Recombinant Bdellovibrio bacteriovorus UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase (lpxC)

Basic Research Questions

• What is the function of LpxC in Bdellovibrio bacteriovorus?

  LpxC (UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase) catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate . This reaction represents the committed step in lipid A biosynthesis, which is essential for the production of lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria . In B. bacteriovorus, LpxC likely plays a critical role in maintaining cellular envelope integrity during both free-living and intraperiplasmic growth phases of its predatory lifecycle .

  Methodologically, LpxC function can be assessed through:

  • Enzyme activity assays measuring the production of UDP-3-O-(R-hydroxymyristoyl)glucosamine

  • Radiolabeling studies tracking lipid A biosynthesis

  • Conditional gene expression systems to manipulate lpxC levels during different predatory phases

• How is recombinant B. bacteriovorus LpxC purified for structural and functional studies?

  Purification of recombinant B. bacteriovorus LpxC involves:

  1. Gene cloning and vector construction:

     • PCR amplification of the lpxC gene from B. bacteriovorus genomic DNA

     • Insertion into expression vector with His-tag or other affinity tag

  2. Protein expression optimization:

     • Testing multiple E. coli expression strains (BL21(DE3), Rosetta, etc.)

     • Optimizing induction conditions (temperature, IPTG concentration, time)

  3. Purification strategy:

     • Immobilized metal affinity chromatography (IMAC) using histidine tag

     • Ion exchange chromatography for further purification

     • Size exclusion chromatography for final polishing

  4. Critical considerations:

     • Adding Zn²⁺ to all buffers to maintain metalloenzyme activity

     • Avoiding metal chelators like EDTA that would inhibit activity

     • Testing enzyme activity after each purification step

• What methods are used to measure B. bacteriovorus LpxC enzymatic activity?

  Enzymatic activity of B. bacteriovorus LpxC can be assessed through:

  1. Spectrophotometric assays:

     • Monitoring release of acetate using coupled enzyme systems

     • Following absorbance changes at 340 nm when NAD⁺ is reduced to NADH

  2. Radiochemical assays:

     • Using ¹⁴C-labeled UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine

     • Separating substrate and product by thin-layer chromatography

  3. Mass spectrometry:

     • Direct detection of substrate depletion and product formation

     • Allows for detailed kinetic analysis

  4. pH-dependency studies:

     • Constructing pH-rate profiles to determine optimal conditions

     • E. coli LpxC shows a bell-shaped curve with pKa values of 6.4±0.1 and 9.1±0.1

  5. Metal-dependency analysis:

     • Testing activity after treatment with metal chelators

     • Reconstitution with different metal ions to confirm zinc specificity

• How does B. bacteriovorus LpxC expression change during its predatory lifecycle?

  B. bacteriovorus exhibits a biphasic lifecycle with distinct free-living and intraperiplasmic growth phases . LpxC expression likely follows phase-specific patterns:

  1. During free-living phase:

     • Higher LpxC expression expected for maintaining robust cell envelope

     • Required for high motility and environmental resistance

  2. During prey invasion:

     • Potential transient downregulation during initial periplasmic entry

     • Allows for structural flexibility during invasion process

  3. During intraperiplasmic growth:

     • Modulated expression as predator grows within prey periplasm

     • Coordination with prey envelope degradation systems

  4. Prior to prey cell lysis:

     • Increased expression to prepare progeny for free-living phase

     • Ensures newly formed Bdellovibrio cells have intact LPS

  Research approaches include:

  • RNA-seq analysis of synchronized predator populations

  • Fluorescent reporter fusions to monitor lpxC expression in real-time

  • Western blot analysis of LpxC protein levels across lifecycle stages

  • Activity assays from cell extracts at different timepoints

• What structural features differentiate B. bacteriovorus LpxC from other bacterial LpxC enzymes?

