complex interacts with an extended composite site in
the O /O region, with Mor thus functioning as corepressor as well as an antirepressor for CI. A detailed
R
M
analysis by NMR spectroscopy also revealed that Mor, in particular, is rather dynamic in solution and
rearrangement of the structure occurs on complex formation .
[24]
Considering the fundamental importance of lysogeny in pathogen evolution, molecular-level information is
[4]
surprisingly scarce for lysogeny switches of phages infecting Gram-positive bacteria including pathogens.
For S. aureus, the currently best-studied temperate phage switch is that of φ11. It resembles the switch of
phage λ, but has a different arrangement of operator sites and involves an additional protein, gp07, which
enhances Cro activity [4,26] . Furthermore, φ11 CI and Cro have only low sequence similarities with the
corresponding λ proteins and significant functional differences .
[27]
Only recently, we have characterized the lysogeny switch of φ13, a member of the Sa3int family of phages ,
[28]
[29]
which, as stated above, is strongly implicated in promoting human colonization by MRSA . The φ13
switch [Figure 1C] shares features with both phage λ and the TP901-1 switches [Figure 1B], with CI-NTD,
Mor, and the operator sites being highly similar to those of the TP901-1 switch, while the CI-CTD region
shows both a dimerization region reminiscent of TP901-1 and a peptidase domain and auto-cleavage site as
is found in λ. We demonstrated that the genomic φ13 region corresponding to the TP901-1 switch also
functions as a switch, that the lytic state is induced through the SOS response, and that φ13 CI binds to O ,
L
O , and O D [28] . Thus, the φ13 switch combines the auto-cleavage mechanism of lambdoid phages with a
R
TP901-1-like dimerization domain and the Mor-mediated regulation. However, a direct interaction between
CI and Mor was not demonstrated, nor were the switch components characterized structurally. In this
paper, we thus focus further on the structural basis of CI:Mor interaction in the switches of TP901-1 and
φ13, highlighting similarities and differences.