SieA is a small prophage-expressed integral membrane protein of Salmonella phage P22 (162 or 164 residues; the start codon is uncertain) with three predicted transmembrane helices and no recognisable catalytic motif. Expressed from the P22 prophage in a lysogen, it resides exclusively in the host inner membrane and mediates superinfection exclusion: it blocks injection of the DNA of superinfecting P22 and other P22-like phages, and of P22 transducing particles, across the inner membrane. Adsorption of the superinfecting virion, host growth, and progeny production after prophage induction are unaffected, and unrelated tailed phages are not excluded. A single chromosomal copy of sieA is necessary and sufficient for exclusion. Phage mutations that escape the block map to the C-terminal regions of the ejection proteins gp16 and gp20, supporting a model in which SieA interferes with the assembly or function of the trans-envelope DNA delivery conduit built from the ejection proteins; the molecular activity by which it does so is unknown. SieA homologs occur in P22-like prophages of several enteric genera and fall into three sequence types less than 30% identical to one another.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0020002 host cell plasma membrane | EXP PMID:7768804 The superinfection exclusion gene (sieA) of bacteriophage P2... | ACCEPT | Summary: UniProt-assigned experimental row for the host (bacterial) inner membrane location of SieA, from Hofer et al. 1995. The location claim is correct and is now directly demonstrated by inner/outer membrane fractionation in Leavitt et al. 2024. Reason: The cached PMID:7768804 record is abstract-only (PMC returns no full text), and the experimental content described there is recovery of overproduced SieA from a crude membrane fraction, with the inner-membrane assignment inferred from three hydrophobic segments; Leavitt et al. 2024 note explicitly that the 1995 study did not resolve inner from outer membrane. The claim nonetheless holds: FLAG-tagged, exclusion-competent SieA partitions with the inner and not the outer membrane (PMID:38236051, Fig. 3B), and this is where SieA acts on injecting phage DNA. The term is therefore accepted as a core location rather than modified. Supporting Evidence: PMID:7768804 The SieA protein was moderately overproduced from an expression vector in cultures of Escherichia coli and could be recovered from the membrane fraction. PMID:38236051 We conclude that SieA is an exclusively inner membrane protein. |
| GO:0020002 host cell plasma membrane | IDA PMID:38236051 Bacteriophage P22 SieA-mediated superinfection exclusion. | ACCEPT | Summary: CACAO IDA row. Leavitt et al. 2024 fractionated inner and outer membranes of Salmonella carrying a single chromosomal C-terminally FLAG-tagged sieA gene and detected SieA by anti-FLAG immunoblot in whole cells and the inner membrane only (Fig. 3B). The CACAO term matches this result at the right granularity. Reason: What the paper shows for this gene: the tagged allele retains full exclusion activity, so the protein assayed is the functional one; the anti-FLAG band appears at similar intensity in whole-cell and inner-membrane lanes and is absent from the outer-membrane lane, with the untagged strain as specificity control (Fig. 3B); a second, independent fractionation method gave the same result. GO:0020002 host cell plasma membrane is the bacterial host inner membrane, so the CACAO term is neither too general nor too downstream, and the row is not a single-image localization claim. Accepted as the core location. Supporting Evidence: PMID:38236051 This C-terminally tagged SieA has strong superinfection exclusion activity PMID:38236051 These antibodies produced tagged SieA bands of approximately the same intensity in the whole-cell extract and inner membrane (IM) fraction lanes of the FLAG-tagged strain, but no such band was present in the outer membrane fraction. PMID:38236051 We conclude that SieA is an exclusively inner membrane protein. |
| GO:0020002 host cell plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping of the UniProt subcellular location "Host cell inner membrane" (SL-0373) to GO:0020002. The underlying UniProt statement carries experimental evidence (ECO:0000269|PubMed:7768804) and the location is confirmed experimentally by PMID:38236051, so the mapping is correct. Reason: The keyword-to-term mapping is accurate for a bacterial host: the inner membrane is the host plasma membrane. The claim agrees with the two experimental rows for the same term, so the actions are consistent. Supporting Evidence: PMID:38236051 We conclude that SieA is an exclusively inner membrane protein. |