  While B. bacteriovorus LpxC structure hasn't been fully characterized, insights can be inferred from related structures like Aquifex aeolicus LpxC :

  1. Core structural elements likely conserved:

     • Zinc-binding pocket with coordinating histidine and aspartate residues

     • Hydrophobic passage accommodating acyl chain

     • Nucleotide-binding domain with uracil recognition elements

  2. Potential adaptations in B. bacteriovorus LpxC:

     • Modified substrate binding site reflecting unique LPS composition

     • Structural adaptations related to predatory lifecycle

     • Potential protein-protein interaction surfaces for regulation

  3. Nucleotide-binding site features based on A. aeolicus LpxC:

     • Uracil binding via hydrophobic interactions with Phe155 and Arg250

     • Hydrogen-bonding interactions with Glu154, Tyr151, and Lys227

     • Phosphate-ribose interactions with Arg137, Lys156, Glu186, and Arg250

  Structural analysis approaches include:

  • X-ray crystallography of purified B. bacteriovorus LpxC

  • Cryo-electron microscopy for structure determination

  • Homology modeling based on known LpxC structures

  • Molecular dynamics simulations to predict structural differences

Advanced Research Questions

• How does B. bacteriovorus LpxC activity influence prey recognition and invasion processes?

  The predatory lifecycle of B. bacteriovorus involves complex prey recognition and invasion mechanisms that may be influenced by LpxC-generated LPS structures:

  1. Recently discovered prey recognition systems:

     • The mosaic adhesive trimer (MAT) superfamily proteins concentrate on the predator surface before prey encounter

     • These proteins show various binding capabilities for recognizing diverse prey epitopes

     • MAT family members localize to a vesicular compartment deposited in prey periplasm during invasion

  2. Potential LpxC involvement in predation:

     • LPS structure likely influences initial attachment to prey surfaces

     • LpxC-generated LPS components may interact with MAT proteins

     • Modified LPS could facilitate invasion through prey cell envelope

  3. Experimental approaches to investigate this relationship:

     • Conditional lpxC mutants to observe effects on predation efficiency

     • Pull-down assays to detect interactions between LPS and MAT proteins

     • Fluorescence microscopy to co-localize LpxC with invasion structures

     • Comparative lipid A analysis between attack-phase and growth-phase cells

• What is the relationship between B. bacteriovorus LpxC and chromosomal replication during predation?

  B. bacteriovorus must coordinate chromosomal replication with its predatory lifecycle, suggesting potential regulatory links with LpxC activity:

  1. Chromosomal replication in B. bacteriovorus:

     • Regulated primarily at the initiation step, involving DnaA protein and oriC region

     • B. bacteriovorus oriC can be bound by both predator and prey DnaA proteins

     • Replication occurs only during intraperiplasmic growth phase

  2. Potential LpxC connections to replication:

     • Cell envelope restructuring (LpxC-dependent) may be coordinated with replication

     • LPS synthesis rates might be synchronized with chromosome copy number

     • Common regulatory pathways may control both processes

  3. Research methodologies:

     • ChIP-seq to identify potential transcription factors regulating both systems

     • Proteomic analysis of protein-protein interactions between pathways

     • Flow cytometry to correlate LPS composition with DNA content

     • Imaging studies to track LpxC and replication proteins during predation

• How do pH fluctuations during prey invasion affect B. bacteriovorus LpxC activity?

  During invasion and growth within prey periplasm, B. bacteriovorus experiences pH changes that could impact LpxC function:

  1. pH dependency of LpxC activity:

     • E. coli LpxC exhibits a bell-shaped pH-rate profile with optima between pH 6.4-9.1

     • Catalytic mechanism involves at least two ionizations important for activity

  2. pH dynamics during predation:

     • Initial prey periplasm pH may differ from external environment

     • Prey cell degradation likely creates localized pH changes

     • Predator metabolism within bdelloplast alters internal pH

  3. Methodological approaches:

     • Activity assays across pH range with purified B. bacteriovorus LpxC

     • pH-sensitive fluorescent probes to measure bdelloplast internal pH

     • Site-directed mutagenesis of key catalytic residues affecting pH sensitivity

     • Mathematical modeling of pH effects on enzyme kinetics during predation

  pH	Relative LpxC Activity (%)	Predatory Phase
  5.5	~30*	Early bdelloplast formation
  6.5	~80*	Active prey degradation
  7.5	~100*	Optimal growth conditions
  8.5	~90*	Late predatory cycle
  9.5	~40*	Not physiologically relevant

  *Predicted values based on E. coli LpxC behavior; specific B. bacteriovorus data to be determined experimentally

• What are the kinetic differences between B. bacteriovorus LpxC and LpxC from its common prey bacteria?

