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 |
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Download this section (compressed HTML)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
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