Recombinant Photorhabdus luminescens subsp. laumondii 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA)

What is the genomic context of gpmA in Photorhabdus luminescens subsp. laumondii?

The gpmA gene is located within the 5.27-Mbp genome of Photorhabdus luminescens subsp. laumondii, which has a G+C content of 42.4% and contains 4,243 candidate protein-coding genes . As a glycolytic enzyme, gpmA would be part of the central metabolic pathways. The complete genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession number LJPB00000000, which provides researchers with the ability to examine the genomic context and potential regulatory elements associated with gpmA expression .

What is the biological function of the gpmA enzyme in P. luminescens?

The 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA) catalyzes the reversible conversion of 3-phosphoglycerate to 2-phosphoglycerate in the glycolytic pathway. This isomerization reaction is critical for energy metabolism in P. luminescens during its complex lifecycle, which includes symbiosis with Heterorhabditis nematodes and pathogenicity against insects . The enzyme likely plays a vital role in ensuring sufficient energy production during different life stages of the bacterium, including both the primary (1°) phenotypic variant that maintains symbiosis with nematodes and the secondary (2°) variant that cannot reassociate with nematodes .

How does the gpmA enzyme from P. luminescens differ from homologous enzymes in other organisms?

While the specific differences of the P. luminescens gpmA are not fully characterized in the provided search results, the evolutionary context suggests potential adaptations to the bacterium's unique lifestyle. Given that P. luminescens undergoes phenotypic switching between primary and secondary forms , the gpmA enzyme may have evolved specific regulatory mechanisms or structural features that support energy metabolism during this transition. Comparative sequence analysis with homologous enzymes from other bacterial species would be required to identify unique amino acid residues or domains that might confer specific properties relevant to P. luminescens' entomopathogenic lifestyle.

What expression systems are recommended for recombinant production of P. luminescens gpmA?

For expressing recombinant proteins from P. luminescens, several expression systems can be considered:

• Prokaryotic expression systems: While E. coli is commonly used for expressing bacterial proteins, it may not be optimal for all P. luminescens proteins depending on folding requirements and post-translational modifications.

• Eukaryotic expression systems: For complex proteins requiring specific post-translational modifications, eukaryotic hosts like Komagataella phaffii (formerly Pichia pastoris) have proven effective for recombinant enzyme production . K. phaffii has been successfully used for other complex recombinant enzymes that require correct folding and disulfide bridge formation .

The choice depends on the specific properties of gpmA. Methodology would include gene cloning into an appropriate vector, transformation of the host, and optimization of expression conditions including temperature, inducer concentration, and cultivation duration.

What are the optimal conditions for fed-batch cultivation of recombinant gpmA?

For recombinant enzyme production using fed-batch cultivation, which is currently state-of-the-art for microbial recombinant protein production , the following parameters should be considered:

For K. phaffii expression systems, fed-batch cultivations have yielded protein concentrations around 0.7 g/L for other recombinant enzymes . The methodology would involve carefully controlling nutrient feeding to maintain optimal growth while inducing protein expression at the appropriate time point.

How can I prevent pseudohyphae formation during continuous cultivation of P. luminescens gpmA in K. phaffii?

When using K. phaffii as an expression host for recombinant proteins, pseudohyphae growth can occur at specific growth rates (μ) below 0.075 h⁻¹ in chemostat cultivations . This morphological change can significantly hinder protein secretion and reduce productivity . To prevent this issue:

• Maintain dilution rates (D) above 0.075 h⁻¹ if possible

• Monitor culture morphology regularly using microscopy

• Consider using fed-batch cultivation instead of continuous cultivation if pseudohyphae formation persists

• Evaluate the effect of PDI co-expression, which may impact maintenance metabolism and potentially influence pseudohyphae formation

Be aware that once pseudohyphae growth begins, it may be irreversible and can persist even at increased dilution rates, as it represents a heritable phenotype .