  Comparing enzyme kinetics between predator and prey LpxC could reveal evolutionary adaptations:

  1. Substrate specificity considerations:

     • Differences in acyl chain preference reflecting membrane composition

     • The ester-linked R-3-hydroxymyristoyl chain increases kcat/KM by ~5×10⁶-fold in E. coli LpxC

     • B. bacteriovorus may show different substrate preferences

  2. Methodological approach:

     • Parallel purification of LpxC from B. bacteriovorus and prey bacteria

     • Determination of kinetic parameters (kcat, KM) with various substrates

     • Inhibitor sensitivity profiling

     • Effects of temperature, pH, and ionic strength on activity

  3. Significance of kinetic differences:

     • May reflect adaptation to predatory lifestyle

     • Could reveal potential for selective inhibition strategies

     • Provides insights into evolution of lipid A biosynthesis

  LpxC Source	KM (μM)	kcat (s⁻¹)	kcat/KM (M⁻¹s⁻¹)	Optimal Temperature (°C)
  B. bacteriovorus	TBD	TBD	TBD	TBD
  E. coli	8.0	3.0	3.75×10⁵	37
  P. aeruginosa	12.3	2.8	2.28×10⁵	37
  A. aeolicus	7.5	4.2	5.60×10⁵	55

• How does zinc coordination in B. bacteriovorus LpxC contribute to its function during predation?

  As a zinc-dependent metalloenzyme, B. bacteriovorus LpxC requires proper metal coordination for activity:

  1. Zinc coordination in LpxC:

     • Essential for catalytic activity

     • Typically involves two histidine residues, an aspartate, and a water molecule

     • Metal-chelating reagents like dipicolinic acid completely inhibit activity

  2. Zinc acquisition during predation:

     • B. bacteriovorus may obtain zinc from prey during intraperiplasmic growth

     • Competition with prey metalloproteins for available zinc

     • Potential zinc storage mechanisms for attack-phase cells

  3. Research approaches:

     • X-ray absorption spectroscopy to determine zinc coordination geometry

     • Site-directed mutagenesis of predicted zinc-coordinating residues

     • Metal reconstitution studies with purified enzyme

     • Zinc content analysis across predatory lifecycle stages

  4. Potential adaptations:

     • Modified zinc affinity optimized for fluctuating zinc availability

     • Structural features protecting zinc site during prey invasion

     • Potential for utilizing alternative metals in zinc-limited conditions

• How can structural knowledge of B. bacteriovorus LpxC inform the development of selective inhibitors?

  Understanding B. bacteriovorus LpxC structure has implications for developing selective inhibitors:

  1. Current state of LpxC inhibitor development:

     • Hydroxamate compounds effectively inhibit LpxC activity

     • Various inhibitors in preclinical development target pathogenic bacteria

     • None have yet reached clinical trials

  2. Structural considerations for selective inhibition:

     • Nucleotide-binding site features (uracil recognition elements)

     • Hydrophobic passage accommodating acyl chain

     • Zinc coordination geometry differences

  3. Dual application potential:

     • Inhibitors that affect pathogenic bacteria but spare B. bacteriovorus

     • Allow combination therapies of antibiotics with predatory bacteria

     • Enable controlled use of B. bacteriovorus as living antibiotic

  4. Methodological approach:

     • Structure-based drug design targeting unique features

     • High-throughput screening with differential inhibition readouts

     • Structure-activity relationship studies with existing inhibitors

     • In vivo testing in predator-prey-inhibitor three-component systems

• What role does LpxC play in B. bacteriovorus resistance to host immune responses?

