Recombinant Burkholderia multivorans Imidazole glycerol phosphate synthase subunit HisF (hisF)

What is the biological function of Imidazole glycerol phosphate synthase in Burkholderia multivorans?

Imidazole glycerol phosphate synthase (IGPS) in Burkholderia multivorans is a heterodimeric bienzyme complex that operates at a central branch point of metabolism. It consists of two subunits: HisF (the cyclase subunit) and HisH (the glutaminase subunit). This enzyme plays a crucial role in the histidine biosynthesis pathway. The HisH subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia, and the HisF subunit then uses this ammonia for a cyclase reaction that produces imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) .

How are the his genes organized in Burkholderia multivorans compared to other bacterial species?

In Burkholderia multivorans, all nine his genes are clustered on the 3.4-Mb chromosome, forming a "his core" of genes belonging to histidine biosynthesis (hisBHAF). This organization differs from other bacterial species, as B. multivorans has a complex genome with three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. Studies have revealed that the majority of amino acid biosynthetic genes (20 out of 23 studied) are located on the 3.4-Mb chromosome. This organization suggests that the proteobacterial his operon was assembled piecewise, through accretion of smaller units containing only some of the genes involved in the biosynthetic route .

What is the evolutionary significance of HisF in Burkholderia species?

HisF in Burkholderia species shows evidence of strong conservation throughout evolution, indicating its essential role in bacterial metabolism. Comparative genomic studies of Burkholderia species have revealed that the histidine biosynthetic core (hisBHAF) has a monophyletic origin without evidence of horizontal gene transfer events. This conservation suggests that HisF and related histidine biosynthesis genes represent ancient and fundamental metabolic machinery. Additionally, research has identified specific mutations in key regulatory genes that may enable adaptation to different environments, particularly in the cystic fibrosis lung environment where B. multivorans is a significant pathogen .

What are the most effective methods for expressing and purifying recombinant Burkholderia multivorans HisF protein?

For effective expression and purification of recombinant Burkholderia multivorans HisF, researchers typically employ the following methodology:

• Expression System: E. coli is the preferred host organism for heterologous expression, as demonstrated with related Burkholderia species HisF proteins .

• Vector Selection: Plasmid vectors containing inducible promoters (such as T7 or araB) allow controlled expression.

• Purification Protocol:

  • Immobilized metal affinity chromatography (IMAC) using His-tags

  • Size exclusion chromatography to improve purity (>85% purity is typically achievable)

  • Ion exchange chromatography as a polishing step

• Storage Conditions: The purified protein should be stored with 5-50% glycerol at -20°C/-80°C to maintain stability. The shelf life is approximately 6 months for liquid form and 12 months for lyophilized form .

• Quality Control: SDS-PAGE analysis confirms purity, while activity assays verify functionality.

To avoid repeated freeze-thaw cycles that can compromise protein integrity, working aliquots should be stored at 4°C for no more than one week .

How can researchers effectively study the allosteric regulation of the HisFH complex?

Studying the allosteric regulation of the HisFH complex requires a multi-technique approach:

• Structural Analysis:

  • X-ray crystallography to determine static structures of different functional states

  • NMR spectroscopy to capture dynamics in solution

• Molecular Dynamics Simulations:

  • Extensive simulations to describe the allosteric activation pathway

  • Enhanced sampling techniques to overcome barriers between functional states

  • Dynamic network analysis to identify allosteric communication pathways

• Kinetic Analysis:

  • Enzyme kinetic assays with varying substrate concentrations

  • Stopped-flow measurements to capture transient intermediates

• Mutagenesis:

  • Site-directed mutagenesis of key residues in allosteric pathways

  • Analysis of effects on catalysis and substrate binding

Research has revealed that the HisFH complex is allosterically regulated through a mechanism where the catalytically active conformation is only formed when substrates of both HisH and HisF are bound. This prevents wasteful turnover of glutamine in the absence of the HisF ligand .

