| Claim / feature | Evidence type | Key quantitative data | Primary source with year and URL |
|---|---|---|---|
| **Gene identity and primary function:** T4 **rI** encodes **RI**, a **t-specific antiholin** that inhibits holin **T** and imposes lysis inhibition (LIN) | Genetics, functional complementation, cross-linking | Plasmid-borne **rI** alone can impose LIN on **T**-mediated lysis; LIN-defective **rI57** fails to block lysis; cross-linked **T–RI** species at ~**38 kDa** | Ramanculov & Young 2001, *Molecular Microbiology*, https://doi.org/10.1046/j.1365-2958.2001.02491.x (pqac-00000004, pqac-00000039) |
| **Protein size / gene product:** RI is a small acidic protein with a short N-terminal export segment and periplasmic C-terminal domain | Genetics, sequence analysis, biochemistry | Reported as **97 aa**, ~**11.1 kDa**, pI **4.8**; soluble periplasmic domain **sRI ~75 aa**, ~**8.8–9.7 kDa** | Paddison et al. 1998, *Genetics*, https://doi.org/10.1093/genetics/148.4.1539; Moussa et al. 2012, *Protein Science*, https://doi.org/10.1002/pro.2042 (pqac-00000006, pqac-00000040, pqac-00000044) |
| **Localization:** RI acts through its **periplasmic domain**; the C-terminal periplasmic region is necessary and sufficient for LIN | Genetics, domain swaps, fractionation | ssPhoA-RICTD chimera is processed, periplasmic, and functionally supports LIN; periplasmic TCTD can block/subvert LIN | Tran et al. 2005, *Journal of Bacteriology*, https://doi.org/10.1128/jb.187.19.6631-6640.2005 (pqac-00000002, pqac-00000038) |
| **Signal peptide processing:** RI has a **cleavable Sec signal peptide**, not a classical SAR domain, in the 2021 revision | Bioinformatics, mutagenesis, fractionation, reporter fusion | SignalP predicted cleavage after **Ala24** (~**95%** probability); **A24P** blocks processing; RI signal peptide exports **PhoA** efficiently | Mehner-Breitfeld et al. 2021, *Frontiers in Microbiology*, https://doi.org/10.3389/fmicb.2021.712460 (pqac-00000016, pqac-00000018, pqac-00000019) |
| **Membrane-anchored RI can still inhibit holin:** cleavage is not required for LIN in a reconstituted system | Reconstitution, mutagenesis, fractionation | **A24P** abolished detectable processing yet preserved LIN; RI(A24P) remained membrane-localized | Schwarzkopf et al. 2024, *Frontiers in Microbiology*, https://doi.org/10.3389/fmicb.2024.1419106 (pqac-00000008, pqac-00000010, pqac-00000011) |
| **Interaction partner:** RI binds the **periplasmic C-terminal domain of holin T** rather than affecting T synthesis | Genetics, biochemistry, cross-linking | T continues to accumulate in inner membrane during LIN; excess periplasmic **TCTD** antagonizes LIN | Ramanculov & Young 2001, *Molecular Microbiology*, https://doi.org/10.1046/j.1365-2958.2001.02491.x; Tran et al. 2005, *Journal of Bacteriology*, https://doi.org/10.1128/jb.187.19.6631-6640.2005 (pqac-00000039, pqac-00000038) |
| **Stoichiometry / oligomeric state (biochemical view):** predominant soluble complex is a **1:1 RI:T heterodimer** | Biochemistry, SEC, analytical ultracentrifugation | SEC apparent mass ~**45.6 kDa**, but sedimentation supports a **heterodimer** near predicted **29.7–30 kDa**; sRI monomer ~**9.2 kDa**, **1.4S**; complex **4.1S** | Moussa et al. 2012, *Protein Science*, https://doi.org/10.1002/pro.2042 (pqac-00000005, pqac-00000044) |
| **Stoichiometry / oligomeric state (structural 2020 model):** soluble domains can form **sRI2–sT2 heterotetramers** and RI homotetramers at high concentration | X-ray crystallography, cryo-EM, solution biophysics | RI tetramer at **10 mg/mL**; PDBePISA buried area ~**8618.8 Ų**, ΔGint ~**−87 kcal/mol**, ΔGdiss ~**13.8 kcal/mol**; cryo-EM map ~**9.4 Å** | Krieger et al. 2020, *Journal of Molecular Biology*, https://doi.org/10.1016/j.jmb.2020.06.013 (pqac-00000012, pqac-00000013, pqac-00000015) |
