AimR Adopts Preexisting Dimer Conformations for Specific Target Recognition in Lysis-Lysogeny Decisions of Bacillus Phage phi3T

Abstract

A bacteriophage switches between lytic and lysogenic life cycles. The AimR-AimP-AimX communication system is responsible for phage lysis-lysogeny decisions during the infection of Bacillus subtilis. AimX is a regulator biasing phage lysis, AimR is a transcription factor activating AimX expression, and AimP is an arbitrium peptide that determines phage lysogeny by deactivating AimR. A strain-specific mechanism for the lysis-lysogeny decisions is proposed in SPbeta and phi3T phages. That is, the arbitrium peptide of the SPbeta phage stabilizes the SPbeta AimR (spAimR) dimer, whereas the phi3T-derived peptide disassembles the phi3T AimR (phAimR) dimer into a monomer. Here, we find that phAimR does not undergo dimer-to-monomer conversion upon arbitrium peptide binding. Gel-filtration, static light scattering (SLS) and analytical ultracentrifugation (AUC) results show that phAimR is dimeric regardless of the presence of arbitrium peptide. Small-angle X-ray scattering (SAXS) reveals that the arbitrium peptide binding makes an extended dimeric conformation. Single-molecule fluorescence resonance energy transfer (smFRET) analysis reveals that the phAimR dimer fluctuates among two distinct conformational states, and each preexisting state is selectively recognized by the arbitrium peptide or the target DNA, respectively. Collectively, our biophysical characterization of the phAimR dynamics underlying specific target recognition provides new mechanistic insights into understanding lysis-lysogeny decisions in Bacillus phage phi3T.

The cytoplasmic synthesis and coupled membrane translocation of eukaryotic polyphosphate by signal-activated VTC complex

“…smFRET data were collected using our MicroTime 200 system (PicoQuant) as previously described 67 . A pulsed interleaved excitation (PIE) scheme at a repetition of 32 MHz was employed for data collection to exclude the emitted photons resulted from donor only or acceptor only species 68 .…”

“…F Dex/Aem represents the photo count for the donor excitation and acceptor emission channel, F Dex/Dem represents the photo count for the donor excitation and donor emission channel, LK represents the donor leakage into the acceptor channel, Di represents the acceptor emission excited directly by the donor excitation wavelength, and γ represents the detection-correction factor. Using the established methods by Shimon Weiss group 72 , we have previously calibrated theses parameters for our instrumentation and fluorophores 67 , with LK = 0.13, Di = 0.06, and γ = 0.47, respectively 67 .…”

Host control by SPβ phage regulatory switch as potential manipulation strategy

“…Interestingly, homologs of AimR were computationally found as harbored also by other categories of phages within Bacilli genomes [18]. Further studies on AimR shed light on biophysical properties of this system [55]. Moreover, it was shown that AimR is not only important during first infection, but it may play a key role as a regulator of genes involved in the activation of the lytic cycle in lysogenic host population.…”

Structural and functional analysis of YopR and identification of an additional key component of the SPβ phage lysis-lysogeny management system

“…In the early stage of infection, the number of phages and the concentration of AimP is low. Apo-AimR binds as a homodimer via its helix-turn-helix domain to the operator in the P aimX promoter and activates transcription of the AimX ncRNA that promotes the lytic cycle (Figure 1) [Erez et al, 2017; Gallego Del Sol et al, 2019, 2022; Guan et al, 2019; Pei et al, 2021]. During the lytic cycle, accumulating AimP is secreted via the Sec pathway and processed by extracellular proteases.…”

“…The formation of an AimP-hexapeptide/AimR complex inhibits the DNA-binding activity of AimR and promotes the switch to the lysogenic cycle [Erez et al, 2017]. While the molecular details of the AimP-hexapeptide/AimR complex have been elucidated [Wang et al, 2018; Dou et al, 2018; Zhen et al, 2019; Gallego Del Sol et al, 2019, 2022; Guan et al, 2019; Pei et al, 2021], the components that act downstream of the AimX ncRNA remain to be identified (Figure 1). However, the AimP-hexapeptide-dependent control of AimR is crucial to prevent the killing of the entire bacterial population by SPβ, thereby ensuring long-term prophage replication.…”