Recombinant Geobacter sulfurreducens Cell division protein FtsL (ftsL), partial

What is the role of FtsL in Geobacter sulfurreducens cell division?

FtsL is an essential membrane protein involved in bacterial cell division, forming part of the divisome complex responsible for septum formation. In G. sulfurreducens, FtsL (like in other bacteria) likely interacts with other divisome proteins such as FtsB and FtsQ to coordinate cell division. Transcript analysis has shown that FtsL expression in G. sulfurreducens appears to be downregulated under certain conditions with a log2 fold change of -1.451 (p-value 0.133), suggesting its expression is condition-dependent . To investigate FtsL function, researchers typically employ gene deletion studies followed by phenotypic characterization, fluorescent protein tagging for localization studies, and protein-protein interaction assays to identify binding partners within the divisome.

How does G. sulfurreducens cell division differ from model organisms like E. coli?

G. sulfurreducens exhibits several unique characteristics that may influence its cell division process:

• Unlike E. coli, G. sulfurreducens has a distinctive metabolism focused on extracellular electron transfer, with extensive cytochrome networks that may integrate with division machinery .

• G. sulfurreducens has approximately 111 c-type cytochromes, many localized to the cell envelope where division occurs .

• Cell composition analysis reveals G. sulfurreducens has unusually high iron content (5-10 times higher than typical bacteria) and elevated lipid content (>20% of dry weight) , which may affect membrane properties where FtsL functions.

• Cell division timing and efficiency may vary based on electron acceptor availability (soluble vs. insoluble) .

To study these differences, researchers should employ comparative genomics, differential expression analysis under varying electron acceptor conditions, and direct observation of division site formation using fluorescence microscopy.

What methods are effective for expressing recombinant G. sulfurreducens FtsL?

Based on successful expression of other G. sulfurreducens proteins, the following methodology is recommended:

• Vector selection: pET21d vectors have been successfully used for G. sulfurreducens proteins .

• Tag placement: C-terminal His-tags are preferable, as N-terminal tags have been shown to be detrimental for proper protein folding in other G. sulfurreducens proteins like cytochrome c7 .

• Expression conditions:

  • Host: BL21(DE3) E. coli cells

  • Induction: 1mM IPTG for 3 hours at 37°C (for cytoplasmic proteins)

  • For membrane proteins like FtsL, lower temperatures (16-25°C) and longer induction times may improve folding

• Verification: SDS-PAGE with expected size of approximately 13 kDa for partial FtsL .

• Purification: His-Bind column chromatography with imidazole gradient elution .

Success has been reported with similar approaches for G. sulfurreducens cytochrome c7, yielding up to 6 mg/L of properly folded protein .

How can researchers investigate FtsL interactions with the extracellular electron transfer machinery?

FtsL's potential interaction with G. sulfurreducens' distinctive electron transfer systems can be investigated through:

• Co-immunoprecipitation studies: Using antibodies against FtsL to identify co-precipitating proteins, particularly cytochromes or electron transfer components.

• Growth phenotype analysis: Compare growth rates and division patterns when grown with different electron acceptors:

  • Fe(III) citrate (soluble)

  • Fe(III) oxide (insoluble)

  • Mn(IV) oxide

  • Electrode surfaces at different potentials (+0.24V vs. -0.1V)

• Fluorescent co-localization: Simultaneously tag FtsL and key electron transfer proteins (such as ExtABCD components) to observe potential spatial relationships during division.

• Comparative expression analysis: Monitor FtsL expression levels across different electron acceptor conditions using qRT-PCR or transcriptomics.

Based on data from studies of ext gene clusters in G. sulfurreducens .

What approaches can determine if FtsL function is affected by G. sulfurreducens' high iron content?

G. sulfurreducens contains significantly higher iron concentration than most bacteria, primarily in its numerous cytochromes . To investigate effects on FtsL:

• Iron limitation experiments: Grow G. sulfurreducens under iron-limited conditions and assess:

  • FtsL expression levels

  • Cell division patterns and frequency

  • FtsL localization using fluorescent tagging

• Protein stability assays: Compare FtsL stability in normal vs. iron-limited conditions using pulse-chase experiments.

• Interaction screening: Perform bacterial two-hybrid assays with FtsL and iron-binding proteins under varying iron concentrations.

• Structural analysis: Use circular dichroism or thermal shift assays to determine if iron availability affects FtsL folding or stability.

Research indicates that limiting iron content inhibits extracellular electron transfer in G. sulfurreducens , which may indirectly affect cell division timing and FtsL function through metabolic changes.

How might conjugative plasmids affect FtsL and cell division in G. sulfurreducens?