| **Current mechanistic interpretation (2024):** physiologically relevant inhibitory species is likely a **dimeric T/RI complex**, not the full tetramer seen in crystals | Reconstitution, mutagenesis, AlphaFold-guided analysis | Only one conserved interface is required; mutations **Y42A**, **Y42L**, **F56A** abolish LIN | Schwarzkopf et al. 2024, *Frontiers in Microbiology*, https://doi.org/10.3389/fmicb.2024.1419106 (pqac-00000008, pqac-00000009, pqac-00000010) |
| **Mechanism of inhibition:** RI prevents **holin oligomerization / triggering**, thereby delaying endolysin release and host lysis | Genetics, biochemistry, structural inference | Holin triggering in T4rI occurs around **~4000 T molecules**; under LIN ~**8000 T molecules** accumulate by **60 min** | Moussa et al. 2012, *Protein Science*, https://doi.org/10.1002/pro.2042; Moussa et al. 2014, *Journal of Bacteriology*, https://doi.org/10.1128/jb.01548-14 (pqac-00000043, pqac-00000045) |
| **Reversibility / triggering:** LIN can be reversed by membrane depolarization or energy poisons, indicating RI blocks T function rather than endolysin availability | Genetics, physiological assays | **KCN** relieves RI block; **CHCl3** causes immediate lysis; energy poisons override LIN | Ramanculov & Young 2001, *Molecular Microbiology*, https://doi.org/10.1046/j.1365-2958.2001.02491.x; Tran et al. 2005, *Journal of Bacteriology*, https://doi.org/10.1128/jb.187.19.6631-6640.2005; Moussa et al. 2014, *Journal of Bacteriology*, https://doi.org/10.1128/jb.01548-14 (pqac-00000039, pqac-00000038, pqac-00000045) |
| **Disulfide-stabilized periplasmic fold:** RI and T require Cys pairs for proper folded interaction | Biochemistry | RI disulfide **Cys69–Cys75**; T disulfide **Cys175–Cys207**; no free thiols detected in purified proteins/complex | Moussa et al. 2012, *Protein Science*, https://doi.org/10.1002/pro.2042; Krieger et al. 2020, *Journal of Molecular Biology*, https://doi.org/10.1016/j.jmb.2020.06.013 (pqac-00000043, pqac-00000042) |
| **Lysis timing / ecological effect:** superinfection-triggered LIN can greatly increase progeny yield and be sustained by repeated superinfection | Infection kinetics, modeling | Repeated superinfection at **<10 min** intervals can sustain inhibition; intracellular virions can increase by about **10-fold**; modeled visible plaque radius ~**85–86%** of rapid-lysis mutant, infection-zone radius ~**96%** | Moussa et al. 2012, *Protein Science*, https://doi.org/10.1002/pro.2042; Hvid & Mitarai 2024, *PLOS Computational Biology*, https://doi.org/10.1101/2024.02.07.579269 (pqac-00000040, pqac-00000024, pqac-00000028) |
| **DNA-binding signal model (2020 structural proposal):** sRI–sT complex may bind superinfection-derived nucleic acids non-specifically to stabilize LIN | Structure, binding assays, modeling | Reported ligand affinities: **guanosine Kd 43 µM**, **AMP 72.4 µM**, **ADP 213.6 µM**, **PRPP 225.9 µM**; DNA shifts with ss **70 bp**, ds **30/58 bp** tested | Krieger et al. 2020, *Journal of Molecular Biology*, https://doi.org/10.1016/j.jmb.2020.06.013 (pqac-00000012, pqac-00000015) |
| **Status of DNA-binding model after 2024 work:** DNA may still stabilize complexes, but the key inhibitory unit is now argued to be membrane-compatible **T/RI dimer** | Structure-informed functional reinterpretation | No new Kd reported in 2024; emphasis shifted from tetramer to membrane-anchored dimer/interface-1 model | Schwarzkopf et al. 2024, *Frontiers in Microbiology*, https://doi.org/10.3389/fmicb.2024.1419106 (pqac-00000009, pqac-00000010) |


*Table: This table compiles the main experimentally supported features of bacteriophage T4 rI/RI, including identity, localization, mechanism, stoichiometry, and lysis-control phenotypes. It is designed as a compact evidence map for the final research report, with quantitative findings and primary-source URLs.*