Rv3660c encodes the septum site determining protein Ssd, a member of the ParA/MinD-like WACA (Walker A Cytoskeletal ATPase) family. Ssd inhibits division septum formation in M. tuberculosis; overexpression leads to filamentous cells devoid of septa, while transposon disruption produces minicells. Ssd is functionally distinct from the true E. coli MinD homologue in M. tuberculosis (Rv1708). Increased Ssd expression induces the dormancy (Dos) regulon and alternative sigma factors (SigF, SigG, SigH, SigI, SigL, SigM), establishing the first reported link between a septum regulatory protein and the adaptive metabolic response associated with non-replicating persistence and virulence. The protein contains an N-terminal CheY-like domain (protein interaction module) and a C-terminal ParA/MinD ATPase domain with a deviant Walker A motif. Essential for growth.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0005524
ATP binding
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: IBA annotation for ATP binding inferred from PANTHER phylogeny (PTN000344128, with E. coli MinD P0AEZ3 as reference). Rv3660c contains a conserved deviant Walker A motif within its P-loop NTPase domain (IPR027417, IPR050625). While direct ATP binding has not been demonstrated for Rv3660c itself, ATPase activity was confirmed for MSMEG_3743 (the M. smegmatis orthologue of Rv1708, a paralog in the same PANTHER family). The domain architecture strongly supports ATP binding capacity.
Reason: The P-loop NTPase domain with Walker A motif is well conserved in Rv3660c. Although direct biochemical evidence for Rv3660c ATP binding is lacking, the domain architecture and membership in the ParA/MinD ATPase family (IPR050625) strongly support this annotation.
Supporting Evidence:
PMID:37526955
MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif
file:MYCTU/Rv3660c/Rv3660c-deep-research-bioreason-sft.md
The P-loop core (IPR050625 with IPR027417) confers ATP binding and ATPase activity, enabling nucleotide-controlled assembly and disassembly cycles
|
|
GO:0016887
ATP hydrolysis activity
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: IBA annotation for ATP hydrolysis activity inferred from PANTHER phylogeny. Rv3660c has the conserved P-loop NTPase fold and Walker A/B motifs characteristic of ParA/MinD ATPases. ATPase activity was demonstrated for the M. smegmatis orthologue MSMEG_3743 (Rv1708 family). However, the Walker A motif in Rv3660c is classified as a deviant Walker A, and direct ATPase activity for Rv3660c has not been experimentally confirmed.
Reason: The deviant Walker A motif and ParA/MinD ATPase domain architecture support ATP hydrolysis capacity. The IBA transfer from the PANTHER family is appropriate given the conserved catalytic residues, though direct demonstration of Rv3660c ATPase activity would strengthen this annotation.
Supporting Evidence:
PMID:37526955
MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif
|
|
GO:0005829
cytosol
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: IBA annotation for cytosolic localization inferred from PANTHER phylogeny (reference protein E. coli MinD P0AEZ3). Rv3660c/Ssd is expected to be a soluble cytosolic protein based on the absence of transmembrane domains and its membership in the ParA/MinD family of soluble ATPases. The UniProt record also carries an HDA annotation from MTBBASE for cytosol. Functional data showing that Ssd expression modulates transcription of the dormancy regulon is consistent with cytoplasmic localization.
Reason: Cytosolic localization is consistent with Ssd being a soluble ParA/MinD-like ATPase that functions in septum site determination and regulation of gene expression. Both IBA and HDA evidence support this.
Supporting Evidence:
PMID:21504606
Transcriptional mapping in M. tuberculosis showed that increased ssd expression elicited a unique response including the dormancy regulon and alternative sigma factors that are thought to play a role in adaptive metabolism
|
|
GO:0009898
cytoplasmic side of plasma membrane
|
IBA
GO_REF:0000033 |
MARK AS OVER ANNOTATED |
Summary: IBA annotation for cytoplasmic side of plasma membrane, transferred from E. coli MinD (P0AEZ3) via PANTHER phylogeny. E. coli MinD oscillates between the cytosol and the cytoplasmic face of the inner membrane via its C-terminal membrane targeting sequence. However, Rv3660c is not the true MinD homologue in M. tuberculosis (that is Rv1708), and Kishore et al. 2023 showed structural divergence between Rv3660c and E. coli MinD, particularly the unique N-terminal domain. There is no experimental evidence for membrane association of Rv3660c/Ssd.