What analytical methods are recommended for assessing the purity and structural integrity of recombinant P. luminescens gpmA?

To thoroughly characterize the recombinant gpmA enzyme, a multi-method approach is recommended:

• Purity assessment:

  • SDS-PAGE with densitometry (>95% purity desired for kinetic studies)

  • Size-exclusion chromatography (SEC)

  • Mass spectrometry to confirm molecular weight

• Structural characterization:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure

  • Differential scanning fluorimetry (DSF) for thermal stability assessment

  • Limited proteolysis to assess domain organization

• Activity confirmation:

  • Enzymatic assay measuring conversion of 3-phosphoglycerate to 2-phosphoglycerate

  • Kinetic parameters determination (Km, kcat, kcat/Km)

The methodology should include multiple orthogonal techniques to provide comprehensive characterization of the enzyme's properties before proceeding to detailed functional studies.

What are the expected kinetic parameters of P. luminescens gpmA and how do they compare to homologs from other bacteria?

While specific kinetic parameters for P. luminescens gpmA are not provided in the search results, researchers should expect to determine:

The methodology should include coupled enzyme assays that can monitor the production of 2-phosphoglycerate, potentially linking to enolase and pyruvate kinase reactions with spectrophotometric detection of NADH oxidation.

How does gpmA activity correlate with the phenotypic switching observed in P. luminescens?

P. luminescens undergoes phenotypic switching between primary (1°) cells that maintain symbiosis with nematodes and secondary (2°) cells that cannot reassociate with nematodes but may interact with plant roots in the rhizosphere . Research methodologies to investigate gpmA's role in this switching could include:

• Comparative expression analysis of gpmA in 1° and 2° phenotypic variants using RT-qPCR

• Creation of deletion mutants through in-frame deletion via double homologous recombination similar to methods used for other P. luminescens genes

• Metabolic flux analysis to determine differences in glycolytic flux between phenotypic variants

• Complementation studies with controlled expression of gpmA to assess rescue of potential metabolic defects

The question addresses whether changes in central carbon metabolism, particularly at the phosphoglycerate mutase step, might contribute to the phenotypic differences observed in the bacterial lifecycle.

What role might gpmA play in the insect pathogenicity of P. luminescens?

P. luminescens is an entomopathogenic bacterium , and central metabolism enzymes like gpmA may contribute to its virulence. To investigate this relationship, researchers could:

• Generate gpmA knockout mutants through methods similar to those described for sdiA gene deletion

• Compare virulence of wild-type and ΔgpmA strains in insect models

• Conduct transcriptomic analysis to identify potential regulatory connections between gpmA and known virulence factors

• Perform metabolomic studies to identify changes in metabolite profiles that might affect virulence factor production

This research question explores the potential dual role of metabolic enzymes as both housekeeping proteins and contributors to pathogenicity, a concept increasingly recognized in bacterial pathogenesis research.

How does environmental stress affect the expression and activity of gpmA in P. luminescens?

During its complex lifecycle, P. luminescens encounters various environmental stresses including oxidative stress in the insect hemolymph and nutrient limitation. To investigate the relationship between stress and gpmA function:

• Expose P. luminescens cultures to different stressors (oxidative, nutrient limitation, temperature shifts)

• Quantify gpmA expression changes using RT-qPCR or RNA-seq

• Measure enzyme activity in cell extracts under varied stress conditions

• Analyze promoter elements for stress-responsive transcription factor binding sites

This research would provide insights into how central metabolism adapts during the different ecological niches that P. luminescens occupies throughout its lifecycle.

What approaches are recommended for resolving the crystal structure of P. luminescens gpmA?