Recent research has shown that conjugative plasmids inhibit extracellular electron transfer in G. sulfurreducens . To investigate impacts on FtsL and cell division:

• Comparative growth studies: Track division rates and morphology in plasmid-bearing vs. plasmid-free cells grown with different electron acceptors.

• Transcriptional analysis: Compare ftsL expression levels between:

  • Wild-type G. sulfurreducens

  • G. sulfurreducens carrying pKJK5 plasmid

  • G. sulfurreducens carrying RP4 plasmid

  • G. sulfurreducens carrying pB10 plasmid

• Division protein localization: Monitor FtsL-fluorescent protein fusions in plasmid-bearing cells to assess divisome assembly.

• Mechanistic investigation: Determine if effects are mediated through:

  • Direct interference with FtsL by plasmid-encoded proteins

  • Indirect effects via metabolic changes

  • Competition for membrane space between type IV secretion systems and the divisome

Data shows that conjugative plasmids significantly reduce Fe(III) oxide reduction rates (p<0.05) , which may alter energy availability for division processes.

What genetic approaches can be used to study essential division genes like ftsL in G. sulfurreducens?

Since complete deletion of essential division genes would be lethal, specialized approaches are needed:

• Markerless deletion system: Utilize the pk18mobsacB vector system successfully used in G. sulfurreducens for:

  • Conditional depletion strains

  • Domain deletion studies

  • Point mutations in functional regions

• Complementation strategies:

  • Use the pRK2-Geo2 plasmid with constitutive acpP (GSU1604) promoter for stable expression

  • Include careful controls like DL100 (which maintains wild-type genes but has same marker insertions)

• Site-directed mutagenesis:

  • Target conserved residues using recombinant PCR approaches

  • Introduce BsmI or similar restriction sites as markers

  • Use homologous recombination for chromosomal integration

• Fluorescent protein fusions:

  • C-terminal fusions preferable given N-terminal sensitivity

  • Verify function through complementation assays

  • Consider photoactivatable fluorescent proteins for dynamic studies

For experimental verification, G. sulfurreducens mutants can be validated by PCR mapping, restriction digestion, and sequencing as demonstrated with pilA mutants .

How might FtsL be involved in G. sulfurreducens' adaptation to different growth substrates?

G. sulfurreducens shows remarkable adaptive capabilities, including evolution to utilize new substrates like lactate . To investigate FtsL's role in adaptation:

• Adaptive laboratory evolution experiments:

  • Serial transfers in challenging conditions (new electron donors/acceptors)

  • Whole genome sequencing to identify mutations

  • Specific monitoring of ftsL and divisome gene expression

• Comparative analysis across adaptation stages:

  • Early adaptation (impaired growth)

  • Mid-adaptation (improving growth)

  • Fully adapted state

• Time-lapse microscopy:

  • Track cell division timing and morphology during adaptation

  • Correlate with metabolic activity (e.g., electron transfer rates)

• Transcriptional regulation analysis:

  • Identify if FtsL is regulated by substrate-responsive transcription factors

  • Investigate if cell division timing is altered during metabolic adaptation

Previous research on lactate adaptation in G. sulfurreducens revealed single-nucleotide polymorphisms in GSU0514, a transcriptional regulator . Similar mechanisms may affect cell division gene expression during adaptation to new conditions.

How does the unique lipid composition of G. sulfurreducens affect FtsL membrane integration and function?

G. sulfurreducens has approximately 22.4% lipid content by weight , significantly higher than most bacteria, which may affect membrane protein function:

• Membrane composition analysis:

  • Compare phospholipid profiles between G. sulfurreducens and model organisms

  • Identify specific lipids interacting with FtsL using lipidomics coupled with immunoprecipitation

  • Assess membrane fluidity using fluorescence anisotropy

• Reconstitution experiments:

  • Express FtsL in liposomes of varying lipid composition

  • Compare protein structure, stability, and interaction capabilities

  • Measure lateral diffusion rates in different membrane environments

• Computational modeling:

  • Molecular dynamics simulations of FtsL in G. sulfurreducens-like membranes

  • Predict lipid-binding sites and conformational changes

• Correlation with electron transfer:

  • Investigate if membrane reorganization during division affects electron transfer capacity

  • Monitor cytochrome distribution during division using super-resolution microscopy

Research shows that G. sulfurreducens has high C:O and H:O ratios (approximately 1.7:1 and 0.25:1) , indicative of reduced cell composition consistent with high lipid content, which would create a distinctive environment for membrane proteins like FtsL.

What molecular mechanisms coordinate cell division with extracellular electron transfer in G. sulfurreducens?