| GO:0033644 host cell membrane | IDA PMID:7768804 The superinfection exclusion gene (sieA) of bacteriophage P2... | MODIFY | Summary: CACAO IDA row on the broad host cell membrane term, from the 1995 recovery of overproduced SieA in the membrane fraction. The claim is true but is the parent of the host cell plasma membrane rows accepted above, which are supported by direct inner/outer membrane separation. Reason: What the cited paper shows for this gene: SieA overproduced from a vector in E. coli is recovered from the membrane fraction, with no separation of inner from outer membrane - so the CACAO student's choice of the generic host cell membrane term is a faithful reading of that experiment, and this is a granularity issue rather than an unsupported claim. Since the specific location is now established, the term should be refined to GO:0020002 host cell plasma membrane, which this gene already carries from PMID:38236051 and from UniProt. Not REMOVE: the membrane claim is correct and reinforces the specific rows. Proposed replacements: host cell plasma membrane Supporting Evidence: PMID:7768804 The SieA protein was moderately overproduced from an expression vector in cultures of Escherichia coli and could be recovered from the membrane fraction. PMID:38236051 That study did not determine whether SieA is in the inner or the outer membrane. PMID:38236051 We conclude that SieA is an exclusively inner membrane protein. |
| GO:0046596 regulation of viral entry into host cell | IMP PMID:4610992 Superinfection exclusion by P22 prophage in lysogens of Salm... | MODIFY | Summary: CACAO IMP row from Susskind, Botstein & Wright 1974, the paper that showed superinfecting phage DNA fails to enter sieA+ lysogens. The direction is right and the claim is true, but the same paper and the same experiment yield GO:0098669 superinfection exclusion, which names the process the protein actually executes. Reason: What the cited paper shows for this gene: by its title, and by the account of it in PMID:38236051, a sieA+ prophage leaves adsorption intact while superinfecting P22 DNA never reaches the cytoplasm - it is neither cut by cytoplasmic restriction enzymes nor expressed. The cached record for PMID:4610992 is title-and-metadata only (no abstract text), so the figures cannot be inspected; per the curator-deference rule this is not grounds for REMOVE, and the result is in any case corroborated by direct assay in 2024. The CACAO term is not too downstream and is not a phenotype read as a process, but it is a parent-level restatement: SieA blocks entry of a superinfecting relative of the resident prophage, which is exactly GO:0098669, a term this gene already carries from the same reference. Replaced by that term. This is a lateral correction rather than a descendant refinement, since GO:0098669 sits under viral process rather than under GO:0046596. Proposed replacements: superinfection exclusion Supporting Evidence: PMID:4610992 Failure of superinfecting phage DNA to enter sieA+ lysogens. PMID:38236051 In a sieA+ lysogen, superinfecting P22 DNA is not cleaved by cytoplasmic host restriction endonucleases, and superinfecting P22 genes are not expressed (20). PMID:38236051 Previous studies have shown that a sieA+ P22 prophage does not affect the adsorption of superinfecting P22 phage virions (18, 23, 24) or the production of progeny after P22 prophage induction (24). |
| GO:0098669 superinfection exclusion | IDA PMID:38236051 Bacteriophage P22 SieA-mediated superinfection exclusion. | ACCEPT | Summary: CACAO row from Leavitt et al. 2024, which shows that one chromosomal copy of sieA is sufficient to lower P22 plaque formation by at least seven orders of magnitude (Fig. 1) and that SieA blocks DNA injection itself, measured as loss of infection-triggered K+ efflux (Fig. 2A). This is the core function. Reason: What the paper shows for this gene: sieA alone, at single copy under its own promoter and with no effect on host growth, confers the exclusion phenotype (Fig. 1); infection of the sieA+ strain releases almost no K+ while the isogenic sieA- strain releases nearly all of it, placing the block at DNA entry rather than at adsorption or at a later step (Fig. 2A); escape mutants map to the ejection proteins gp16 and gp20, tying the block mechanistically to the DNA delivery conduit. The CACAO term matches the assay exactly and is the correct granularity for a prophage-encoded exclusion protein, so this is not a phenotype read as a process. Accepted as core. Supporting Evidence: PMID:38236051 Thus, the sieA gene in one copy per Salmonella genome is sufficient to mediate robust superinfection exclusion. PMID:38236051 Figure 2A shows that P22 causes very little, if any, K+ release from the sieA+ strain, while nearly all of the cellular K+ is released from the isogenic sieA– strain, suggesting that DNA injection per se is blocked by SieA. PMID:38236051 We conclude from the above results that SieA alone blocks injection of P22 DNA into Salmonella cells and has no other additional effect on P22 growth. |