The allosteric activation occurs on a millisecond timescale and involves correlated time-evolving dynamic networks connecting the effector and substrate binding sites. The combined binding of effector and substrate dramatically decreases the conformational barrier associated with oxyanion hole formation, leading to a 4500-fold increase in glutamine hydrolysis activity .

What CRISPR/Cas9 approaches are most suitable for genomic editing of hisF in Burkholderia multivorans?

For genomic editing of hisF in Burkholderia multivorans, a modified two-plasmid CRISPR/Cas9 system has proven effective. The methodology involves:

• Plasmid System:

  • pCasPA: Contains Cas9 and λ-Red system encoding genes under an arabinose-inducible promoter (araB)

  • pACRISPR: Carries the guide RNA and homology repair templates

• Optimization Steps:

  • Selection marker replacement for Burkholderia compatibility

  • Optimized araB promoter induction for controlled Cas9 expression

  • Establishment of plasmid curing procedures based on sacB gene or growth at sub-optimal temperature (18-20°C)

• Implementation Process:

  • Design a 20-nucleotide spacer targeting hisF

  • Develop repair arms (0.6-0.8 kbp each) flanking the target region

  • Transform L-arabinose-induced electrocompetent B. multivorans cells containing pCasPA with the modified pACRISPR plasmid

  • Select transformants and confirm gene editing by PCR and sequencing

  • Cure plasmids through serial passages at 18-20°C

This system has demonstrated high efficiency in precise unmarked deletions and targeted gene insertions in B. multivorans, with the advantage of requiring only a single homologous recombination event, making it faster than conventional allelic exchange methods .

How does the structure of HisF contribute to the ammonia tunneling mechanism in the HisFH complex?

The structure of HisF plays a critical role in the ammonia tunneling mechanism within the HisFH complex:

• Barrel Structure and Tunnel Formation:
  HisF forms a beta-strand barrel structure that creates a central tunnel spanning approximately 25 Å from the HisH active site to the HisF cyclase active site. This tunnel allows ammonia, produced by glutamine hydrolysis in HisH, to migrate to the opposite face of the barrel without being released into the bulk solvent .

• Conformational Changes and Tunnel Gating:
  The allosteric activation of the complex induces conformational changes that are essential for tunnel formation and regulation. When both the HisF substrate (PrFAR) and HisH substrate (glutamine) are bound, the complex attains a closed HisF:HisH interface, enabling efficient ammonia transfer .

• Ammonia Transit Mechanism:
  Molecular dynamics simulations have revealed that:

  • The tunnel contains specific hydrophilic residues that facilitate ammonia passage

  • The movement of ammonia through the tunnel is controlled by a series of hydrogen-bonding interactions

  • The tunnel prevents the wasteful release of ammonia into the surrounding environment

• Catalytic Coordination:
  The structure ensures that glutamine hydrolysis in HisH is synchronized with cyclase activity in HisF, preventing wasteful turnover of glutamine in the absence of the HisF substrate .

This sophisticated tunneling mechanism represents an evolutionary solution to the challenge of transferring a reactive intermediate (ammonia) between distant active sites without diffusion losses, highlighting the intricate structural basis for catalytic efficiency in this enzyme complex.

What are the molecular mechanisms underlying the allosteric activation of Imidazole glycerol phosphate synthase in Burkholderia multivorans?