Reason: This annotation is transferred from E. coli MinD, which has a characterized membrane targeting sequence for inner membrane association. Rv3660c is a distinct Ssd-family protein, not the true MinD homologue (Rv1708 is). The structural analysis in Kishore et al. 2023 showed that the Rv3660c structure differs significantly from E. coli MinD, with an N-terminal extension absent in MinD. No experimental evidence supports membrane localization for Rv3660c. The IBA transfer may be inappropriate for this specific localization feature.
Supporting Evidence:
PMID:37526955
Comparative structural analyses showed Rv1708 to be closer in similarity to Ec MinD than Rv3660c
|
|
GO:0010974
negative regulation of division septum assembly
|
IDA
PMID:21504606 Mycobacterium tuberculosis septum site determining protein, ... |
NEW |
Summary: Rv3660c/Ssd overexpression inhibits septum formation, producing filamentous cells devoid of septa; transposon disruption produces minicells with reduced bacterial length. This directly demonstrates a role in negative regulation of division septum assembly.
Reason: This BP annotation captures the core experimentally demonstrated function of Rv3660c/Ssd from the England et al. 2011 study. Both overexpression and disruption phenotypes support this annotation.
Supporting Evidence:
PMID:21504606
Increased expression of ssd in M. smegmatis and M. tuberculosis inhibited septum formation resulting in elongated cells devoid of septa
PMID:21504606
Disruption of rv3660c by transposon insertion negated the unique transcriptional response and led to a reduced bacterial length
|
|
GO:0000918
division septum site selection
|
IDA
PMID:21504606 Mycobacterium tuberculosis septum site determining protein, ... |
NEW |
Summary: Rv3660c is a member of the septum site determining protein family. Bioinformatics analysis identified it as an ortholog of Ssd proteins in actinobacteria, and gene dosage studies showed it regulates septum formation. The transposon disruption minicell phenotype is characteristic of septum site selection defects.
Reason: Rv3660c was identified as a septum site determining protein by consensus modeling and functional characterization. The minicell phenotype on disruption and filamentation on overexpression are classic hallmarks of septum site selection regulators.
Supporting Evidence:
PMID:21504606
Septum site determining protein (Ssd), encoded by rv3660c was discovered to be an ortholog of septum site regulating proteins in actinobacteria by bioinformatics analysis
PMID:21504606
Disruption of rv3660c by transposon insertion negated the unique transcriptional response and led to a reduced bacterial length
|
|
GO:0051782
negative regulation of cell division
|
IMP
PMID:21504606 Mycobacterium tuberculosis septum site determining protein, ... |
NEW |
Summary: Overexpression of Rv3660c/Ssd inhibits cell division in both M. smegmatis and M. tuberculosis, resulting in filamentous cells. This effect links septum regulation to cell cycle arrest and is associated with induction of the dormancy regulon. The protein functions as a negative regulator of cell division through inhibition of septum formation.
Reason: The filamentation phenotype on Ssd overexpression directly demonstrates negative regulation of cell division. This is further supported by the connection to dormancy regulon induction, which is associated with cell cycle arrest in M. tuberculosis.
Supporting Evidence:
PMID:21504606
Increased expression of ssd in M. smegmatis and M. tuberculosis inhibited septum formation resulting in elongated cells devoid of septa
PMID:21504606
This study establishes the first connection between a septum regulatory protein and induction of alternative metabolism consisting of alternative sigma factors and the dormancy regulon that is associated with establishing a non-replicating persistent intracellular lifestyle
|
Q: Does Rv3660c/Ssd have direct ATPase activity, and if so, is it stimulated by a specific activating partner analogous to MinE?
Suggested experts: Tirumalai R Raghunand, Richard A Slayden
Q: What is the direct mechanism by which Ssd inhibits FtsZ polymerization - does it interact directly with FtsZ or act through an intermediary?
Suggested experts: Tirumalai R Raghunand
Q: Is the link between Ssd expression and dormancy regulon induction direct (Ssd acts as a transcriptional regulator) or indirect (downstream consequence of cell division arrest)?
Suggested experts: Richard A Slayden, Kathleen England
Experiment: Express and purify recombinant Rv3660c and measure ATPase activity in vitro using a malachite green phosphate release assay, with and without potential activating partners from the same genomic neighborhood.
Hypothesis: Rv3660c/Ssd possesses intrinsic ATPase activity mediated by its deviant Walker A motif
Type: biochemical assay
Experiment: Perform bacterial two-hybrid or co-immunoprecipitation experiments to test for direct Ssd-FtsZ interaction in M. tuberculosis. If positive, use light scattering assays to measure FtsZ polymerization kinetics in the presence and absence of purified Ssd.
