Revealing the mechanism of repressor inactivation during switching of a temperate bacteriophage

Abstract

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.

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

“…MOR (UniprotKB accession number http://www.uniprot.org/uniprot/O48504) was expressed and purified as previously described [15]. CIΔ58 (UniprotKB accession number http://www.uniprot.org/uniprot/O48503, residue 1–122) variants were purchased as purified plasmid DNA samples cloned into expression vector pET‐30a(+) with kanamycin resistance (Genscript Biotech, Leiden, the Netherlands).…”

“…Recently, the crystal structure of CI‐NTD in complex with MOR was determined using X‐ray crystallography, suggesting the mechanism by which MOR relieves repression of P L through binding to one CI‐NTD and preventing interaction of the other CI‐NTD with DNA on the O L site. As differences in K D for MOR binding to CI‐NTD or CIΔ58 are insignificant (672 ± 43 and 633 ± 38 n m , respectively), the additional linker and CI‐CTD 1 do not seem to play a role in the CI/MOR interaction, also supported by NMR titration using MOR and isotope labelled CI‐NTD or CI∆58 [15].…”

“…The flexible interdomain linker in phage CI repressors can play a crucial role in the way they function. A linkerless mutant of the phage lambda CI lost the ability to bind DNA cooperatively at certain distances [9,12,15]. (C) Computational models used as input for molecular dynamics simulations.…”

“…The colors correspond to the color scheme in (A). The crystal structures of NTD and CTD 1 of CI and MOR are shown, as determined previously[9,12,15]. (C) Computational models used as input for molecular dynamics simulations.…”

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).…”