Recombinant Mannheimia succiniciproducens Cell division protein FtsB (ftsB), partial

What is the biological function of FtsB in Mannheimia succiniciproducens?

FtsB is an integral membrane protein of the bacterial divisome that plays a critical role in cell division. As part of the divisome complex, FtsB works alongside other division proteins to facilitate the steps required for bacterial cell division. FtsB is a single-pass membrane protein with a periplasmic coiled-coil domain that forms an essential heterologous association with its partner FtsL, which represents a crucial event for the recruitment of late components to the division site . In M. succiniciproducens, like in other bacteria, FtsB is likely essential for proper cell envelope remodeling during division, particularly in coordinating processes across the gram-negative cell envelope layers.

How does FtsB interact with other cell division proteins in bacterial systems?

FtsB interacts primarily with FtsL and FtsQ to form a critical subcomplex within the divisome. Research has shown that FtsB and FtsL are mutually dependent for their recruitment to the division site, and both proteins depend on the localization of FtsQ . Structural analyses indicate that FtsB self-associates through its transmembrane domain where a polar residue (Gln 16) plays a critical role through the formation of an inter-helical hydrogen bond . The periplasmic domain forms a canonical coiled-coil structure. It's hypothesized that the transmembrane helices of FtsB form a stable dimeric core for association with FtsL into a higher-order oligomer, and FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex competent for binding to FtsQ .

What genomic and structural features characterize FtsB in M. succiniciproducens?

While the specific genomic context of FtsB in M. succiniciproducens is not directly detailed in the available research, general structural features of FtsB include a single transmembrane domain and a periplasmic domain that forms a coiled-coil structure. X-ray crystallography has revealed that approximately 30 juxta-membrane amino acids of FtsB form a canonical coiled coil . A conserved glycine residue in the linker region suggests that flexibility between the transmembrane and coiled-coil domains is functionally important . Based on research in other bacterial systems, FtsB likely contains conserved interaction interfaces that facilitate both homo-oligomerization and heterologous association with other divisome proteins.

What are the optimal conditions for expressing recombinant M. succiniciproducens FtsB in heterologous systems?

For optimal expression of recombinant M. succiniciproducens FtsB in heterologous systems, researchers should consider the following methodological approach:

• Expression system selection: E. coli BL21(DE3) is often suitable for membrane protein expression, using vectors containing T7 promoters.

• Induction conditions: For membrane proteins like FtsB, lower induction temperatures (16-25°C) and reduced IPTG concentrations (0.1-0.5 mM) typically yield better results by allowing proper folding and insertion into membranes.

• Media optimization: M9 minimal medium supplemented with glucose as carbon source can be used, mirroring the efficient glucose utilization seen in M. succiniciproducens . The medium should be buffered appropriately, considering M. succiniciproducens is capnophilic (CO₂-loving) .

• Fusion tags: C-terminal His-tags are generally preferred for membrane proteins, though fusion with solubility enhancers like MBP may improve yield. For structural studies, approaches similar to those used in previous FtsB research where the periplasmic domain was expressed as a fusion with Gp7 could be employed .

• Extraction conditions: Mild detergents like DDM or LDAO are typically effective for extracting membrane proteins while maintaining their native structure.

How can researchers confirm the proper folding and functionality of recombinant FtsB?

Confirming proper folding and functionality of recombinant FtsB requires multiple complementary approaches:

• Circular dichroism (CD) spectroscopy: This technique can verify the secondary structure content of the purified protein, particularly the alpha-helical content expected in the coiled-coil periplasmic domain.

• Size exclusion chromatography: This can assess the oligomeric state of the protein, as FtsB is known to self-associate and form higher-order complexes with FtsL . The expected elution profile should be consistent with the predicted molecular weight of FtsB oligomers.

• In vitro interaction assays: Pull-down assays or surface plasmon resonance can verify whether the recombinant FtsB can bind to its known partners, particularly FtsL and FtsQ. These interaction studies can be designed based on previously identified interaction hot spots, such as those in the conserved membrane-distal part of FtsQ's β domain around residue Y248, which primarily interacts with FtsB .

• Complementation assays: Testing whether the recombinant FtsB can complement an FtsB-deficient bacterial strain provides functional validation. Growth and division phenotypes should be monitored microscopically.

