Recombinant Bdellovibrio bacteriovorus UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD)

What is the role of MurD in the predatory lifecycle of Bdellovibrio bacteriovorus?

MurD (UDP-N-acetylmuramoylalanine--D-glutamate ligase) in B. bacteriovorus catalyzes the addition of D-glutamate to UDP-N-acetylmuramoyl-L-alanine in peptidoglycan biosynthesis, following the reaction: UDP-MurNAc-L-Ala + D-Glu + ATP ⇔ UDP-MurNAc-L-Ala-D-Glu + ADP + Pi . This enzymatic activity is particularly significant during B. bacteriovorus predation cycle when the bacterium modifies its cell wall to accommodate filamentous growth within the prey. During the bdelloplast stage, after prey invasion, B. bacteriovorus undergoes extensive peptidoglycan remodeling that likely requires MurD activity for progeny cell wall synthesis prior to septation .

The reaction mechanism proceeds via phosphorylation of the C-terminal carboxylate of UDP-MurNAc-L-alanine by ATP's γ-phosphate to form an acyl phosphate intermediate, followed by nucleophilic attack by the D-glutamate amide group . While detailed characterization of B. bacteriovorus MurD is limited, the enzyme likely functions similarly to other bacterial MurD enzymes but may have adapted specific features for the predatory lifestyle.

How does B. bacteriovorus MurD differ structurally from homologous enzymes in other bacteria?

Despite sharing the core catalytic function with other bacterial MurD proteins, the B. bacteriovorus MurD exhibits distinct structural features reflecting its specialized predatory lifestyle:

• Domain architecture: While E. coli MurD comprises three domains (two with Rossmann fold architecture and one with mononucleotide-binding fold observed in GTPase family) , B. bacteriovorus MurD likely maintains this arrangement but with sequence variations in non-catalytic regions.

• Active site geometry: The active site located in the cleft between the GTPase domain and domain 3 may show adaptations specific to B. bacteriovorus metabolism during predation .

• Conserved residues: Critical residues likely preserved include those involved in substrate binding and catalysis, such as homologs to E. coli MurD's Gly114, Lys115, Ser/Thr116, Glu157, His183, Asn268, Asn271, Arg302, and Asp317 .

Methodological approach for structural comparison: Perform sequence alignment using CLUSTALW with MurD sequences from diverse bacteria, followed by homology modeling using E. coli MurD crystal structure (PDB: available from data in reference 2) as template. Validate models through molecular dynamics simulations to identify conserved structural elements versus B. bacteriovorus-specific adaptations.

What regulatory mechanisms control MurD expression during the host-dependent lifecycle of B. bacteriovorus?

B. bacteriovorus employs sophisticated regulatory mechanisms for MurD expression that coordinate with its predatory lifecycle phases:

• Transcriptional regulation: Expression likely increases during the growth phase within the bdelloplast when cell wall synthesis is required for multiple progeny formation. Methodology: Quantitative RT-PCR analysis of murD transcript levels across predation timepoints.

• Post-translational modifications: Potential phosphorylation or other modifications may rapidly activate/deactivate MurD based on predatory phase requirements. Methodology: Phosphoproteomic analysis of MurD isolated from different predation stages.

• Integration with signaling networks: Secondary messenger systems, particularly cGAMP signaling (produced by Bd0367) which regulates B. bacteriovorus motility and predation cycle completion , may coordinate with MurD activity. The cGAMP signaling regulates gliding motility necessary for prey exit , suggesting potential coordination with cell wall modification enzymes like MurD.

Experimental approach: Create fluorescent transcriptional and translational reporter fusions to track murD expression and protein localization throughout the predation cycle using time-lapse microscopy.

What expression systems yield optimal soluble recombinant B. bacteriovorus MurD?

Optimizing recombinant B. bacteriovorus MurD expression requires consideration of several factors:

Expression systems comparison:

Methodological approach:

• Clone the B. bacteriovorus murD gene into multiple vectors with different fusion tags (His6, MBP, SUMO, GST)

• Test expression in E. coli at various temperatures (16°C, 25°C, 30°C, 37°C) and IPTG concentrations (0.1-1.0 mM)

• Analyze protein solubility by SDS-PAGE of soluble and insoluble fractions

• For difficult expressions, employ an auto-induction media system with slow protein production

• Validate protein activity using the enzymatic assay measuring ADP formation or UDP-MurNAc-L-Ala-D-Glu production

Based on studies with E. coli MurD, the recombinant protein requires careful handling to maintain structural integrity related to its three-domain architecture that includes Rossmann fold domains .

What purification strategies preserve the catalytic activity of B. bacteriovorus MurD?

