pppL (SP-STP) — Streptococcus pyogenes M1 GAS — curation notes

UniProt Q99YM9 (Q99YM9_STRP1, unreviewed), 246 aa, locus SPy_1626, gene pppL,
commonly called SP-STP in the literature. Co-transcribed with and immediately
downstream of the eukaryote-type Ser/Thr kinase SP-STK (SPy_1625).

Provenance note: no deep-research provider was available in this container
(no API keys), so just deep-research was not run and no -deep-research-*.md
file exists for this gene. Literature work here was done directly: the three
cited PMIDs were re-fetched with full text where PMC allowed
(cache_publication(force=True), which upgraded PMID:21917918 and PMID:22262847
from abstract-only to full text; PMID:16487973 remains abstract-only, J Mol Biol
is not in PMC). The GONUTS wiki page for PMID:21917918 was checked for the
students' recorded evidence but gowiki.tamu.edu is currently serving only
"temporarily offline while we perform upgrades", so the CACAO-side reasoning
could not be inspected.

Family and catalytic machinery

PPM/PP2C-type metal-dependent Ser/Thr phosphatase: InterPro IPR015655 (PP2C),
IPR001932, Pfam PF13672 (PP2C_2), PROSITE PS51746 (PPM_2 domain, residues
2–240), EC 3.1.3.16 [file:STRP1/pppL/pppL-uniprot.txt "DOMAIN 2..240"].
UniProt's PROSITE-ProRule cofactor note: "Binds 2 magnesium or manganese ions
per subunit." The family assignment is stated in the primary literature too
PMID:21917918.

Metal preference is experimental: PMID:16487973 Mg(2+) is only a UniRule/ProRule
expectation for the fold, not tested for this protein.

Catalysis and substrates

• Endogenous substrate work in the founding paper: the kinase/phosphatase couple
  acts on a histone-like protein (SP-HLP), and only threonine was reversibly
  modified PMID:16487973
• Response regulators of two-component systems are the physiologically important
  substrates. SP-STK phosphorylates WalR on Thr, and SP-STP removes it
  [PMID:21917918 "SP-STK was able to phosphorylate SP-WalR ( Fig. 3 C )
  specifically at threonine residues, as is evident by TLC"; PMID:21917918 "The
  SP-STP was able to dephosphorylate the SP-STK-phosphorylated SP-WalR"].
• Same for CovR [PMID:21917918 "The specificity of SP-STK mediated CovR
  phosphorylation was confirmed by its subsequent dephosphorylation by the
  cognate SP-STP"; PMID:21917918 "the TLC revealed that SP-STK mediates
  phosphorylation of CovR specifically at its threonine residues"].
• Host substrate: histone H1. In vitro dephosphorylation of CDK1-phosphorylated
  H1 PMID:22262847 and loss of the Thr146 phospho-epitope in nuclei of treated cells
  PMID:22262847.

So every mapped dephosphorylation event in this protein's literature is on a
threonine; no serine site has been demonstrated, although the PPM fold and
EC 3.1.3.16 cover both.

Localization

Cytoplasmic plus secreted, by antibody detection PMID:16487973 (the 2011 paper later showed stp is
not in fact essential for survival, only for virulence). Secreted SP-STP enters
host pharyngeal cells and reaches the nucleus PMID:22262847, and
ectopically expressed SP-STP is nuclear PMID:22262847.

Bacterial phenotypes of the non-polar stp mutants (PMID:21917918)

Two M1 backgrounds, M1SF370 (M1ΔSTP) and M1T15448 (M1T1ΔSTP), complemented from
a low-copy plasmid.

• Chains and septa. Long chains, thickened walls, and
  PMID:21917918; the authors' own conclusion is regulatory
  PMID:21917918. The proposed mechanism runs
  through WalR phosphorylation and CdhA up-regulation PMID:21917918. No evidence that SP-STP is itself part of a
  division-site-selection machinery (no Min/Noc/DivIVA-like activity shown).
• Capsule. PMID:21917918, with 14–100-fold lower has transcript
  and restoration on complementation — i.e. SP-STP positively regulates capsule
  synthesis, via CovR. Note the contrast with the kinase: deleting SP-STK
  increases capsule.
• Fatty acids. PMID:21917918 with
  PMID:21917918. Lipid analysis
  (Table 1) was done for M1SF370 only. Direction: loss of STP → more fatty acid,
  so STP is a negative regulator of fatty acid biosynthesis — but indirectly,
  through the FabT repressor and the STK/STP couple, which the authors state as
  speculation.
• Adherence/invasion. PMID:21917918 and
  PMID:21917918, with the discussion stating
  PMID:21917918.
  Entry into host therefore goes up, not down, when stp is deleted, and the
  adherence effect is strain-specific — so "positive regulation by symbiont of
  entry into host" has the sign backwards for the one measurable entry readout.
• Mutants were efficiently phagocytosed and attenuated in mice.

Host apoptosis (PMID:22262847)

Strong, multi-assay: PMID:22262847, restored by complementation; purified protein sufficient and
specific versus the E. faecalis homolog EF-STP PMID:22262847; annexin-V, JC-1 mitochondrial depolarization, caspase
cleavage and inhibitor blockade PMID:22262847; in vivo TUNEL PMID:22262847; and requirement for
catalytic activity/structure PMID:22262847
(D36A/D192A/D231A). Caveats: two cell lines of four responded, and most work is
on Detroit 562.

CACAO-specific observations

All eight experimental rows in the GOA file are assigned_by: CACAO; the only
non-CACAO rows are two UniProt IEAs. Judgments and the reason each lands where it
does are in the review file. Patterns worth recording for the project page:

1. Phenotype read as process, with wrong granularity — GO:0000918 division
   septum site selection from the septation phenotype. The paper's own wording is
   "regulation of septa formation"; SP-STP is a signalling phosphatase upstream of
   WalR/CdhA, not a component of the site-selection machinery.
2. Sign inverted — GO:0075294 positive regulation by symbiont of entry into
   host. The mutant is more invasive (6-fold), and the adherence decrease is
   seen in only one of the two strains. Neither direction is established, so the
   sign-free parent (GO:0052372) is what the data support. This is a new shape for
   the taxonomy: not "phenotype as process" but phenotype as process with the
   sign read off the wrong readout (adherence standing in for entry).
3. Right claim, weaker paper than the one that shows it — GO:0062085 positive
   regulation of capsule polysaccharide biosynthetic process is keyed to
   PMID:16487973, whose isogenic mutants were kinase mutants
   PMID:16487973 and in which capsule went up; the abstract does assert
   that these proteins modulate capsule
   PMID:16487973, and the full
   text is unavailable, so the row is kept — but the quantified stp-mutant evidence
   for the positive direction is in PMID:21917918.
4. Correct but general MF — GO:0004721 from the 2006 biochemistry, where the
   same paper restricts the modified residue to threonine; the specific child
   GO:0004722 is already annotated.
5. Rows 3–5 of the taxonomy aside, the three best CACAO rows here (GO:0004722 IDA,
   GO:0035970 IDA, GO:0052151 IMP) are accurate and well chosen; this gene is not
   an instance of "paper not about the gene".