• Mutagenesis of key residues: Targeted mutations of conserved residues, such as Gln 16 in the transmembrane domain, which is known to be critical for FtsB homo-oligomerization through inter-helical hydrogen bonding , can confirm structural integrity.

What structural features of M. succiniciproducens FtsB are critical for its function in cell division?

Several structural features are likely critical for M. succiniciproducens FtsB function based on research on FtsB in other bacterial systems:

• Transmembrane domain: The transmembrane domain contains conserved residues that facilitate homo-oligomerization, with particular importance of the polar residue Gln 16, which forms inter-helical hydrogen bonds . This self-association likely provides a stable dimeric core for subsequent complex formation.

• Periplasmic coiled-coil domain: The approximately 30 juxta-membrane amino acids form a canonical coiled-coil structure essential for interactions with other divisome components .

• Linker region: A conserved glycine residue in the linker between the transmembrane and coiled-coil domains suggests that flexibility between these domains is functionally important , potentially allowing conformational changes during divisome assembly.

• Interaction interfaces: The periplasmic domain contains specific interfaces for interaction with FtsL and FtsQ. These interactions are essential for the recruitment and stabilization of late divisome components .

• Oligomerization potential: The ability to form higher-order oligomers with FtsL is likely critical for creating a competent complex that can bind to FtsQ and facilitate recruitment to the divisome .

How does the structure of FtsB in M. succiniciproducens compare to homologous proteins in other bacterial species?

While specific comparative structural data for M. succiniciproducens FtsB is limited in the available research, general patterns of conservation and divergence can be inferred:

The fundamental structural organization of FtsB is likely conserved across bacterial species, consisting of a single transmembrane domain and a periplasmic coiled-coil domain. In E. coli and other well-studied systems, FtsB has been characterized as a single-pass membrane protein with a periplasmic coiled coil . This architecture is probably maintained in M. succiniciproducens FtsB.

Key functional residues, particularly those involved in protein-protein interactions, tend to be conserved. The transmembrane domain of FtsB contains an evolutionarily conserved interaction interface where polar residues like Gln 16 play critical roles through inter-helical hydrogen bonding . Similar conservation patterns would be expected in M. succiniciproducens FtsB.

The periplasmic domain structure, which forms a canonical coiled coil, is likely conserved, though sequence variations may exist that affect interaction specificity with divisome partners. Differences in sequence may reflect adaptation to specific cellular environments or co-evolution with interaction partners.

A detailed comparative analysis would require experimental structural determination of M. succiniciproducens FtsB and comparison with existing structures from other bacterial species.

How can recombinant M. succiniciproducens FtsB be used in screening for novel cell division inhibitors?

Recombinant M. succiniciproducens FtsB can serve as a valuable tool for screening novel cell division inhibitors through the following methodological approaches:

• In vitro binding assays: Purified recombinant FtsB can be used in high-throughput binding assays to identify small molecules that disrupt its interaction with FtsL or FtsQ. These assays could employ techniques such as fluorescence polarization, where fluorescently labeled FtsB is monitored for changes in polarization signal upon compound binding.

• Structure-based virtual screening: Using the structural data of FtsB, particularly the critical interaction interfaces, computational screening can identify compounds predicted to bind at these sites. The interactions between FtsQ and FtsB have been recognized as an excellent target for cell division inhibitor searches , and structural data can guide rational drug design efforts.

• Competitive displacement assays: Assays that measure the displacement of labeled interaction partners (such as FtsL or FtsQ) from immobilized FtsB can identify compounds that disrupt these essential protein-protein interactions.

• Bacterial two-hybrid systems: Modified versions of bacterial two-hybrid systems incorporating recombinant FtsB can screen for compounds that disrupt its interactions with other divisome components in a cellular context.

• Phenotypic rescue assays: Compounds identified through primary screens can be validated by testing their ability to induce division defects in bacterial cultures and whether these defects phenocopy specific mutations in the FtsB interaction interface.

This approach is particularly promising as the divisome represents an excellent target for antibacterial compounds, with structural research showing that mutations disrupting the interface between FtsQ and FtsB effectively inhibit cell division .

What approaches can be used to investigate the dynamic assembly of FtsB into the divisome in living M. succiniciproducens cells?