Effective purification of B. bacteriovorus MurD requires multiple chromatography steps while maintaining conditions that preserve the catalytic site integrity:

Purification protocol recommendation:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged MurD

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol

  • Include 10-20 mM imidazole in binding buffer to reduce non-specific binding

• Intermediate purification: Ion exchange chromatography

  • Theoretical pI-based selection between cation or anion exchange

  • Salt gradient elution (0-500 mM NaCl)

• Polishing step: Size exclusion chromatography

  • Buffer optimization: 25 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • Critical for removing aggregates and ensuring homogeneity

Activity preservation factors:

• Include Mg²⁺ ions (5 mM) in all buffers as MurD requires magnesium for full activity

• Maintain reducing environment (1-5 mM DTT or β-mercaptoethanol) throughout purification

• Add 10% glycerol to prevent protein denaturation

• Process samples quickly at 4°C to minimize degradation

• Avoid freezing/thawing cycles; flash-freeze aliquots in liquid nitrogen

The presence of phosphate may enhance enzyme stability based on E. coli MurD studies where enzymatic activity was maximal in the presence of magnesium and phosphate ions .

What analytical methods can be used to characterize the enzymatic activity of purified B. bacteriovorus MurD?

Comprehensive characterization of B. bacteriovorus MurD activity requires multiple complementary approaches:

Primary activity assays:

• Coupled enzyme assay: Measure ADP production by coupling to pyruvate kinase and lactate dehydrogenase, monitoring NADH oxidation at 340 nm

• Radiometric assay: Using [¹⁴C]-labeled D-glutamate to quantify incorporation into UDP-MurNAc-L-Ala-D-Glu

• HPLC-based assay: Separation and quantification of reaction products

Kinetic parameter determination:

• Vary substrate concentrations systematically to determine Km and Vmax values for each substrate (UDP-MurNAc-L-Ala, D-Glu, ATP)

• Evaluate effect of divalent cations (Mg²⁺, Mn²⁺) on activity

• Determine optimal pH and temperature profiles

Structural analysis:

• Circular dichroism spectroscopy to assess secondary structure

• Thermal shift assays to evaluate protein stability

• Surface plasmon resonance to measure substrate binding kinetics

Activity validation:

• Mass spectrometry confirmation of reaction products

• Inhibition studies using phosphinate transition-state analogs, which are effective MurD inhibitors

The reaction likely follows the same mechanism proposed for E. coli MurD: phosphorylation of the C-terminal carboxylate of UDP-MurNAc-L-alanine followed by nucleophilic attack by the D-glutamate amide group .

How do the three domains of B. bacteriovorus MurD coordinate substrate binding and catalysis?

B. bacteriovorus MurD likely maintains the three-domain architecture observed in E. coli MurD, with each domain playing specific roles in catalysis:

Domain organization and functions:

• N-terminal domain: Exhibits Rossmann fold architecture for nucleotide binding, potentially interacting with the UDP portion of UDP-MurNAc-L-Ala

• Central domain: Contains a mononucleotide-binding fold resembling the GTPase family, likely involved in ATP binding

• C-terminal domain: Second Rossmann fold domain, likely participates in D-glutamate binding

Interdomain communication:
The active site forms at the interface between the central GTPase-like domain and domain 3, with substrate UMA entering from the side closest to domain 1 and ATP from the opposite side . This arrangement facilitates sequential binding and precise positioning of substrates for catalysis.

Key residues in substrate coordination:
Based on E. coli MurD structural data, critical residues likely include homologs to:

• Leu15, Thr16, Thr36, Arg37, Gly73, Asn138, His183 for UMA binding

• Gly114, Lys115, Ser/Thr116 (P-loop), Glu157 for ATP binding and magnesium coordination

Methodological approach for domain function investigation:

• Generate individual domain constructs and assess binding capabilities

• Perform site-directed mutagenesis of key residues at domain interfaces

• Use FRET-based assays to monitor domain movements during catalysis

• Apply hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon substrate binding

What structural adaptations in B. bacteriovorus MurD might reflect its predatory lifestyle compared to non-predatory bacteria?

B. bacteriovorus MurD likely exhibits specific adaptations that support its unique predatory lifecycle:

Potential structural adaptations:

• Substrate specificity modifications: Altered binding pocket architecture to accommodate variations in prey-derived precursors

• Catalytic efficiency: Potentially enhanced catalytic rates to support rapid cell wall synthesis during filamentous growth inside prey

• Regulatory interfaces: Additional surfaces for interaction with B. bacteriovorus-specific regulatory proteins

• Stability features: Enhanced stability in the changing intracellular environment of the prey cell

Methodological approach for comparative structural analysis:

• Crystallize B. bacteriovorus MurD and solve structure using methods similar to those used for E. coli MurD (multiple anomalous dispersion and multiple isomorphous replacement)

• Perform molecular dynamics simulations comparing B. bacteriovorus MurD with non-predatory bacterial homologs

• Use differential scanning calorimetry to compare thermal stability profiles

• Conduct hydrogen-deuterium exchange mass spectrometry to identify regions with altered solvent accessibility

• Perform cross-linking mass spectrometry to map protein interaction surfaces

The unique lifecycle of B. bacteriovorus, involving entry into prey periplasm, modification of prey cell wall, and filamentous growth followed by septation , may have driven evolutionary adaptations in its MurD enzyme.

