Repression of the lysogenic PR promoter in bacteriophage TP901-1 through binding of a CI-MOR complex to a composite OM-OR operator

Margit Pedersen,Jesper Tvenge Neergaard,Johan Cassias,Kim Sneppen,Leila Lo Leggio,Mogens Kilstrup,Karin Hammer,Kim Krighaar Rasmussen

doi:10.1038/s41598-020-65493-0

Margit Pedersen, Jesper Tvenge Neergaard + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-65493-0

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Abstract

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the PR and PL promoters respectively. CI can repress the transcription of the mor gene at three operator sites (OR, OL, and OD), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (OM sites), and a common topology of three OM sites adjacent to the OR motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The OR site and all putative OM sites are important for establishment of the anti-immune repression of PR, and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the PR promoter.

Highlights

Genetic switches are used by temperate bacteriophages to decide between their two alternative life cycles following injection of their DNA into the host bacterium[1]; the lytic cycle where the phage multiplies and kills the bacterium at once or the lysogenic cycle where killing is suspended
In the Lactococcus lactis laboratory strains MG136322 belonging to subspecies cremoris we have likewise found a small region corresponding the C-terminal domain of the CI repressor in the T712 prophage of MG1363
We have presented two models for establishment of the anti-immune repression of the PR promoter in TP901-1 and related bacteriophages against bacteria of Lactococcus, Streptococcus, Enterococcus, and Staphylococcus species

Summary

Introduction

Genetic switches are used by temperate bacteriophages to decide between their two alternative life cycles following injection of their DNA into the host bacterium[1]; the lytic cycle where the phage multiplies and kills the bacterium at once or the lysogenic cycle where killing is suspended. Overlapping the lytic mor (PL) promoter is a second CI operator (OL), and a third (OD) is located in the distal part of the mor gene This overall operator topology appears to be shared among many lactococcal and streptococcal bacteriophage switches, despite the fact that the operator sequences do not follow the same consensus[7]. Upon binding of CI dimers to each of these operators in the lysogenic state of the TP901-1 switch, the PL promoter is almost fully repressed ensuring low concentrations of MOR and lytic proteins, while the PR promoter is only partially auto-repressed. The apparent simplicity of the system is deceiving as genetic analysis has revealed that the CI protein is required in collaboration with MOR for repression of its own promoter[3], so the cI gene needs to be transcribed at a certain level in the lytic state of the switch. The suggestion that CI should function as a co-repressor for MOR is not exceptional, and would add an interesting twist to the P1 paradigm

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