Investigating dynamic assembly of FtsB into the divisome in living M. succiniciproducens cells requires sophisticated live-cell imaging and molecular techniques:

• Fluorescent protein fusions: Creating functional FtsB-fluorescent protein fusions (such as FtsB-GFP or FtsB-mCherry) allows visualization of protein localization during cell division using time-lapse fluorescence microscopy. Care must be taken to ensure the fusion does not disrupt protein function.

• FRET/BRET analyses: Förster/Bioluminescence Resonance Energy Transfer between fluorescently tagged divisome components can measure real-time protein-protein interactions during divisome assembly. For example, tagging FtsB and FtsL with appropriate donor/acceptor pairs can track their interaction dynamics.

• Photo-activatable or photo-convertible tags: These allow pulse-chase experiments to track specific populations of newly synthesized FtsB molecules as they incorporate into the divisome.

• Super-resolution microscopy: Techniques like STORM, PALM, or structured illumination microscopy overcome the diffraction limit and provide nanoscale resolution of divisome assembly.

• Single-molecule tracking: This approach can reveal the recruitment sequence, stoichiometry, and exchange dynamics of individual FtsB molecules.

• Cross-linking coupled with mass spectrometry: In vivo cross-linking at different cell division stages followed by mass spectrometry analysis can capture transient interactions and conformational changes. Similar to the scanning photo-cross-linking approach used to map interactions of FtsQ with FtsB at the amino acid level .

• Microfluidics-based synchronization: This technique allows the study of divisome assembly in synchronized cell populations, enhancing the signal-to-noise ratio for subtle dynamic changes.

How does FtsB function integrate with the metabolic network of M. succiniciproducens?

The integration of FtsB function with M. succiniciproducens metabolism likely involves complex regulatory networks that coordinate cell division with metabolic status:

• Metabolic state sensing: The assembly and function of the divisome, including FtsB, may respond to the metabolic state of the cell. M. succiniciproducens has a specialized metabolism optimized for succinic acid production, featuring strong PEP carboxylation, a branched TCA cycle, and non-PTS glucose uptake . The divisome might integrate signals from these metabolic pathways to coordinate division with nutrient availability.

• Energy requirement coupling: Cell division is an energy-intensive process. In M. succiniciproducens, which has been metabolically engineered for efficient succinic acid production , the energy allocation between production pathways and cell division processes likely involves coordinated regulation.

• CO₂-dependent regulation: M. succiniciproducens is capnophilic, requiring CO₂ for optimal growth and succinic acid production . FtsB function and divisome assembly may be influenced by CO₂ levels, potentially through indirect effects on membrane properties or direct regulatory mechanisms.

• Growth rate coordination: The expression and assembly of divisome components need to be coordinated with growth rate, which is directly influenced by the metabolic network state. The genome-scale metabolic network of M. succiniciproducens consists of 686 reactions and 519 metabolites , providing multiple potential regulatory inputs to the cell division machinery.

• Membrane composition effects: The phospholipid composition of the membrane, which is a product of cellular metabolism, can affect the function of membrane proteins like FtsB. The transmembrane domain of FtsB, which is critical for its homo-oligomerization , may be sensitive to membrane composition changes resulting from metabolic shifts.

What experimental approaches can link FtsB function to the unique metabolic characteristics of M. succiniciproducens?

To investigate links between FtsB function and M. succiniciproducens metabolism, researchers can employ several experimental strategies:

• Conditional FtsB expression under varying metabolic conditions: By creating strains with controllable FtsB expression, researchers can examine how divisome assembly responds to different carbon sources, CO₂ levels, or metabolic inhibitors. M. succiniciproducens can utilize various carbon sources including sucrose through its phosphotransferase system (PTS) , providing opportunities to test division coordination under different metabolic states.

• Metabolomic profiling of FtsB mutants: Comparing the metabolite profiles of wild-type cells with those expressing mutant FtsB (with alterations in key structural features ) can reveal metabolic adaptations to divisome dysfunction.

• 13C flux analysis in division-perturbed cells: Using labeled carbon sources combined with mass spectrometry can track metabolic flux changes when division is perturbed through FtsB mutations or depletion.

• Integration with genome-scale metabolic models: The existing genome-scale metabolic model of M. succiniciproducens can be expanded to include constraints related to cell division processes, enabling in silico predictions of how metabolic perturbations affect division and vice versa.