How does B. bacteriovorus MurD activity coordinate with other peptidoglycan synthesis enzymes during predation?

B. bacteriovorus MurD functions within a complex network of cell wall synthesis enzymes that undergo precise temporal regulation during predation:

Coordination with other Mur ligases:

• Sequential activity with MurC (adds L-Ala), MurE (adds diaminopimelate/lysine), and MurF (adds D-Ala-D-Ala)

• Potential physical interaction in a multienzyme complex, similar to what has been observed in other bacteria

Integration with predation-specific processes:

• Coordination with lysozymes and deacetylases mentioned in search result that are involved in prey entry and exit

• Potential interaction with DivIVA-centered network that regulates cell division processes during the predatory cycle

Regulatory interconnections:

• Possible connection with cGAMP signaling pathway, which regulates B. bacteriovorus gliding motility necessary for prey exit

• Temporal regulation linked to predator cell division within the bdelloplast

Methodological approaches:

• Bacterial two-hybrid (BTH) assays to identify protein-protein interactions between MurD and other cell wall synthesis enzymes

• Co-immunoprecipitation followed by mass spectrometry to identify in vivo interaction partners

• Super-resolution microscopy using fluorescently tagged MurD to track localization during different predation stages

• Time-course transcriptomics and proteomics to correlate MurD expression with other cell wall synthesis genes

• Chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors regulating murD expression

What strategies can be employed to create conditional murD mutants in B. bacteriovorus for functional studies?

Creating conditional murD mutants in B. bacteriovorus requires specialized approaches due to this gene's likely essential nature and the predator's unique lifecycle:

Genetic manipulation strategies:

Experimental workflow:

• Generate constructs using suicide vectors compatible with B. bacteriovorus

• Introduce genetic modifications via conjugation with E. coli donor strains

• Select for integration events with appropriate antibiotics

• Verify conditional phenotypes by assessing predation efficiency under permissive vs. restrictive conditions

• Analyze peptidoglycan composition under depletion conditions using HPLC analysis

• Monitor morphological changes using electron microscopy and fluorescent d-amino acid labeling

Phenotypic analysis focus:

• Effects on prey invasion efficiency

• Impact on bdelloplast formation and stability

• Consequences for filamentous growth

• Influence on progeny septation and release

• Changes in peptidoglycan composition and structure

How has the evolutionary history of murD in predatory bacteria shaped its functional properties?

The evolutionary trajectory of murD in predatory bacteria like B. bacteriovorus reflects adaptations to their unique ecological niche:

Phylogenetic analysis approach:

• Construct maximum-likelihood phylogenetic trees using murD sequences from diverse bacterial lineages

• Calculate selection pressures (dN/dS ratios) across different bacterial lifestyles

• Identify sites under positive selection in predatory lineages

• Map selection hotspots onto protein structure models

• Perform ancestral sequence reconstruction to trace evolutionary changes

Potential evolutionary adaptations:

• Sequence modifications that enhance catalytic efficiency during rapid intracellular growth

• Alterations in regulatory regions for precise temporal control during predation

• Co-evolution with other peptidoglycan synthesis genes specific to predatory bacteria

• Potential horizontal gene transfer events that contributed to predatory adaptations

Structural-functional consequences:

• Changes in substrate binding affinities to accommodate predatory lifestyle

• Modifications in allosteric regulation mechanisms

• Altered protein stability parameters for function in prey cytoplasm

• Evolution of protein-protein interaction interfaces specific to predatory bacteria

Investigations should consider that B. bacteriovorus has a MurD homologue but lacks a MurL homologue, suggesting potential pathway adaptations compared to non-predatory bacteria .

What compensatory mechanisms might exist in B. bacteriovorus for peptidoglycan synthesis given the apparent absence of some conventional Mur proteins?