| GO:0098669 superinfection exclusion | IMP PMID:4610992 Superinfection exclusion by P22 prophage in lysogens of Salm... | ACCEPT | Summary: CACAO IMP row on the same term from the 1974 genetic study: sieA+ lysogens exclude superinfecting P22 because the superinfecting DNA fails to enter the cell. The term matches the paper's conclusion. Reason: What the cited paper shows for this gene: comparison of sieA+ and sieA- lysogens shows that superinfecting phage DNA does not enter the sieA+ cell (paper title; the supporting detail - DNA not cleaved by cytoplasmic restriction enzymes, genes not expressed, adsorption unaffected - is recounted in PMID:38236051). The cached record has no abstract or full text, so the figures cannot be checked; by the curator-deference rule the experimental annotation stands, and here it is independently confirmed by the 2024 direct assays, so ACCEPT rather than UNDECIDED. This row and the IDA row to the same term reinforce each other. Supporting Evidence: PMID:4610992 Failure of superinfecting phage DNA to enter sieA+ lysogens. PMID:38236051 The SieA protein blocks superinfection by P22-like phages (including P22 itself) at an early stage of infection (19, 22–24). |
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Download this section (compressed HTML)Q: Does SieA act by direct protein-protein contact with the P22 ejection proteins gp16 and gp20, or indirectly by modifying the inner membrane environment in which the injection conduit assembles?
Suggested experts: Sherwood R. Casjens, Justin C. Leavitt
Q: What is the relationship between SieA and the host inner membrane protein YajC, which is itself essential for P22 DNA injection? SieA-resistant phages still fail to plate on a yajC mutant host, so the two requirements appear separable.
Suggested experts: Sherwood R. Casjens
Q: Would GO benefit from a molecular function term for prophage-encoded inhibition of phage genome translocation across the host inner membrane, so that exclusion proteins such as SieA can be annotated at the activity level and not only to the process term GO:0098669?
Q: Which of the two possible start codons is used in vivo, and what is the membrane topology of the conserved cytoplasmic-side motif FxRYPxEAxxFNK (P22 SieA residues 145-157) shared by all three SieA sequence types?
Suggested experts: Carolyn M. Teschke
Experiment: Co-purify FLAG-tagged SieA from inner membranes of infected sieA+ Salmonella and probe for gp16 and gp20, comparing wild-type ejection proteins with the escape variants gp16 P546S, gp16 G532W and gp20 G350D; complement with in vivo crosslinking or a bacterial two-hybrid assay suited to membrane-embedded partners.
Hypothesis: SieA binds the P22 ejection proteins gp16 and/or gp20, and escape mutations weaken that interaction.
Type: protein interaction assay
Experiment: Reconstitute purified SieA into proteoliposomes or inverted inner membrane vesicles and assay ejection-protein conduit formation and DNA translocation in vitro, distinguishing a direct steric or binding activity from an indirect membrane effect.
Hypothesis: SieA is itself sufficient to block conduit formation or DNA translocation, with no other phage or host factor required.
Type: in vitro reconstitution
Experiment: Construct chimeras between the three SieA sequence types (A, B, C), including swaps of the N-terminal extensions unique to types B and C, and assay exclusion of P22, of P22 hybrids carrying heterologous ejection protein genes (HK620, Sf6, L, CUS-3) and of non-P22-like phages such as SP6.
Hypothesis: Target specificity is determined by a defined region of SieA, likely in the membrane-embedded or periplasmic segments rather than the type-specific N-terminal extensions.
Type: chimera / structure-function analysis
Experiment: Determine SieA topology experimentally by substituted-cysteine accessibility or dual-reporter (PhoA/LacZ) fusions at defined positions, then mutate the conserved motif and measure exclusion efficiency and K+ efflux upon P22 infection.
Hypothesis: The conserved FxRYPxEAxxFNK motif lies on the cytoplasmic face and is required for exclusion.
Type: membrane topology mapping and mutagenesis
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