• Proteome-wide interaction screens: Techniques like BioID or APEX proximity labeling with FtsB as bait can identify unexpected interactions with metabolic enzymes, particularly under different growth conditions.

• Growth synchronization experiments: Synchronizing cell populations and measuring both metabolite concentrations and FtsB localization/activity can reveal temporal relationships between metabolic shifts and divisome assembly.

• Genetic interaction mapping: Systematic genetic interaction analysis between FtsB mutations and metabolic gene deletions can identify functional connections between division and metabolism.

What are common challenges in the purification of recombinant M. succiniciproducens FtsB, and how can they be addressed?

Purification of membrane proteins like FtsB presents several challenges that can be addressed with specific methodological approaches:

• Low expression levels:

  • Solution: Optimize codon usage for expression host, use strong promoters, and consider fusion partners that enhance expression.

  • Method: Test multiple expression constructs with different fusion tags (MBP, SUMO, Gp7) in parallel; the Gp7 fusion approach has been successfully used for FtsB structural studies .

• Protein aggregation:

  • Solution: Modify buffer conditions and use appropriate detergents for membrane protein stabilization.

  • Method: Screen detergents systematically (DDM, LDAO, FC-12) at different concentrations; perform thermal stability assays to identify optimal conditions.

• Improper membrane insertion:

  • Solution: Slow down protein production to allow proper membrane targeting.

  • Method: Reduce induction temperature to 16-20°C and use lower inducer concentrations; consider using specialized E. coli strains like C41(DE3) designed for membrane protein expression.

• Protein degradation:

  • Solution: Include protease inhibitors and optimize purification speed.

  • Method: Add a complete protease inhibitor cocktail throughout purification; use cooled chambers for FPLC systems; consider adding stabilizing agents like glycerol (10-15%).

• Poor solubilization efficiency:

  • Solution: Optimize detergent type, concentration, and solubilization conditions.

  • Method: Test a matrix of conditions varying detergent type, concentration (0.5-2% w/v), temperature (4-25°C), and duration (1-16 hours).

• Co-purification of binding partners:

  • Solution: Use high-salt washes or chaotropic agents to disrupt interactions, if desired.

  • Method: Include washing steps with buffers containing 300-500 mM NaCl or low concentrations of urea (1-2 M) if pure FtsB is required; alternatively, co-purify with natural binding partners like FtsL for functional studies.

How can researchers troubleshoot experiments investigating FtsB-dependent divisome assembly?

Troubleshooting FtsB-dependent divisome assembly experiments requires systematic approaches to address common issues:

• Non-functional fusion proteins:

  • Diagnostic: Aberrant cell morphology (filamentation) when expressing FtsB fusions.

  • Solution: Try alternative fusion positions (N-terminal vs. C-terminal) or use smaller tags; create linker libraries with varying flexibility and length.

  • Validation: Complement an FtsB depletion strain with the fusion construct and verify normal division.

• Mislocalized FtsB constructs:

  • Diagnostic: Diffuse or punctate localization instead of mid-cell rings.

  • Solution: Verify that fusion constructs maintain critical interaction interfaces; ensure expression levels are not too high, which can saturate normal localization machinery.

  • Method: Use an inducible promoter to titrate expression levels; compare localization patterns with other divisome components like FtsZ.

• Inconsistent protein-protein interaction results:

  • Diagnostic: Variable or contradictory results in pull-down or two-hybrid assays.

  • Solution: Control for detergent effects on interactions; ensure proper protein folding; consider that some interactions may require multiple divisome components simultaneously.

  • Method: Try alternative interaction assays like microscale thermophoresis or bio-layer interferometry; reconstruct minimal complexes with purified components.

• Poor temporal resolution in dynamic studies:

  • Diagnostic: Inability to distinguish assembly sequence or transient interactions.

  • Solution: Improve synchronization of division events; increase imaging frequency; use faster-maturing fluorophores.

  • Method: Implement microfluidic growth chambers for long-term imaging; use photoactivatable fluorophores for pulse-chase experiments.

• Metabolic state interference:

  • Diagnostic: Inconsistent divisome assembly under seemingly identical conditions.

  • Solution: Strictly control growth conditions and media composition; monitor cellular metabolic state.

  • Method: Include metabolic sensors in imaging experiments; standardize culture optical density and growth phase for experiments.