B. bacteriovorus exhibits interesting adaptations in its peptidoglycan synthesis pathway, potentially compensating for missing components:

Known pathway peculiarities:

• Presence of MurD homologue but absence of MurL homologue

• Potential modifications in precursor utilization pathways

• Possible dual-function proteins compensating for missing enzymes

Potential compensatory mechanisms:

• Enzyme promiscuity: Existing enzymes with broadened substrate specificity

• Alternative pathways: Non-canonical routes for peptidoglycan precursor synthesis

• Prey-derived components: Utilization of host cell wall precursors or enzymes

• Novel enzymes: B. bacteriovorus-specific proteins fulfilling functions of missing enzymes

Methodological investigation approach:

• Metabolomic profiling to identify unusual peptidoglycan precursors

• Activity-based protein profiling to discover novel peptidoglycan-modifying enzymes

• Heterologous complementation studies with known Mur proteins from model organisms

• Structural analysis of B. bacteriovorus peptidoglycan to identify unique modifications

• Comparative genomics across predatory and non-predatory deltaproteobacteria

This research direction connects to the broader question of how B. bacteriovorus has adapted conventional bacterial processes for its predatory lifestyle, potentially revealing novel enzymatic activities and pathway organizations.

How might recombinant B. bacteriovorus MurD be utilized as a tool for antimicrobial development?

B. bacteriovorus MurD offers unique opportunities for antimicrobial research beyond conventional applications:

Research applications in antimicrobial development:

• Structural template: B. bacteriovorus MurD structure could reveal unique features for designing inhibitors that specifically target pathogen MurD

• Mechanism insights: Understanding predator-specific adaptations may reveal new inhibition strategies

• Substrate analogs: Development of modified substrates based on B. bacteriovorus MurD specificity as potential antimicrobials

• Combination approaches: Synergistic targeting of multiple peptidoglycan synthesis steps guided by B. bacteriovorus predation strategies

Methodological approaches:

• High-throughput screening of compound libraries against recombinant MurD

• Structure-based drug design using crystallographic data

• Fragment-based screening to identify novel binding scaffolds

• Peptidoglycan precursor analogs design and testing

Potential advantages of B. bacteriovorus MurD-inspired approaches:

• Novel inhibition mechanisms derived from predatory adaptations

• Insights into bacterial peptidoglycan vulnerability from a natural bacterial predator

• Potential for narrow-spectrum targeting based on structural differences

This research direction focuses on academic understanding of inhibition mechanisms rather than commercial product development, providing fundamental insights that could inform future antimicrobial strategies.

What techniques can resolve conformational changes in B. bacteriovorus MurD during catalysis?

Advanced biophysical techniques can elucidate the dynamic structural changes in B. bacteriovorus MurD during its catalytic cycle:

Time-resolved structural analysis approaches:

Methodological workflow:

• Generate labeled MurD variants with minimal functional impact

• Establish reaction conditions that allow synchronized initiation

• Perform time-resolved measurements capturing different catalytic states

• Integrate data from multiple techniques to build a comprehensive model

• Develop computational simulations that incorporate experimental constraints

Specific transitions to investigate:

• Conformational changes upon UDP-MurNAc-L-Ala binding

• Structural rearrangements during ATP binding and phosphoryl transfer

• Domain movements accompanying D-glutamate binding and incorporation

• Release of products and return to initial state

These approaches could reveal whether B. bacteriovorus MurD employs unique conformational mechanisms compared to non-predatory bacteria, potentially related to its specialized function during predation.

How does B. bacteriovorus MurD activity interface with cyclic nucleotide signaling pathways during predation?

Recent research has revealed the importance of cyclic nucleotide signaling in B. bacteriovorus, suggesting potential coordination with cell wall synthesis enzymes like MurD:

Known signaling pathway connections:

• B. bacteriovorus Bd0367 produces cGAMP and c-di-GMP, with cGAMP regulating gliding motility necessary for prey exit

• Cell wall modification is crucial during both prey entry and exit stages

• Coordinated regulation likely exists between signaling pathways and peptidoglycan synthesis

Potential interfaces between MurD and signaling networks:

• Transcriptional regulation: Cyclic nucleotide-responsive transcription factors controlling murD expression

• Post-translational modifications: Direct or indirect modification of MurD activity by signaling proteins

• Protein-protein interactions: Physical interactions between MurD and components of signaling pathways

• Metabolic coordination: Synchronized regulation of energy allocation between motility and cell wall synthesis

Methodological investigation approaches:

• ChIP-seq to identify transcription factors binding murD promoter region

• Phosphoproteomics to detect signaling-dependent modifications of MurD

• Bacterial two-hybrid screening against components of known signaling pathways

• Fluorescence microscopy with dual-labeled proteins to track co-localization

• Genetic epistasis experiments combining mutations in signaling and murD genes

This research direction connects fundamental predatory mechanisms, potentially revealing how B. bacteriovorus coordinates complex processes like motility (regulated by cGAMP) and cell wall synthesis (requiring MurD) during its predatory lifecycle.