The allosteric activation of Imidazole glycerol phosphate synthase in Burkholderia multivorans involves several coordinated molecular events:

• Substrate-Induced Conformational Changes:

  • The binding of PrFAR (the HisF substrate) induces conformational changes that propagate to the HisH subunit

  • These changes transform the glutaminase active site from an inactive to a catalytically competent state

  • The active conformation includes proper formation of the oxyanion hole necessary for glutamine hydrolysis

• Dynamic Allosteric Networks:

  • Time-evolving networks of residues connect the effector binding site (in HisF) to the substrate binding site (in HisH)

  • These networks transmit allosteric signals over a distance of approximately 25 Å

  • The signal transmission occurs on a millisecond timescale

• Interface Dynamics:

  • The HisF:HisH interface undergoes significant rearrangements during activation

  • Initially, the complex captures glutamine in a catalytically inactive HisH conformation

  • Subsequently, it attains a closed interface configuration that enables glutamine hydrolysis

• Energy Landscape Modification:

  • Combined binding of effector and substrate dramatically decreases the conformational barrier for oxyanion hole formation

  • This energy landscape modification explains the experimentally observed 4500-fold increase in glutamine hydrolysis activity

Molecular dynamics simulations have been instrumental in uncovering these mechanisms, revealing how IGPS spontaneously captures glutamine, undergoes interface closure, and forms the oxyanion hole required for efficient catalysis. These findings provide a molecular basis for understanding the long-range allosteric communication in this important enzyme complex.

How have parallel adaptations in HisF contributed to Burkholderia multivorans evolution in cystic fibrosis environments?

Genomic analysis of Burkholderia multivorans isolates from cystic fibrosis patients has revealed significant insights into how parallel adaptations in histidine biosynthesis genes, including hisF, contribute to bacterial evolution in this specialized environment:

• Genomic Diversity Patterns:
  Endemic B. multivorans strains infecting different CF patients show peculiar patterns of genomic diversity. While isolates may share identical sequence types (e.g., ST-742), whole genome analysis reveals significant differences between patients. This suggests divergent evolutionary trajectories driven by host-specific selection pressures .

• Parallel Adaptations:
  Research has identified sets of parallel adaptations across multiple CF patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the CF lung. A study of 13 isolates from an endemic B. multivorans strain found 30 such parallel adaptations across multiple patients .

• Evolutionary Mechanisms:
  The evolution of B. multivorans in the CF lung involves:

  • Low rates of adaptive evolution within individual patients

  • High between-patient strain diversity

  • Specific mutations in key metabolic genes, including those involved in histidine biosynthesis

  • Structural genomic variations including plasmid content differences and transposase activity

• Environmental Adaptations:
  Histidine biosynthesis genes may be particularly important for adaptation to the CF lung environment due to:

  • Nutrient limitations in the CF lung microenvironment

  • Need to respond to oxidative stress from host immune responses

  • Adaptation to low oxygen and iron concentrations

These findings suggest that an environmental microdiverse reservoir must exist for endemic B. multivorans strains, where active diversification takes place. The histidine biosynthetic pathway, including HisF, appears to be subject to specific selective pressures in the CF lung environment, contributing to the remarkable adaptability of this pathogen.

What role does HisF play in the pathogenesis of Burkholderia multivorans in cystic fibrosis patients?

The role of HisF in Burkholderia multivorans pathogenesis in cystic fibrosis (CF) patients involves several interconnected aspects:

How do genomic variations in hisF genes affect the evolution of Burkholderia multivorans during cystic fibrosis infection?

Genomic variations in hisF genes contribute significantly to the evolution of Burkholderia multivorans during cystic fibrosis infection through several mechanisms:

These observations indicate that genomic variations in histidine biosynthesis genes, including hisF, are an important component of B. multivorans adaptation during CF infection. The limited within-patient evolution but high between-patient diversity suggests that an environmental microdiverse reservoir must exist for endemic strains, where active diversification takes place before lung colonization.

What are the most effective methods for analyzing the allosteric conformational changes in the HisFH complex?