Hypothesis: Ssd directly interacts with FtsZ to inhibit its polymerization
Type: protein-protein interaction
Experiment: Compare transcriptional profiles of Ssd overexpression with those of other cell division inhibitors (e.g., SulA) to determine whether the dormancy response is specific to Ssd or a general consequence of division arrest. Use a catalytically dead Ssd Walker A mutant to test whether ATPase activity is required for the transcriptional response.
Hypothesis: The dormancy regulon induction by Ssd is an indirect consequence of cell division arrest rather than direct transcriptional regulation
Type: transcriptomics
I begin with the domain architecture. The N-terminus (residues 1β109) carries IPR059050, a CheY-like N-terminal domain. CheY-like modules are receiver-like folds that create a compact, Ξ²/Ξ± architecture optimized for proteinβprotein interaction and for coupling to partner surfaces rather than catalysis. From residue ~100 onward, the sequence is assigned to IPR050625 (ParA/MinD ATPase family, residues 100β334), which defines a deviant P-loop NTPase that uses ATP binding and hydrolysis to drive self-assembly and surface association. This is reinforced by two overlapping P-loop NTPase superfamily signatures, IPR027417 (P-loop containing nucleoside triphosphate hydrolase, residues 116β337 and 117β318), which specify the Walker A/B and switch elements that coordinate Mg2+-ATP and catalyze hydrolysis. The entire polypeptide is also encompassed by IPR022521 (Rv3660c family, residues 2β338), indicating a specialized lineage of ParA/MinD-like proteins. The ordered layoutβan N-terminal CheY-like interaction module followed by a C-terminal ParA/MinD-type P-loop ATPase coreβcreates a two-part machine: the N-terminus provides a docking interface for regulatory partners, while the C-terminus supplies an ATP-dependent polymerization/oligomerization engine.
This architecture causes specific molecular functions. The P-loop core (IPR050625 with IPR027417) confers ATP binding and ATPase activity, enabling nucleotide-controlled assembly and disassembly cycles. The CheY-like N-terminus (IPR059050) contributes a broad protein-binding surface that recognizes transcriptional regulators. Together, these features support protein binding (GO:0005515) and ATP binding/ATPase activity as the operative molecular functions that modulate partner behavior.
The biological process follows from how these activities influence transcriptional control. ATP-driven polymerization of ParA/MinD-like ATPases often repositions or sequesters regulatory factors. Here, the N-terminal interaction module is poised to bind a transcriptional activator and the ATPase cycle is used to restrain its activityβeither by direct occlusion, by trapping the activator in an inactive complex, or by spatially segregating it. This mechanism implements negative regulation of DNA-templated transcription (GO:0045892), consistent with inhibition of a transcriptional activator such as Rv3658c.
Cellular location is dictated by the absence of transmembrane segments and the soluble nature of CheY-like and ParA/MinD folds. These assemblies operate in the cytosol, where they can access transcription factors and nucleoid-proximal regions. Thus, the protein resides in the cytosol (GO:0005829), with likely enrichment near the inner membrane or nucleoid periphery when engaging partners, but without integral membrane features.
Mechanistically, I propose the following model. The N-terminal CheY-like domain binds the transcriptional activator Rv3658c and possibly other regulators encoded in the same operon. The C-terminal ParA/MinD-like ATPase binds ATP and cycles between monomeric and oligomeric states; ATP-bound oligomers could scaffold or cage the bound activator, reducing its ability to recruit RNA polymerase. Hydrolysis to ADP would trigger disassembly, potentially releasing the activator or resetting the complex. The operon partnersβRv3656c (a small conserved hypothetical protein), Rv3657c (a conserved membrane-like hypothetical protein), and Rv3658c (the transcriptional activator)βlikely form a local regulatory module: the small cytosolic factor (Rv3656c) may chaperone or stabilize the ATPaseβactivator complex, the membrane-like protein (Rv3657c) could tether the complex to the inner membrane to spatially restrict activator function, and the transcriptional activator (Rv3658c) is the direct target of inhibition. Additional conserved hypothetical proteins may act as adaptors that tune oligomerization or localization. In sum, the domain architecture drives ATP-dependent assembly that binds and inhibits a transcriptional activator in the cytosol, thereby down-modulating transcriptional output.
## Functional Summary
A cytosolic, ATP-dependent assembly factor that binds regulatory proteins and inhibits a nearby transcriptional activator. Its N-terminal interaction module engages the activator, while a C-terminal P-loop ATPase core drives nucleotide-controlled oligomerization that cages or sequesters the activator, reducing its ability to stimulate transcription. Within an operon of conserved hypothetical proteins, it likely collaborates with small cytosolic and membrane-associated partners to position and stabilize the inhibitory complex at the cytosolic face of the cell, thereby tuning transcriptional output.
## UniProt Summary
Inhibits the transcriptional activator Rv3658c.
## InterPro Domains
- IPR059050: Rv3660c-like, CheY-like N-terminal domain (domain) [1-109]
- IPR022521: Rv3660c (family) [2-338]
- IPR050625: ParA/MinD ATPase (family) [100-334]
- IPR027417: P-loop containing nucleoside triphosphate hydrolase (homologous_superfamily) [116-337]
- IPR027417: P-loop containing nucleoside triphosphate hydrolase (homologous_superfamily) [117-318]
## GO Term Predictions
### Molecular Function
### Biological Process
### Cellular Component
Rv3660c encodes a septum site determining protein (Ssd) in M. tuberculosis. It is a member of the ParA/MinD/Ssd WACA (Walker A Cytoskeletal ATPase) family but is distinct from the true E. coli MinD homologue in M. tuberculosis (Rv1708).
UniProt: P9WKX7. 350 amino acids. COG0455 (ATPases involved in chromosome partitioning). Contains a deviant Walker A motif (P-loop NTPase fold).
This is the primary characterization study. Rv3660c was identified as encoding a septum site determining protein (Ssd) by consensus-modeling bioinformatics.
Kishore et al. showed that Rv1708 (not Rv3660c) is the true E. coli MinD homologue in M. tuberculosis:
Rv3660c was identified as essential for optimal growth by transposon site hybridization (TraSH) PMID:12657046
Rv3660c was induced by albendazole and thiabendazole (GTPase inhibitors of FtsZ), linking it to septum formation regulation PMID:16735741
Identified as a high-confidence drug target through interactome, reactome, and genome-scale structural analysis pipeline (targetTB).
PANTHER family PTHR43384 classifies Rv3660c as related to "septum site-determining protein MinD homolog" (SF11). The IBA annotations are transferred from this family via PANTHER:PTN000344128, with E. coli MinD (P0AEZ3) and a plant homologue (G3XD64) as reference proteins.
Rv3660c/Ssd is a septum site determining protein that inhibits cell division septum formation in M. tuberculosis. It belongs to the ParA/MinD ATPase superfamily but is functionally distinct from the true MinD homologue (Rv1708). Its key biological roles are:
The protein acts in the cytosol and contains ATP binding/hydrolysis capacity via a conserved Walker A motif, though direct ATPase activity has only been demonstrated for the M. smegmatis Rv1708 homologue (MSMEG_3743), not for Rv3660c itself.
Source: Rv3660c-deep-research-bioreason-sft.md
The BioReason SFT functional summary states:
A cytosolic, ATP-dependent assembly factor that binds regulatory proteins and inhibits a nearby transcriptional activator. Its N-terminal interaction module engages the activator, while a C-terminal P-loop ATPase core drives nucleotide-controlled oligomerization that cages or sequesters the activator, reducing its ability to stimulate transcription. Within an operon of conserved hypothetical proteins, it likely collaborates with small cytosolic and membrane-associated partners to position and stabilize the inhibitory complex at the cytosolic face of the cell, thereby tuning transcriptional output.
This summary fabricates a specific molecular mechanism -- direct inhibition of a transcriptional activator Rv3658c through physical sequestration -- that has no experimental support. The curated review, based on England et al. 2011 (PMID:21504606) and Kishore et al. 2023 (PMID:37526955), establishes that Rv3660c encodes Ssd, a septum site determining protein that inhibits division septum formation through regulation of FtsZ polymerization. The actual biology is cell division regulation, not transcriptional repression.
Major errors:
The summary claims Rv3660c "inhibits a nearby transcriptional activator" (Rv3658c) through physical caging. There is no evidence for direct Rv3660c-Rv3658c interaction or for transcriptional activator sequestration. The actual experimentally supported function is negative regulation of division septum assembly -- overexpression produces filamentous cells devoid of septa, and transposon disruption produces minicells.
The summary describes the mechanism as "nucleotide-controlled oligomerization that cages or sequesters the activator." This is an invented mechanism. The published data shows Ssd functions in septum site determination, analogous to the MinD/Ssd family in other bacteria, affecting FtsZ polymerization.
The summary entirely misses the dormancy connection -- the most biologically significant finding for this protein. England et al. 2011 showed that Ssd expression induces the dormancy (Dos) regulon and alternative sigma factors, establishing "the first connection between a septum regulatory protein and induction of alternative metabolism...associated with establishing a non-replicating persistent intracellular lifestyle." This is absent from the BioReason output.
The summary does not recognize that Rv3660c is functionally distinct from E. coli MinD. Kishore et al. 2023 demonstrated that Rv1708 (not Rv3660c) is the true MinD homologue, while Rv3660c belongs to the Ssd subfamily with a unique N-terminal domain.
What is correct:
Comparison with interpro2go:
There are no GO_REF:0000002 (interpro2go) annotations in the GOA for this protein. The existing annotations are all IBA from GO_REF:0000033 (PANTHER phylogeny). The BioReason reasoning trace correctly identifies the InterPro domains (IPR059050, IPR022521, IPR050625, IPR027417) and derives ATP binding and ATPase activity from them, which parallels what interpro2go would produce from the P-loop NTPase signatures. However, BioReason goes far beyond interpro2go by fabricating a specific transcriptional repression mechanism involving Rv3658c that has no basis in either domain architecture or published literature. The interpro2go-equivalent predictions (ATP binding, ATPase, P-loop NTPase fold) are correct and modest. The BioReason "value-added" narrative is where the errors occur -- it hallucinates a biological mechanism (transcriptional activator sequestration) rather than identifying the actual experimentally characterized function (septum site determination and dormancy regulon induction).
The thinking trace correctly identifies the domain architecture and the ParA/MinD ATPase family membership. It accurately describes the P-loop NTPase fold, Walker A/B motifs, and the CheY-like N-terminal domain.
However, the trace makes a critical inferential leap without evidence when it states: "the N-terminal interaction module is poised to bind a transcriptional activator and the ATPase cycle is used to restrain its activity." This appears to be confabulation driven by the operon context (seeing Rv3658c annotated as a transcriptional activator) rather than any domain-based or literature-based reasoning. The trace fabricates an entire molecular mechanism -- "ATP-bound oligomers could scaffold or cage the bound activator, reducing its ability to recruit RNA polymerase" -- that is pure speculation presented as mechanistic deduction.
The trace also incorrectly frames the biology as "negative regulation of DNA-templated transcription (GO:0045892)" when the actual function is negative regulation of division septum assembly (GO:0010974). This is a fundamental misattribution of biological process. Notably, the GO term predictions section of the BioReason output is empty, suggesting the model generated no actual GO term predictions despite the narrative.
The UniProt summary line in the BioReason output states "Inhibits the transcriptional activator Rv3658c" -- this does not appear in the actual UniProt record for P9WKX7, which says "May play a role in septum formation." This appears to be fabricated content.
id: P9WKX7
gene_symbol: Rv3660c
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:83332
label: Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
description: Rv3660c encodes the septum site determining protein Ssd, a member of
the ParA/MinD-like WACA (Walker A Cytoskeletal ATPase) family. Ssd inhibits division
septum formation in M. tuberculosis; overexpression leads to filamentous cells devoid
of septa, while transposon disruption produces minicells. Ssd is functionally distinct
from the true E. coli MinD homologue in M. tuberculosis (Rv1708). Increased Ssd
expression induces the dormancy (Dos) regulon and alternative sigma factors (SigF,
SigG, SigH, SigI, SigL, SigM), establishing the first reported link between a septum
regulatory protein and the adaptive metabolic response associated with non-replicating
persistence and virulence. The protein contains an N-terminal CheY-like domain (protein
interaction module) and a C-terminal ParA/MinD ATPase domain with a deviant Walker
A motif. Essential for growth.
references:
- id: PMID:21504606
title: Mycobacterium tuberculosis septum site determining protein, Ssd encoded by
rv3660c, promotes filamentation and elicits an alternative metabolic and dormancy
stress response
findings:
- statement: Rv3660c encodes a septum site determining protein (Ssd) that inhibits
septum formation when overexpressed
supporting_text: Increased expression of ssd in M. smegmatis and M. tuberculosis
inhibited septum formation resulting in elongated cells devoid of septa
- statement: Transposon disruption of rv3660c leads to minicell morphology
supporting_text: Disruption of rv3660c by transposon insertion negated the unique
transcriptional response and led to a reduced bacterial length
- statement: Ssd expression induces the dormancy regulon and alternative sigma factors
supporting_text: Transcriptional mapping in M. tuberculosis showed that increased
ssd expression elicited a unique response including the dormancy regulon and
alternative sigma factors that are thought to play a role in adaptive metabolism
- statement: First link between septum regulation and dormancy/persistence response
supporting_text: This study establishes the first connection between a septum
regulatory protein and induction of alternative metabolism consisting of alternative
sigma factors and the dormancy regulon that is associated with establishing
a non-replicating persistent intracellular lifestyle
- id: PMID:37526955
title: Septum site placement in Mycobacteria - identification and characterisation
of mycobacterial homologues of Escherichia coli MinD.
findings:
- statement: Rv1708 (not Rv3660c) is the true E. coli MinD homologue in M. tuberculosis
supporting_text: Comparative structural analyses showed Rv1708 to be closer in
similarity to Ec MinD than Rv3660c
- statement: Rv3660c has an N-terminal region absent in E. coli MinD
supporting_text: The N-terminal region encircled in Rv3660c is absent in E
- statement: The M. smegmatis Rv1708 orthologue MSMEG_3743 has confirmed ATPase
activity
supporting_text: MSMEG_3743 displayed ATPase activity, consistent with its containing
a conserved Walker A motif
- id: PMID:16735741
title: Identification of cell cycle regulators in Mycobacterium tuberculosis by
inhibition of septum formation and global transcriptional analysis
findings:
- statement: Rv3660c is induced by FtsZ inhibitors albendazole and thiabendazole
supporting_text: Inhibition of septum formation prevented cell division and led
to bacterial filamentation...Microarray-based transcriptional profiling allowed
the evaluation of multiple metabolic processes in response to inhibition of
septum formation
- id: PMID:12657046
title: Genes required for mycobacterial growth defined by high density mutagenesis
findings:
- statement: Rv3660c is essential for optimal growth of M. tuberculosis
supporting_text: comprehensively identify the genes required by the causative
agent, Mycobacterium tuberculosis, for optimal growth
- id: PMID:19099550
title: targetTB - a target identification pipeline for Mycobacterium tuberculosis
through an interactome, reactome and genome-scale structural analysis
findings:
- statement: Rv3660c identified as a high-confidence drug target
supporting_text: the identification of 451 high-confidence targets
- id: file:MYCTU/Rv3660c/Rv3660c-deep-research-bioreason-sft.md
title: BioReason SFT reasoning trace for Rv3660c
findings:
- statement: BioReason identifies ParA/MinD ATPase domain architecture with
CheY-like N-terminal domain
- id: PMID:9634230
title: Deciphering the biology of Mycobacterium tuberculosis from the complete genome
sequence
findings: []
- id: GO_REF:0000033
title: Annotation inferences using phylogenetic trees
findings: []
existing_annotations:
- term:
id: GO:0005524
label: ATP binding
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: IBA annotation for ATP binding inferred from PANTHER phylogeny (PTN000344128,
with E. coli MinD P0AEZ3 as reference). Rv3660c contains a conserved deviant
Walker A motif within its P-loop NTPase domain (IPR027417, IPR050625). While
direct ATP binding has not been demonstrated for Rv3660c itself, ATPase activity
was confirmed for MSMEG_3743 (the M. smegmatis orthologue of Rv1708, a paralog
in the same PANTHER family). The domain architecture strongly supports ATP binding
capacity.
action: ACCEPT
reason: The P-loop NTPase domain with Walker A motif is well conserved in Rv3660c.
Although direct biochemical evidence for Rv3660c ATP binding is lacking, the
domain architecture and membership in the ParA/MinD ATPase family (IPR050625)
strongly support this annotation.
supported_by:
- reference_id: PMID:37526955
supporting_text: MSMEG_3743 displayed ATPase activity, consistent with its containing
a conserved Walker A motif
- reference_id: file:MYCTU/Rv3660c/Rv3660c-deep-research-bioreason-sft.md
supporting_text: The P-loop core (IPR050625 with IPR027417) confers ATP binding
and ATPase activity, enabling nucleotide-controlled assembly and disassembly
cycles
- term:
id: GO:0016887
label: ATP hydrolysis activity
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: IBA annotation for ATP hydrolysis activity inferred from PANTHER phylogeny.
Rv3660c has the conserved P-loop NTPase fold and Walker A/B motifs characteristic
of ParA/MinD ATPases. ATPase activity was demonstrated for the M. smegmatis
orthologue MSMEG_3743 (Rv1708 family). However, the Walker A motif in Rv3660c
is classified as a deviant Walker A, and direct ATPase activity for Rv3660c
has not been experimentally confirmed.
action: ACCEPT
reason: The deviant Walker A motif and ParA/MinD ATPase domain architecture support
ATP hydrolysis capacity. The IBA transfer from the PANTHER family is appropriate
given the conserved catalytic residues, though direct demonstration of Rv3660c
ATPase activity would strengthen this annotation.
supported_by:
- reference_id: PMID:37526955
supporting_text: MSMEG_3743 displayed ATPase activity, consistent with its containing
a conserved Walker A motif
- term:
id: GO:0005829
label: cytosol
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: IBA annotation for cytosolic localization inferred from PANTHER phylogeny
(reference protein E. coli MinD P0AEZ3). Rv3660c/Ssd is expected to be a soluble
cytosolic protein based on the absence of transmembrane domains and its membership
in the ParA/MinD family of soluble ATPases. The UniProt record also carries
an HDA annotation from MTBBASE for cytosol. Functional data showing that Ssd
expression modulates transcription of the dormancy regulon is consistent with
cytoplasmic localization.
action: ACCEPT
reason: Cytosolic localization is consistent with Ssd being a soluble ParA/MinD-like
ATPase that functions in septum site determination and regulation of gene expression.
Both IBA and HDA evidence support this.
supported_by:
- reference_id: PMID:21504606
supporting_text: Transcriptional mapping in M. tuberculosis showed that increased
ssd expression elicited a unique response including the dormancy regulon and
alternative sigma factors that are thought to play a role in adaptive metabolism
- term:
id: GO:0009898
label: cytoplasmic side of plasma membrane
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: IBA annotation for cytoplasmic side of plasma membrane, transferred from
E. coli MinD (P0AEZ3) via PANTHER phylogeny. E. coli MinD oscillates between
the cytosol and the cytoplasmic face of the inner membrane via its C-terminal
membrane targeting sequence. However, Rv3660c is not the true MinD homologue
in M. tuberculosis (that is Rv1708), and Kishore et al. 2023 showed structural
divergence between Rv3660c and E. coli MinD, particularly the unique N-terminal
domain. There is no experimental evidence for membrane association of Rv3660c/Ssd.
action: MARK_AS_OVER_ANNOTATED
reason: This annotation is transferred from E. coli MinD, which has a characterized
membrane targeting sequence for inner membrane association. Rv3660c is a distinct
Ssd-family protein, not the true MinD homologue (Rv1708 is). The structural
analysis in Kishore et al. 2023 showed that the Rv3660c structure differs significantly
from E. coli MinD, with an N-terminal extension absent in MinD. No experimental
evidence supports membrane localization for Rv3660c. The IBA transfer may be
inappropriate for this specific localization feature.
supported_by:
- reference_id: PMID:37526955
supporting_text: Comparative structural analyses showed Rv1708 to be closer
in similarity to Ec MinD than Rv3660c
- term:
id: GO:0010974
label: negative regulation of division septum assembly
evidence_type: IDA
original_reference_id: PMID:21504606
review:
summary: Rv3660c/Ssd overexpression inhibits septum formation, producing
filamentous cells devoid of septa; transposon disruption produces
minicells with reduced bacterial length. This directly demonstrates a
role in negative regulation of division septum assembly.
action: NEW
reason: This BP annotation captures the core experimentally demonstrated
function of Rv3660c/Ssd from the England et al. 2011 study. Both
overexpression and disruption phenotypes support this annotation.
supported_by:
- reference_id: PMID:21504606
supporting_text: Increased expression of ssd in M. smegmatis and M.
tuberculosis inhibited septum formation resulting in elongated cells
devoid of septa
- reference_id: PMID:21504606
supporting_text: Disruption of rv3660c by transposon insertion negated
the unique transcriptional response and led to a reduced bacterial
length
- term:
id: GO:0000918
label: division septum site selection
evidence_type: IDA
original_reference_id: PMID:21504606
review:
summary: Rv3660c is a member of the septum site determining protein
family. Bioinformatics analysis identified it as an ortholog of Ssd
proteins in actinobacteria, and gene dosage studies showed it
regulates septum formation. The transposon disruption minicell
phenotype is characteristic of septum site selection defects.
action: NEW
reason: Rv3660c was identified as a septum site determining protein by
consensus modeling and functional characterization. The minicell
phenotype on disruption and filamentation on overexpression are
classic hallmarks of septum site selection regulators.
supported_by:
- reference_id: PMID:21504606
supporting_text: Septum site determining protein (Ssd), encoded by
rv3660c was discovered to be an ortholog of septum site regulating
proteins in actinobacteria by bioinformatics analysis
- reference_id: PMID:21504606
supporting_text: Disruption of rv3660c by transposon insertion negated
the unique transcriptional response and led to a reduced bacterial
length
- term:
id: GO:0051782
label: negative regulation of cell division
evidence_type: IMP
original_reference_id: PMID:21504606
review:
summary: Overexpression of Rv3660c/Ssd inhibits cell division in both
M. smegmatis and M. tuberculosis, resulting in filamentous cells.
This effect links septum regulation to cell cycle arrest and is
associated with induction of the dormancy regulon. The protein
functions as a negative regulator of cell division through inhibition
of septum formation.
action: NEW
reason: The filamentation phenotype on Ssd overexpression directly
demonstrates negative regulation of cell division. This is further
supported by the connection to dormancy regulon induction, which is
associated with cell cycle arrest in M. tuberculosis.
supported_by:
- reference_id: PMID:21504606
supporting_text: Increased expression of ssd in M. smegmatis and M.
tuberculosis inhibited septum formation resulting in elongated cells
devoid of septa
- reference_id: PMID:21504606
supporting_text: This study establishes the first connection between
a septum regulatory protein and induction of alternative metabolism
consisting of alternative sigma factors and the dormancy regulon
that is associated with establishing a non-replicating persistent
intracellular lifestyle
core_functions:
- description: Septum site determining protein that inhibits division septum assembly.
Overexpression causes cell filamentation devoid of septa; disruption causes minicell
formation. Functions as a negative regulator of FtsZ polymerization to control
the timing and placement of septum formation.
molecular_function:
id: GO:0016887
label: ATP hydrolysis activity
directly_involved_in:
- id: GO:0010974
label: negative regulation of division septum assembly
- id: GO:0000918
label: division septum site selection
- id: GO:0051782
label: negative regulation of cell division
locations:
- id: GO:0005829
label: cytosol
supported_by:
- reference_id: PMID:21504606
supporting_text: Increased expression of ssd in M. smegmatis and M. tuberculosis
inhibited septum formation resulting in elongated cells devoid of septa
- reference_id: PMID:21504606
supporting_text: Disruption of rv3660c by transposon insertion negated the unique
transcriptional response and led to a reduced bacterial length
- reference_id: PMID:16735741
supporting_text: Inhibition of septum formation prevented cell division and led
to bacterial filamentation...Microarray-based transcriptional profiling allowed
the evaluation of multiple metabolic processes in response to inhibition of
septum formation
suggested_questions:
- question: Does Rv3660c/Ssd have direct ATPase activity, and if so, is it stimulated
by a specific activating partner analogous to MinE?
experts:
- Tirumalai R Raghunand
- Richard A Slayden
- question: What is the direct mechanism by which Ssd inhibits FtsZ polymerization
- does it interact directly with FtsZ or act through an intermediary?
experts:
- Tirumalai R Raghunand
- question: Is the link between Ssd expression and dormancy regulon induction direct
(Ssd acts as a transcriptional regulator) or indirect (downstream consequence
of cell division arrest)?
experts:
- Richard A Slayden
- Kathleen England
suggested_experiments:
- hypothesis: Rv3660c/Ssd possesses intrinsic ATPase activity mediated by its deviant
Walker A motif
description: Express and purify recombinant Rv3660c and measure ATPase activity
in vitro using a malachite green phosphate release assay, with and without potential
activating partners from the same genomic neighborhood.
experiment_type: biochemical assay
- hypothesis: Ssd directly interacts with FtsZ to inhibit its polymerization
description: Perform bacterial two-hybrid or co-immunoprecipitation experiments
to test for direct Ssd-FtsZ interaction in M. tuberculosis. If positive, use light
scattering assays to measure FtsZ polymerization kinetics in the presence and
absence of purified Ssd.
experiment_type: protein-protein interaction
- hypothesis: The dormancy regulon induction by Ssd is an indirect consequence of
cell division arrest rather than direct transcriptional regulation
description: Compare transcriptional profiles of Ssd overexpression with those of
other cell division inhibitors (e.g., SulA) to determine whether the dormancy
response is specific to Ssd or a general consequence of division arrest. Use a
catalytically dead Ssd Walker A mutant to test whether ATPase activity is required
for the transcriptional response.
experiment_type: transcriptomics