Understanding how G. sulfurreducens coordinates division with its unique metabolism requires examining:

• Metabolic synchronization:

  • How cytochrome synthesis and assembly coordinate with cell division

  • Whether division is triggered by specific redox states

• Division site selection:

  • If electron transfer structures influence division plane positioning

  • Whether cytochrome-rich membrane domains affect FtsZ assembly (similar to MreB-FtsZ interaction in E. coli)

• Regulatory networks:

  • Transcriptomic analysis to identify co-regulated genes between division and electron transfer

  • Construction of regulatory network models connecting these processes

• Proteomics approaches:

  • Quantitative proteomics across growth phases

  • Protein-protein interaction networks between divisome and electron transfer machinery

• Electron flow measurements:

  • Correlate electron transfer rates with division stages

  • Measure local redox potentials during division using redox-sensitive probes

The connection between FtsL and outer membrane electron conduits like ExtABCD, which are essential for electrode respiration , represents a critical area for investigation of division-respiration coordination.

What are the critical quality control steps for confirming authentic recombinant G. sulfurreducens FtsL?

To ensure proper expression and folding of recombinant FtsL:

• Expression verification:

  • Western blot with anti-His antibodies

  • Mass spectrometry confirmation of protein identity and size

  • N-terminal sequencing to verify correct processing

• Structural integrity assessment:

  • Circular dichroism to confirm proper secondary structure (expected to be primarily α-helical)

  • Size exclusion chromatography to verify monomeric/oligomeric state

  • Limited proteolysis to assess domain folding

• Functional validation:

  • Binding assays with known division protein partners

  • Complementation of E. coli ftsL temperature-sensitive mutants

  • Membrane integration assessment using fractionation studies

• Purity assessment:

  • SDS-PAGE with Coomassie and silver staining

  • Analytical ultracentrifugation

  • Endotoxin testing for any therapeutic applications

Based on the successful expression of other G. sulfurreducens proteins, yields of 6 mg/L of culture can be achieved for properly folded proteins .

How can researchers design experiments to address contradictory findings regarding FtsL expression in G. sulfurreducens?

When addressing conflicting data about FtsL expression:

• Standardize growth conditions:

  • Carefully control electron donor/acceptor pairs:

    • Acetate/fumarate (standard laboratory conditions)

    • Acetate/Fe(III) citrate (soluble metal reduction)

    • Acetate/Fe(III) oxide (insoluble metal reduction)

    • Acetate/electrode (at defined potentials)

  • Standardize growth phase for sampling (early log, mid-log, late log)

  • Control for biofilm vs. planktonic states

• Multi-method verification:

  • Compare protein levels (Western blot) with transcript levels (qRT-PCR)

  • Use fluorescent reporter fusions to track expression in single cells

  • Perform absolute quantification using targeted proteomics (MRM-MS)

• Strain validation:

  • Sequence verify all strains before experiments

  • Control for spontaneous mutations that might arise during cultivation

  • Create biological replicates from separate colonies

• Data analysis approaches:

  • Use appropriate statistical methods for small differences

  • Consider normalization approaches carefully (internal controls may also vary)

  • Implement blinded analysis where possible

Research has shown that G. sulfurreducens expression patterns differ significantly between growth with soluble electron acceptors (fumarate, Fe(III) citrate) versus insoluble acceptors (Fe(III) oxide, electrodes) , which may explain apparent contradictions in FtsL expression data.

What imaging approaches can overcome challenges in visualizing FtsL localization in G. sulfurreducens?

G. sulfurreducens presents unique imaging challenges due to its high cytochrome content and metal-reducing lifestyle:

• Super-resolution techniques:

  • PALM/STORM microscopy to achieve 20-30 nm resolution

  • Structured illumination microscopy (SIM) for live-cell imaging

  • Stimulated emission depletion (STED) microscopy for precise localization

• Specific labeling strategies:

  • HaloTag or SNAP-tag fusions rather than standard fluorescent proteins

  • Split fluorescent protein complementation to confirm interactions

  • Site-specific labeling with small organic dyes via click chemistry

• Sample preparation optimization:

  • Minimal media to reduce autofluorescence

  • Specialized fixation protocols to preserve membrane structure

  • Thin sectioning techniques for transmission electron microscopy

• Advanced data analysis:

  • Deconvolution algorithms specific for metal-rich bacteria

  • Machine learning approaches to distinguish signal from background

  • Correlative light and electron microscopy to connect protein localization with ultrastructure

• Growth surfaces optimization:

  • Transparent indium tin oxide electrodes for live imaging during electrode respiration

  • Thin Fe(III) oxide coatings on glass for imaging during metal reduction