  B. bacteriovorus is being investigated as a potential "living antibiotic" , making its interaction with host immunity crucial:

  1. Current safety profile findings:

     • No reduction in mouse viability after intranasal or intravenous inoculation

     • Only modest inflammatory response at 1 hour post-exposure

     • Response not sustained at 24 hours

     • Predators cleared quickly and efficiently from the host

  2. LpxC-generated LPS implications:

     • Modified lipid A structure may reduce immunogenicity

     • Could affect recognition by pattern recognition receptors

     • May influence predator clearance rate by immune cells

  3. Research methodologies:

     • LPS structural analysis from B. bacteriovorus versus pathogenic bacteria

     • In vitro immunostimulation assays with purified LPS

     • Cytokine profiling in response to wild-type versus LpxC-modified B. bacteriovorus

     • Tracking predator survival with different LPS compositions in vivo

  Cytokine	Response to B. bacteriovorus (fold change)	Response to E. coli LPS (fold change)	Timepoint
  IL-6	↑ (kidney, spleen)	↑↑↑↑	3h
  TNF	↑ (liver)	↑↑↑↑↑	3h
  CXCL-1/KC	↑ (blood)	↑↑↑	3h
  All measured	Return to baseline	Sustained elevation	18-24h

• How is LpxC activity coordinated with the unique vesicular compartment formation during prey invasion?

  Recent discoveries about B. bacteriovorus invasion mechanisms provide new context for understanding LpxC function:

  1. Vesicular compartment during invasion:

     • B. bacteriovorus protein CpoB (Bd0635) concentrates into a vesicular compartment

     • This vesicle is deposited into prey periplasm during invasion

     • Contains several phage-tail-fiber-like proteins of the MAT superfamily

     • The vesicle remains associated with the entry point in the bdelloplast

  2. Potential LpxC involvement:

     • LpxC-generated LPS may contribute to vesicle membrane composition

     • Lipid A modifications could facilitate vesicle formation or stability

     • LpxC activity might be spatiotemporally regulated during vesicle formation

  3. Research approaches:

     • Co-localization studies of LpxC with CpoB during invasion

     • Lipidomic analysis of isolated vesicular compartments

     • Conditional LpxC inhibition during invasion process

     • Correlating LpxC activity with vesicle formation efficiency

• What genomic and proteomic approaches can reveal LpxC evolutionary adaptations in B. bacteriovorus?

  Understanding evolutionary adaptations in B. bacteriovorus LpxC requires multi-omics approaches:

  1. Comparative genomics strategies:

     • Sequence alignment of lpxC genes across predatory and non-predatory bacteria

     • Analysis of selection pressure (dN/dS ratios) on lpxC gene

     • Identification of co-evolving genes in lipid A biosynthesis pathway

     • Examination of horizontal gene transfer events affecting lpxC

  2. Structural proteomics:

     • Homology modeling based on known LpxC structures

     • Prediction of predator-specific structural adaptations

     • Molecular dynamics simulations to assess functional differences

  3. Functional proteomics:

     • Protein-protein interaction networks involving LpxC

     • Post-translational modifications affecting LpxC regulation

     • Protein turnover rates during different predatory phases

  4. Integrative analysis:

     • Correlation of lpxC sequence variations with prey range

     • Mapping structural differences to predatory efficiency

     • Evolutionary trajectory reconstruction of lipid A biosynthesis

• What biotechnological applications could emerge from engineering B. bacteriovorus LpxC?

  Engineered B. bacteriovorus LpxC could enable several biotechnological applications:

  1. Enhanced predatory bacteria as antimicrobials:

     • Modified LpxC to improve predator survival in therapeutic environments

     • Engineered substrate specificity to target specific pathogens

     • Reduced immunogenicity through lipid A modifications

  2. Environmental applications:

     • Engineered predators for biofilm control in industrial settings

     • Modified LpxC to enhance predator survival in harsh environments

     • Biosensors incorporating predatory bacteria for pathogen detection

  3. Synthetic biology platforms:

     • Cell envelope engineering through modified LpxC activity

     • Creation of bacterial chassis with novel surface properties

     • Development of selective inhibitors for spatial control of predation

  4. Methodological approaches:

     • Directed evolution of lpxC gene for desired properties

     • CRISPR-Cas9 genome editing to introduce specific modifications

     • High-throughput screening for enhanced predatory phenotypes

     • In vivo testing in relevant application environments

Comparative Data for LpxC Research

*Predicted values based on homology; specific experimental data not available in current research

Methodological Approaches for B. bacteriovorus LpxC Research