sieA (Salmonella phage P22) — curation notes

Provenance note: no deep-research API keys were available in this container, so
just deep-research was not run and no -deep-research-<provider>.md file exists. Research
was done directly from the cached publications (publications/PMID_7768804.md,
PMID_4610992.md, PMID_38236051.md), the UniProt record (sieA-uniprot.txt), the PubMed
MCP, and OLS. The GONUTS wiki (gowiki.tamu.edu) was tried for the CACAO student evidence on
both PMID pages (/wiki/index.php/PMID:4610992, /wiki/index.php/PMID:7768804) but the site
is currently offline for upgrades ("temporarily offline for upgrades"), so the student notes
could not be read.

Protein

Q38674, 164 aa (start codon uncertain: 492 or 480 bp ORF, so 164 or 162 aa), encoded between
P22 genes mnt and 16. Three predicted transmembrane helices (UniProt FT TRANSMEM 8–28,
85–105, 121–141, ECO:0000255; TMHMM in Fig. 3A of PMID:38236051). Pfam PF26849 (SIEA),
InterPro IPR059742. UniProt subcellular location: "Host cell inner membrane
{ECO:0000269|PubMed:7768804}; Multi-pass membrane protein {ECO:0000305}". No catalytic motif,
no known molecular activity.

Function

P22 prophages carry four superinfection exclusion systems (c2 repressor, gtrABC, sieA,
sieB). SieA acts early: PMID:38236051
Adsorption is unaffected, and so is progeny production after induction:
PMID:38236051
The classic genetic/physiological evidence that the block is at DNA entry is Susskind,
Botstein & Wright 1974 (PMID:4610992; cache is abstract-only, abstract text itself is not in
the cached record — only title, authors, DOI), summarized by the 2024 paper:
PMID:38236051 and
PMID:38236051 The paper's own title
states the result: "Failure of superinfecting phage DNA to enter sieA+ lysogens."
Exclusion is not DNA-sequence specific — transduction is excluded too:
PMID:38236051

Leavitt et al. 2024 (PMID:38236051, full text cached)

sieA alone, in one chromosomal copy, is sufficient:
PMID:38236051 with plaque formation lowered ≥7 logs (Fig. 1):
PMID:38236051.
DNA injection per se is blocked, by K+ efflux assay (Fig. 2A):
PMID:38236051 and
PMID:38236051
Inner membrane localization (Fig. 3B, anti-FLAG immunoblot of inner vs outer membrane
fractions of a chromosomal sieA-FLAG strain that retains exclusion activity):
PMID:38236051; the 1995
study had not resolved this: PMID:38236051
Mechanism: escape mutants map to ejection (E-) proteins gp16 and gp20:
PMID:38236051
and PMID:38236051 Location of the interference:
PMID:38236051
Specificity: PMID:38236051
Diversity: three sequence types <30% identical;
PMID:38236051

Hofer et al. 1995 (PMID:7768804, abstract-only; PMC full text not retrievable via PMC API)

PMID:7768804 and PMID:7768804 — i.e. the experimental part is a crude membrane-fraction
recovery under overproducing conditions in E. coli; the inner-membrane assignment there is
sequence-based inference, later confirmed experimentally in 2024.

CACAO rows (ASSIGNED BY = CACAO in sieA-goa.tsv)

term	ev	PMID	assessment
GO:0046596 regulation of viral entry into host cell	IMP	4610992	correct direction (negative), but less informative than GO:0098669, which is annotated from the same paper; superinfection exclusion is the process the gene executes
GO:0098669 superinfection exclusion	IMP	4610992	matches the paper exactly (failure of superinfecting DNA to enter sieA+ lysogens)
GO:0098669 superinfection exclusion	IDA	38236051	matches; necessary-and-sufficient plus K+ efflux and escape-mutant data
GO:0020002 host cell plasma membrane	IDA	38236051	matches Fig. 3B inner/outer membrane fractionation
GO:0033644 host cell membrane	IDA	7768804	true but entailed by the host cell plasma membrane rows; the 1995 paper's own evidence is a crude membrane fraction, which is all it can support

No CACAO row here is a phenotype-as-process error, a paper-not-about-the-gene error, or an
unsupported single-image localization: every row is on the one protein the papers are about,
and the localization row is backed by a fractionation with two methods plus a tagged-protein
control. The only granularity issues are the broad GO:0033644 beside GO:0020002, and
GO:0046596, which is a parent-level restatement of the same experiment that yielded the
specific GO:0098669.

Open questions

Does SieA bind gp16/gp20 directly, or alter the inner membrane so the conduit cannot
assemble? No binding assay has been reported.
Why is SieA specific for P22-like phages and not other short-tailed phages (SP6)?
Is the host inner membrane protein YajC, required for P22 DNA injection, a SieA target?
PMID:38236051 — the SieA-resistant triple mutants still did not plate on a
yajC– host, so the two requirements are separable but unexplained.
Which start codon is used in vivo, and what is the membrane topology of the conserved
cytoplasmic motif FxRYPxEAxxFNK (residues 145–157)?