Flexible linker modulates the binding affinity of the TP901‐1 CI phage repressor to DNA

Abstract

Temperate bacteriophages can switch between two life cycles following infection of a host bacterium: the lytic or lysogenic life cycle. The choice between these is controlled by a bistable genetic switch. We investigated the genetic switch of the lactococcal temperate bacteriophage, TP901-1, which is controlled by two regulatory proteins, the Clear 1 (CI) repressor and modulator of repression (MOR) antirepressor. CI consists of a DNAbinding N-terminal domain and a C-terminal domain responsible for oligomerization, connected by a flexible interdomain linker. Full-length CI is hexameric, whereas the truncated version CI with 58 C-terminal residues truncated (CIΔ58), missing the second C-terminal subdomain, is dimeric, but binds with the same affinity as full-length CI to the O L operator site, responsible for lytic genes transcription repression. Three variants of CIΔ58 with shorter, longer, and PP substituted linkers were produced and confirmed by circular dichroism spectroscopy and nanodifferential scanning fluorimetry to be well folded. With small-angle X-ray scattering, we delineated the conformational space sampled by the variants and wild-type in solution and found that shortening and lengthening the linker decrease and increase this, respectively, as also substantiated by molecular dynamics and as intended. Isoelectric focusing electrophoresis confirmed that all variants are able to bind to the MOR antirepressor. However, using electrophoretic mobility shift assays, we showed that shortening and lengthening the linker lead to a 94 and 17 times decrease in affinity to O L , respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNAbinding and subsequent TP901-1 genetic switch function.

CI:Mor interactions in the lysogeny switches of Lactococcus lactis TP901-1 and Staphylococcus aureus φ13 bacteriophages

“…Protein-protein interactions between φ13 CI and Mor were assessed using isoelectric focusing (IEF) electrophoresis under native conditions, similarly as previously for variants of TP901-1 CIΔ58 (a truncated version of CI missing the last 58 residues) and Mor [ 22 ] . Samples were run on a SERVALYT™ PRECOTES™ Wide Range pH 3-10 gel with SERVA IEF Marker 3-10, Liquid Mix as a protein standard.…”

“…Cooperative binding of the TP901-1 CI dimer to O L ensures 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 [ 22 ] to a characteristic all α-helical C-terminal domain [ 20 ] , within which a helical hook subdomain mediates dimerization (CTD 1 ) [ 23 ] . TP901-1 CI does not contain a typical auto-cleavage site or a peptidase domain at its C-terminus.…”

“…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][19][20][21][22][23][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 R and P L promotors, respectively) instead of Cro.…”

Effect of encapsulation and natural polyphenolic compounds on bacteriophage stability and activity on Escherichia coli in Lactuca sativa L. var. longifolia

“…Lambda phage is temperate and requires the repressor protein Cl to begin and maintain the lysogenic cycle. The Cl protein encodes by the cI gene and represses the late gene expression responsible for the lytic cycle (Varming et al, 2021). For this reason, because the two isolated coliphages did not have these genes, they are helpful to the user in phage therapy.…”

Partial Atomic Model of the Tailed Lactococcal Phage TP901-1 as Predicted by AlphaFold2: Revelations and Limitations

“…The temperate lactococcal P335 phage TP901-1 is particularly well characterized with respect to its three-dimensional structure and the interactions with its host [5,[16][17][18]. Furthermore, it has been employed as a model to understand the phenomenon of lysogeny and the factors that underpin the lytic-lysogenic lifestyle decision process [19][20][21]. TP901-1 structural module encompassing 22 genes that are responsible for the biosynthesis of the phage head, tail, baseplate, and DNA packaging machinery (Figure 1).…”