Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1

International audienceThe CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function

Characterization of the genetic switch from phage ɸ13 important for Staphylococcus aureus colonization in humans

Temperate phages are bacterial viruses that after infection either reside integrated into a bacterial genome as prophages forming lysogens or multiply in a lytic lifecycle. The decision between lifestyles is determined by a switch involving a phage‐encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here, we investigate the switch of phage ɸ13 from the human pathogen Staphylococcus aureus. ɸ13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the ɸ13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high‐affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from ɸ13 with the lytic promoter fused to lacZ into S. aureus and Bacillus subtilis. Analysis of β‐galactosidase expression indicated that decision frequency is independent of host factors. The white “lysogenic” phenotype, which relies on the expression of cI, could be switched to a stable blue “lytic” phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage ɸ13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus

Structural basis of the bacteriophage TP901-1 CI repressor dimerization and interaction with DNA

Temperate bacteriophages are known for their bistability, which in TP901‐1 is controlled by two proteins, CI and MOR. Clear 1 repressor (CI) is hexameric and binds three palindromic operator sites via an N‐terminal helix‐turn‐helix domain (NTD). A dimeric form, such as the truncated CI∆58 investigated here, is necessary for high‐affinity binding to DNA. The crystal structure of the dimerization region (CTD$_1$) is determined here, showing that it forms a pair of helical hooks. This newly determined structure is used together with the known crystal structure of the CI‐NTD and small angle X‐ray scattering data, to determine the solution structure of CI∆58 in complex with a palindromic operator site, showing that the two NTDs bind on opposing sides of the DNA helix