Analyzing allosteric conformational changes in the HisFH complex requires a multi-faceted approach combining several advanced biophysical and computational techniques:

• X-ray Crystallography and Cryo-EM:

  • Capturing different functional states of the complex (apo, substrate-bound, effector-bound, and fully activated)

  • Co-crystallization with substrate analogs or transition state mimics to trap intermediate conformations

  • Time-resolved crystallography to capture conformational changes in real-time

• Solution NMR Spectroscopy:

  • Chemical shift perturbation experiments to map binding interfaces and conformational changes

  • Relaxation dispersion methods to characterize millisecond timescale dynamics relevant to allosteric activation

  • Hydrogen/deuterium exchange mass spectrometry to identify regions with altered dynamics upon activation

• Molecular Dynamics Simulations:

  • Extensive simulations (reaching microsecond to millisecond timescales) to observe spontaneous conformational transitions

  • Enhanced sampling techniques (such as umbrella sampling, metadynamics, or replica exchange) to overcome energy barriers between states

  • Dynamic network analysis to identify correlated motions and allosteric communication pathways

• FRET and Single-Molecule Techniques:

  • Förster resonance energy transfer between strategically placed fluorophores to monitor distance changes during activation

  • Single-molecule FRET to observe conformational heterogeneity and transitions between states

  • Single-molecule force spectroscopy to characterize mechanical properties of different conformational states

• Mutagenesis Combined with Kinetic Analysis:

  • Site-directed mutagenesis of residues in proposed allosteric pathways

  • Kinetic characterization of mutants to quantify effects on allosteric activation

  • Double-mutant cycle analysis to identify energetically coupled residues

Recent research has successfully employed molecular dynamics simulations combined with enhanced sampling to reveal how the HisFH complex spontaneously captures glutamine in a catalytically inactive conformation, subsequently attains a closed interface, and finally forms the oxyanion hole required for efficient glutamine hydrolysis. These computational approaches, validated by experimental data, have provided unprecedented insights into the millisecond timescale allosteric activation mechanism .

What structural characteristics distinguish Burkholderia multivorans HisF from homologous proteins in other bacterial species?

The structural characteristics that distinguish Burkholderia multivorans HisF from homologous proteins in other bacterial species include several key features:

While detailed structural information specifically for B. multivorans HisF is limited, comparative analysis with homologous proteins from related species (including B. mallei and B. cenocepacia) suggests these structural specializations play important roles in adapting the enzyme to the specific metabolic requirements and environmental challenges faced by this pathogen during host infection .

How have histidine biosynthesis genes in Burkholderia multivorans evolved compared to other Burkholderia species?

The evolution of histidine biosynthesis genes in Burkholderia multivorans shows both shared patterns and distinct features compared to other Burkholderia species:

• Genomic Organization:

  • B. multivorans, like other Burkholderia species, has a complex genome with multiple chromosomes

  • All nine his genes in B. multivorans are clustered on the 3.4-Mb chromosome, forming a "core" of histidine biosynthesis genes

  • This organization is generally conserved across the Burkholderia genus, suggesting evolutionary stability of this gene cluster

• Phylogenetic Analysis:

  • Phylogenetic studies have shown that histidine biosynthesis genes in Burkholderia have a monophyletic origin

  • There is no evidence of horizontal gene transfer events affecting these genes, indicating vertical inheritance throughout Burkholderia evolution

  • The histidine biosynthetic core shows strong conservation of structure and organization throughout the entire genus

• Evolutionary Lineages:

  • B. multivorans specifically has been shown to separate into two distinct evolutionary clades (lineage 1 and lineage 2)

  • Average nucleotide identity analysis and phylogenetic alignment of core genes demonstrate clear separation between these lineages

  • Comparative genomics has identified lineage-specific genes (ghrB_1 in lineage 1 and glnM_2 in lineage 2)

• Adaptation Signatures:

  • Comparative evolutionary patterns between B. multivorans and B. cenocepacia during coinfection of CF patients reveal both species-specific and shared evolutionary adaptations

  • Both species accumulate mutations at similar rates (2.27 SNPs/year for B. multivorans and 2.08 SNPs/year for B. cenocepacia)

  • Certain orthologous genes shared by B. cenocepacia and B. multivorans have been found to be under strong selection, accumulating mutations associated with lineage diversification

• Regulatory Evolution:

  • The intergenic regions of the his genes show evidence of evolutionary adaptation

  • Analysis of substitution rate, entropy plot, and bendability has suggested the existence of a putative transcription promoter upstream of hisB

  • These findings support the hypothesis that the proteobacterial his operon was assembled piecewise, through accretion of smaller units containing only some of the genes involved in the biosynthetic route

The evidence suggests that while the core histidine biosynthesis machinery is strongly conserved across Burkholderia species, reflecting its essential metabolic role, there are species-specific and lineage-specific adaptations that likely reflect the particular ecological niches and lifestyles of different Burkholderia species, including the adaptation of B. multivorans to the cystic fibrosis lung environment.

What insights can comparative genomics provide about the function and regulation of hisF across different Burkholderia species?

Comparative genomics provides several key insights into the function and regulation of hisF across different Burkholderia species:

These comparative insights reveal that while the core function of HisF is highly conserved across Burkholderia species, subtle variations in sequence, regulation, and adaptive mutations likely reflect the diverse ecological niches these bacteria occupy, from environmental reservoirs to the specialized conditions of the CF lung. The constitutive expression pattern and complex operon organization suggest that Burkholderia species may have evolved unique regulatory mechanisms for histidine biosynthesis compared to other bacterial genera.

What are the main challenges in producing stable and active recombinant Burkholderia multivorans HisF protein, and how can they be addressed?

Producing stable and active recombinant Burkholderia multivorans HisF protein presents several challenges that require specific technical solutions:

• Protein Solubility Issues:

  Challenge: HisF can form inclusion bodies when overexpressed in E. coli.

  Solutions:

  • Use solubility-enhancing fusion tags (MBP, SUMO, or Thioredoxin)

  • Optimize expression temperature (typically 16-20°C) to slow protein synthesis

  • Co-express with chaperones (GroEL/GroES system) to assist proper folding

  • Employ auto-induction media to achieve gradual protein expression

• Protein Stability Concerns:

  Challenge: HisF can exhibit limited stability during purification and storage.

  Solutions:

  • Add 5-50% glycerol to storage buffer to prevent aggregation

  • Store as aliquots at -80°C to avoid freeze-thaw cycles

  • Include reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

  • Optimize buffer composition based on thermal shift assays to maximize stability

• Functional Activity Assessment:

  Challenge: HisF functions as part of a heterodimeric complex with HisH, making activity assessment complex.

  Solutions:

  • Co-express HisF with HisH to produce the functional complex

  • Develop assays that can measure activity either with purified HisH added separately or with synthetic ammonia as a substrate

  • Use circular dichroism and thermal shift assays to confirm proper folding as a proxy for potential activity

• Expression System Selection:

  Challenge: Standard expression systems may not produce protein with native post-translational modifications.

  Solutions:

  • For basic structural and biochemical studies, E. coli expression is sufficient

  • For studies requiring native modifications, consider Burkholderia-based expression systems using the CRISPR/Cas9 genome editing tools recently developed

  • Evaluate expression in closely related non-pathogenic Burkholderia strains

• Protein Purity and Homogeneity:

  Challenge: Obtaining homogeneous preparations for structural studies.

  Solutions:

  • Implement multi-step purification protocols combining IMAC, ion exchange, and size exclusion chromatography

  • Use limited proteolysis to remove flexible regions that might cause heterogeneity

  • Perform dynamic light scattering analysis to confirm sample monodispersity

  • Remove aggregation-prone batches based on analytical size exclusion profiles

Table 1: Optimization Parameters for B. multivorans HisF Expression and Purification

By addressing these challenges with the appropriate technical solutions, researchers can produce stable, active recombinant B. multivorans HisF protein suitable for structural, biochemical, and functional studies.

What are the key considerations when designing experiments to study the interaction between HisF and HisH in Burkholderia multivorans?

When designing experiments to study the interaction between HisF and HisH in Burkholderia multivorans, researchers should consider several key factors: