Recombinant Geobacter sulfurreducens D-alanine--D-alanine ligase (ddl)

What is the biological function of D-alanine-D-alanine ligase in G. sulfurreducens?

D-alanine-D-alanine ligase (Ddl) in G. sulfurreducens catalyzes the ATP-dependent formation of the D-alanyl-D-alanine dipeptide, which serves as a critical substrate for peptidoglycan crosslinking in bacterial cell walls. The enzyme facilitates the dimerization of two D-alanine molecules, generating the terminal dipeptide that becomes incorporated into the peptidoglycan precursor. This reaction is essential for maintaining cell wall integrity and structure in G. sulfurreducens, which is particularly important for this organism given its unique extracellular electron transfer capabilities that require robust cell envelope properties. Unlike some other bacteria that can utilize D-alanyl-D-lactate pathways, G. sulfurreducens relies primarily on the D-alanyl-D-alanine pathway for peptidoglycan synthesis, making Ddl an essential enzyme for its survival and growth .

What is the kinetic profile of recombinant G. sulfurreducens Ddl?

Although specific kinetic parameters for G. sulfurreducens Ddl are not directly reported in the search results, comparable studies on other bacterial Ddl enzymes provide insight into its likely kinetic behavior. Based on studies of Ddl from other bacterial species, recombinant G. sulfurreducens Ddl would be expected to demonstrate Michaelis-Menten kinetics with differential affinities for its substrates.

For example, kinetic studies on Mycobacterium tuberculosis Ddl (Tb-DdlA) revealed a higher affinity for ATP (KmATP: 50.327 ± 4.652 μmol/L) compared to D-alanine (KmAla: 1.011 ± 0.094 mmol/L) . G. sulfurreducens Ddl likely exhibits a similar substrate preference pattern, with ATP binding preceding D-alanine binding in an ordered sequential mechanism. This kinetic profile would be consistent with the enzyme's role in catalyzing the ATP-dependent formation of the D-alanyl-D-alanine dipeptide necessary for peptidoglycan synthesis in G. sulfurreducens cell walls.

What are the optimal conditions for recombinant expression of G. sulfurreducens Ddl?

For optimal heterologous expression of recombinant G. sulfurreducens Ddl, Escherichia coli is typically the preferred expression system due to its ease of genetic manipulation and high protein yield. Based on protocols used for similar bacterial Ddl enzymes, the gene encoding G. sulfurreducens Ddl should be cloned into an expression vector (such as pET series) with a histidine tag for subsequent purification. Expression conditions can be optimized by using E. coli BL21(DE3) or similar strains, with induction using IPTG at a concentration of 0.5-1.0 mM when cultures reach an OD600 of 0.6-0.8.

Temperature optimization is critical, with lower temperatures (16-25°C) during induction often yielding higher amounts of soluble protein compared to standard 37°C incubation. The addition of glucose (0.5-1%) to the culture medium can help reduce basal expression before induction. For G. sulfurreducens proteins specifically, anaerobic expression conditions may yield more native-like protein conformations, given the anaerobic nature of the source organism. Post-induction culture periods typically range from 4-18 hours depending on the temperature, with longer times needed for lower temperature inductions .

What is the most effective purification strategy for recombinant G. sulfurreducens Ddl?

The most effective purification strategy for recombinant G. sulfurreducens Ddl involves a multi-step approach starting with affinity chromatography. If the recombinant protein contains a polyhistidine tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins serves as an efficient first purification step. Cell lysis should be performed in buffer containing 20-50 mM Tris-HCl (pH 7.5-8.0), 300-500 mM NaCl, 5-10% glycerol, and protease inhibitors.

Following IMAC, size exclusion chromatography (SEC) provides further purification and allows assessment of the oligomeric state of the enzyme. For optimal activity, the final buffer composition should include 25-50 mM Tris-HCl (pH 7.5-8.0), 100-200 mM NaCl, and 5-10% glycerol. If higher purity is required, an ion exchange chromatography step can be incorporated between IMAC and SEC.

The purified protein can be identified using Western blot analysis with anti-polyhistidine antibodies, and its identity can be confirmed by mass spectrometry. Enzyme purity should be assessed by SDS-PAGE, aiming for >95% purity for structural and detailed enzymatic studies. For long-term storage, the purified enzyme should be flash-frozen in liquid nitrogen and stored at -80°C in buffer containing 10-20% glycerol to maintain activity .

How can the activity of purified recombinant G. sulfurreducens Ddl be verified?

Verification of purified recombinant G. sulfurreducens Ddl activity can be accomplished through multiple complementary approaches. The primary method involves a colorimetric assay measuring the release of inorganic phosphate (Pi) during the ATP-dependent formation of D-alanyl-D-alanine. This assay typically uses malachite green or similar reagents that form colored complexes with free phosphate, allowing spectrophotometric quantification at 620-650 nm.

Another robust verification method is the direct detection of the D-alanyl-D-alanine product using high-performance thin-layer chromatography (HP-TLC). The reaction mixture containing purified Ddl, D-alanine, and ATP is incubated at optimal conditions (typically 37°C, pH 7.5-8.0), and the formation of the D-alanyl-D-alanine dipeptide is monitored by HP-TLC with appropriate standards .

For more sensitive and quantitative analysis, high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) can be employed to detect and quantify the D-alanyl-D-alanine product. These techniques provide higher resolution and detection sensitivity compared to TLC-based methods.

A functional complementation assay can also be performed by expressing recombinant G. sulfurreducens Ddl in a Ddl-deficient bacterial strain and assessing whether it restores growth or vancomycin sensitivity, providing in vivo verification of enzyme functionality .

How can G. sulfurreducens Ddl be used as a counterselection marker in genetic engineering?

G. sulfurreducens Ddl can be utilized as a counterselection marker in genetic engineering based on the principle demonstrated in Lactobacillus species. Although specific applications in G. sulfurreducens are not directly described in the search results, the mechanism could be adapted for this organism.

The counterselection strategy leverages the ability of dipeptide ligase-type Ddl to generate D-alanyl-D-alanine termini in peptidoglycan, increasing sensitivity to vancomycin. For implementation in G. sulfurreducens, a heterologous dipeptide ligase gene would be incorporated into a suicide vector. When this vector integrates into the genome, the expressed Ddl increases vancomycin sensitivity. Subsequent growth on vancomycin-containing media selects for cells that have lost the vector through a second recombination event, allowing for markerless genetic modifications .

This approach has been demonstrated to reduce the time required for identifying recombinants to approximately 5 days, which is about half the time needed by conventional methods . A liquid-based selection approach further streamlines this process. Phylogenetic analysis of Ddl sequences can predict which bacterial species, including potentially G. sulfurreducens, would be suitable for this counterselection system.

The advantage of this system is that it represents a "plug and play" counterselection approach that does not require prior genome editing or synthetic media, making it potentially valuable for genetic manipulation of G. sulfurreducens, especially for creating markerless mutations or gene deletions .

How does D-cycloserine inhibit G. sulfurreducens Ddl and what are the implications for antimicrobial development?

D-cycloserine (DCS) inhibits D-alanine-D-alanine ligase through competitive binding at the D-alanine binding sites of the enzyme. While specific studies on G. sulfurreducens Ddl inhibition are not directly reported in the search results, the mechanism likely follows that observed in other bacterial Ddl enzymes.

DCS competitively inhibits both D-alanine binding sites of Ddl, with different binding affinities. Studies on D-alanyl-D-alanine synthetase showed a KI value of 2.2 × 10-5 M for the first binding site (E + I ⇌ EI) and a KAI value of 1.4 × 10-4 M for the second site (EA + I ⇌ EAI) . This inhibition is instantaneous and completely reversible, leading to depletion of the D-alanyl-D-alanine dipeptide pool necessary for peptidoglycan synthesis.

Metabolomic studies revealed that DCS treatment causes distinct metabolic changes depending on concentration. At lower concentrations (0.25× and 1× MIC), DCS primarily inhibits Ddl, leading to accumulation of alanine. At higher concentrations (5× MIC), DCS also inhibits alanine racemase (Alr), causing alanine depletion . This dual-target effect makes DCS a potent antimicrobial agent but also contributes to its side effects.

For antimicrobial development targeting G. sulfurreducens Ddl, these findings suggest that designing Ddl-specific inhibitors with improved specificity over DCS could yield effective antimicrobials with fewer side effects. The development of a colorimetric assay for high-throughput screening of Ddl inhibitors provides a valuable tool for discovering such compounds .

What high-throughput screening methods are available for identifying novel G. sulfurreducens Ddl inhibitors?

Several high-throughput screening (HTS) methods can be adapted for identifying novel inhibitors of G. sulfurreducens Ddl. The primary approach is a colorimetric assay that measures the release of inorganic phosphate during the ATP-dependent formation of D-alanyl-D-alanine. This method has been validated for Mycobacterium tuberculosis Ddl and can be adapted for G. sulfurreducens Ddl .

The assay typically uses malachite green or similar phosphate-detecting reagents in a 96-well or 384-well format to enable rapid screening of compound libraries. Compounds that inhibit Ddl activity will show reduced phosphate release compared to uninhibited controls. This approach allows for the screening of thousands of compounds in a short period.

Alternative HTS methods include:

• Coupled enzyme assays that link Ddl activity to a detectable signal, such as fluorescence or luminescence

• Thermal shift assays to identify compounds that bind to and stabilize Ddl, resulting in altered protein thermal denaturation profiles

• Surface plasmon resonance (SPR) or microscale thermophoresis (MST) to directly measure compound binding to purified Ddl

For validation of hits from primary screens, secondary assays including direct product detection methods such as HP-TLC or LC-MS can be employed . Additionally, metabolomic approaches can help distinguish between compounds that target Ddl specifically versus those with multiple targets in the D-alanine pathway, similar to the studies that differentiated the effects of D-cycloserine and L-cycloserine .

How can structural information about G. sulfurreducens Ddl be leveraged for rational drug design?

Structural information about G. sulfurreducens Ddl provides valuable insights for rational drug design approaches targeting this enzyme. While specific structural studies on G. sulfurreducens Ddl are not directly reported in the search results, structural biology approaches can be applied based on homology modeling using related bacterial Ddl structures.

Key strategies for rational drug design targeting G. sulfurreducens Ddl include:

• Active Site Targeting: The enzyme's active site contains highly conserved residues essential for substrate binding and catalysis. Compounds designed to competitively inhibit D-alanine or ATP binding can effectively block Ddl function. Detailed knowledge of the binding pockets can guide the design of inhibitors with improved affinity and specificity.

• Transition State Mimics: Designing compounds that mimic the transition state of the Ddl-catalyzed reaction can yield potent inhibitors. These mimics typically have higher affinity for the enzyme than the natural substrates.

• Allosteric Inhibition: Identifying allosteric sites on G. sulfurreducens Ddl can enable the design of non-competitive inhibitors that bind away from the active site but induce conformational changes that impair catalytic function.

• Fragment-Based Drug Design: This approach involves screening small molecular fragments that bind to different regions of Ddl and then linking or expanding promising fragments to create potent inhibitors.

Computational methods, including molecular docking, molecular dynamics simulations, and virtual screening, can accelerate the drug discovery process by predicting binding modes and affinities of potential inhibitors before experimental validation . Integration of structural information with functional data, such as the kinetic parameters of G. sulfurreducens Ddl, further enhances the rational design of selective inhibitors.

How can protein-protein interaction studies with G. sulfurreducens Ddl provide insights into peptidoglycan synthesis machinery?

Protein-protein interaction (PPI) studies with G. sulfurreducens Ddl can reveal critical insights into the organization and regulation of the peptidoglycan synthesis machinery. Research on Mycobacterium tuberculosis Ddl identified eight putative interaction partners through pull-down assays and MS/MS analysis, suggesting that Ddl functions within a complex network of proteins involved in cell wall biosynthesis .

For G. sulfurreducens Ddl, similar approaches including pull-down assays, co-immunoprecipitation, bacterial two-hybrid systems, or proximity labeling methods can be employed to identify its interaction partners. These studies would likely reveal connections between Ddl and other enzymes involved in peptidoglycan precursor synthesis, cell wall assembly, and potentially proteins unique to G. sulfurreducens' extracellular electron transfer systems.

Understanding these interactions could elucidate:

• How peptidoglycan synthesis is coordinated with other cellular processes

• Regulatory mechanisms that control cell wall remodeling during different growth phases

• Potential connections between cell wall synthesis and extracellular electron transfer machinery

• Species-specific adaptations in the peptidoglycan synthesis pathway that contribute to G. sulfurreducens' unique environmental niche

These insights could lead to the identification of novel targets for antimicrobial development or strategies to enhance G. sulfurreducens' capabilities for bioremediation and bioelectrochemical applications .

What is the relationship between G. sulfurreducens Ddl activity and cyclic dinucleotide signaling pathways?

The relationship between G. sulfurreducens Ddl activity and cyclic dinucleotide signaling pathways represents an intriguing area for advanced research, although direct connections are not extensively documented in the current literature. G. sulfurreducens utilizes cyclic dinucleotide signaling, particularly through cyclic GMP-AMP (3′,3′-cGAMP) regulated by the Hypr GGDEF enzyme GacA, to control gene expression related to attachment and biofilm formation .

While direct interactions between Ddl and cyclic dinucleotide pathways are not explicitly described, several potential relationships can be hypothesized:

• Ddl activity and the resulting peptidoglycan composition may influence membrane properties that affect the localization or function of cyclic dinucleotide synthases or receptors.

• Cyclic dinucleotide signaling may regulate Ddl expression or activity as part of coordinating cell wall remodeling during transitions between planktonic and biofilm lifestyles.

• Both systems may be co-regulated in response to environmental cues, such as the availability of electron acceptors or nutrient conditions.

Research indicates that cGAMP signaling in G. sulfurreducens affects growth on particulate electron acceptors like Fe(III) oxides but is not essential for biofilm growth on electrodes (associated with cyclic di-GMP signaling) . This suggests distinct signaling pathways control different attachment modes. The cell wall composition, influenced by Ddl activity, may play differential roles in these attachment strategies, potentially linking Ddl function to specific cyclic dinucleotide signaling outcomes.

Advanced research techniques such as transcriptomics, metabolomics, and genetic interaction studies could help elucidate the connections between these pathways and reveal how they collectively contribute to G. sulfurreducens' remarkable environmental adaptability .

How might CRISPR-Cas9 genome editing be optimized to study G. sulfurreducens Ddl function in vivo?

Optimizing CRISPR-Cas9 genome editing for studying G. sulfurreducens Ddl function in vivo requires specialized approaches tailored to this organism's genetic characteristics. While specific CRISPR-Cas9 protocols for G. sulfurreducens are not directly described in the search results, the following strategies could be effectively applied:

• Vector and Delivery System Optimization:

  • Develop shuttle vectors compatible with both E. coli (for cloning) and G. sulfurreducens

  • Optimize electroporation protocols specifically for G. sulfurreducens, considering its unique cell wall properties

  • Consider conjugation-based delivery methods if transformation efficiency is low

• Guide RNA Design:

  • Use G. sulfurreducens genome information to design highly specific sgRNAs targeting ddl

  • Employ machine learning algorithms to predict sgRNA efficiency and minimize off-target effects

  • Design multiple sgRNAs targeting different regions of the ddl gene to increase editing success

• Homology-Directed Repair Templates:

  • Design repair templates with 1-2 kb homology arms flanking the ddl modifications

  • Include silent mutations in the PAM site to prevent re-cutting after successful editing

  • Consider introducing marker genes that can be subsequently removed using counterselection

• Counterselection Strategy:

  • Implement a Ddl-based counterselection system similar to that developed for Lactobacillus species

  • This approach would be particularly valuable for creating markerless mutations in ddl

• Phenotypic Validation:

  • Design experiments to assess cell wall integrity, vancomycin sensitivity, and growth on different electron acceptors

  • Implement metabolomic approaches to quantify D-alanyl-D-alanine pools before and after genetic modifications

  • Examine biofilm formation capabilities on various surfaces including electrodes

This optimized CRISPR-Cas9 approach would enable precise genetic manipulation of G. sulfurreducens ddl, facilitating detailed in vivo studies of its function in peptidoglycan synthesis, vancomycin sensitivity, biofilm formation, and extracellular electron transfer capabilities .

What strategies can address poor expression yields of recombinant G. sulfurreducens Ddl?

Poor expression yields of recombinant G. sulfurreducens Ddl can be addressed through multiple optimization strategies:

• Codon Optimization:

  • Analyze the G. sulfurreducens ddl gene for rare codons in the expression host

  • Synthesize a codon-optimized version of the gene for the specific expression host

  • Consider using specialized E. coli strains that contain extra copies of rare tRNAs

• Expression Vector Selection:

  • Test different promoter strengths (T7, tac, araBAD)

  • Evaluate various fusion tags (His, GST, MBP, SUMO) that can enhance solubility

  • Consider dual-tagging strategies for improved purification and detection

• Expression Conditions Optimization:

  • Perform a temperature screen (15°C, 20°C, 25°C, 30°C, 37°C)

  • Test different IPTG concentrations (0.1 mM to 1.0 mM)

  • Evaluate expression in various media formulations (LB, TB, auto-induction media)

  • Consider anaerobic expression conditions to mimic G. sulfurreducens' native environment

• Solubility Enhancement:

  • Add osmolytes (glycerol, sorbitol) or mild detergents to lysis buffers

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Try cell-free expression systems for difficult proteins

• Refolding Strategies:

  • If expression results in inclusion bodies, develop a refolding protocol

  • Use gradual dialysis to remove denaturants

  • Implement on-column refolding during purification

A systematic approach testing combinations of these strategies can significantly improve recombinant G. sulfurreducens Ddl expression yields. Document each condition's effect on both total and soluble protein yield to identify optimal parameters .

How can researchers troubleshoot inactive recombinant G. sulfurreducens Ddl?

Troubleshooting inactive recombinant G. sulfurreducens Ddl requires a systematic investigation of potential causes and appropriate remediation strategies:

• Protein Structure Verification:

  • Confirm protein integrity by mass spectrometry

  • Assess secondary structure using circular dichroism spectroscopy

  • Verify oligomerization state using size exclusion chromatography

  • Check for proper disulfide bond formation if applicable

• Buffer Optimization:

  • Test various pH conditions (pH 6.5-8.5)

  • Evaluate different buffer systems (Tris, HEPES, phosphate)

  • Screen salt concentrations (50-500 mM NaCl)

  • Add stabilizing agents (glycerol, reducing agents)

• Cofactor and Metal Ion Requirements:

  • Ensure ATP is fresh and at optimal concentration

  • Test different divalent cations (Mg²⁺, Mn²⁺, Ca²⁺)

  • Consider dialysis to remove potential inhibitors

  • Add potential allosteric activators

• Substrate Quality:

  • Verify D-alanine purity and stereochemical integrity

  • Prepare fresh substrate solutions before assays

  • Consider potential substrate inhibition at high concentrations

• Assay Optimization:

  • Validate assay components individually with known controls

  • Ensure detection methods are functioning properly

  • Compare multiple activity detection methods (colorimetric, HPLC, TLC)

  • Optimize enzyme concentration and reaction time

If these approaches fail to recover activity, consider that the recombinant protein may require post-translational modifications absent in the expression system or specific folding conditions that were not met during expression. In such cases, switching to an alternative expression system or attempting in vitro reconstitution with potential cofactors could restore activity .

What are the key considerations for designing G. sulfurreducens Ddl mutants to study structure-function relationships?

Designing G. sulfurreducens Ddl mutants for structure-function studies requires careful consideration of several factors to ensure meaningful results:

• Rational Mutation Site Selection:

  • Target conserved active site residues identified through sequence alignment with well-characterized Ddl enzymes

  • Focus on the single amino acid in the active site (tyrosine/phenylalanine) that determines dipeptide versus depsipeptide ligase activity

  • Consider substrate binding residues, catalytic residues, and structural elements

  • Examine residues at dimer interface if oligomerization is important for function

• Mutation Type Strategy:

  • Design conservative mutations to subtly alter activity (e.g., Y→F, D→E)

  • Create non-conservative mutations to dramatically change properties (e.g., charged→hydrophobic)

  • Generate alanine-scanning mutants of specific regions

  • Design chimeric proteins by swapping domains with Ddl from other species

• Functional Validation Approach:

  • Plan complementary assays to measure:

    • Enzyme kinetics (Km, kcat, substrate specificity)

    • Protein stability (thermal denaturation, proteolytic susceptibility)

    • Oligomerization state

    • Inhibitor sensitivity

• In Vivo Phenotype Correlation:

  • Develop assays to measure peptidoglycan composition changes

  • Assess vancomycin sensitivity alterations

  • Evaluate biofilm formation capabilities

  • Measure growth on different electron acceptors

• Structural Analysis Integration:

  • Obtain crystal structures of key mutants if possible

  • Use molecular dynamics simulations to predict mutation effects

  • Validate structural predictions with experimental data

  • Consider hydrogen-deuterium exchange mass spectrometry to detect conformational changes

A particularly informative approach would be to create mutations that switch G. sulfurreducens Ddl between dipeptide and depsipeptide ligase activities, similar to studies in other bacterial species that demonstrated a single amino acid can determine vancomycin sensitivity . Such mutations would provide valuable insights into both enzyme mechanism and the role of peptidoglycan composition in G. sulfurreducens' unique physiological properties .