For structural studies of P. luminescens gpmA, researchers should consider:

• Protein purification optimization:

  • Multi-step chromatography (affinity, ion exchange, size exclusion)

  • Assess protein homogeneity by dynamic light scattering (DLS)

  • Screen buffer conditions using differential scanning fluorimetry

• Crystallization screening:

  • Systematic screening of crystallization conditions (pH, precipitants, additives)

  • Co-crystallization with substrates, cofactors, or inhibitors

  • Seeding techniques for crystal optimization

• Structure determination:

  • X-ray diffraction at synchrotron radiation facilities

  • Molecular replacement using homologous structures as search models

  • Model building and refinement with contemporary software packages

The methodology requires iterative optimization at each step, particularly during crystallization condition screening, which often requires hundreds of different conditions to identify initial crystal hits.

What structural features might distinguish P. luminescens gpmA from other characterized phosphoglycerate mutases?

While specific structural information for P. luminescens gpmA is not provided in the search results, comparative structural analysis with homologous enzymes might reveal:

• Unique active site residues that may affect substrate specificity or catalytic efficiency

• Differences in oligomeric state (many phosphoglycerate mutases function as dimers or tetramers)

• Species-specific surface features that might enable protein-protein interactions relevant to P. luminescens metabolism

• Structural adaptations related to the bacterium's entomopathogenic lifestyle and ecological niche

Methodology would involve homology modeling based on known phosphoglycerate mutase structures, followed by molecular dynamics simulations to assess functional implications of structural differences.

How can genome-scale metabolic modeling be used to understand the role of gpmA in P. luminescens metabolism?

To explore the systemic role of gpmA in P. luminescens metabolism, researchers can employ genome-scale metabolic modeling:

• Model construction:

  • Develop a genome-scale metabolic model based on the annotated genome of P. luminescens subsp. laumondii

  • Include reaction stoichiometry, directionality, and gene-protein-reaction associations

  • Incorporate gpmA-catalyzed reactions with associated kinetic parameters

• Flux analysis:

  • Perform flux balance analysis (FBA) to predict optimal flux distributions

  • Compare flux through gpmA-catalyzed reactions under different growth conditions

  • Simulate gpmA knockouts to predict systemic metabolic effects

• Integration with experimental data:

  • Validate model predictions with experimental metabolomics data

  • Refine the model based on experimental observations

  • Use the model to guide experimental design for further investigations

This systems biology approach provides a computational framework to understand how changes in gpmA activity might propagate through the metabolic network of P. luminescens.

What transcriptomic approaches would best reveal the regulatory networks involving gpmA in P. luminescens?

To understand the regulatory context of gpmA in P. luminescens, researchers should consider:

• RNA-sequencing analysis:

  • Compare transcriptomes of wild-type and gpmA mutant strains

  • Analyze expression patterns across different growth phases and conditions

  • Identify co-expressed genes that may be functionally related

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Identify transcription factors that bind to the gpmA promoter region

  • Map the complete regulon of identified transcription factors

  • Characterize regulatory networks involving gpmA

• Data integration approaches:

  • Combine transcriptomic data with metabolomic profiles

  • Construct gene regulatory networks using machine learning approaches

  • Validate key regulatory interactions with reporter gene assays

This methodology would reveal how gpmA expression is coordinated with other genes during different phases of P. luminescens lifecycle, including during symbiosis with nematodes and pathogenicity against insects .

How might understanding the structure-function relationship of P. luminescens gpmA contribute to biotechnological applications?

Detailed understanding of P. luminescens gpmA structure and function could enable several biotechnological applications:

• Enzyme engineering:

  • Design of gpmA variants with enhanced catalytic efficiency or stability

  • Development of biosensors for metabolic intermediates

  • Creation of novel biocatalysts for industrial processes

• Metabolic engineering:

  • Optimization of glycolytic flux in industrial microorganisms

  • Enhancement of heterologous protein production through metabolic balancing

  • Development of synthetic biology tools based on P. luminescens metabolic components

• Therapeutic applications:

  • Identification of inhibitors targeting pathogen-specific gpmA features

  • Design of antimicrobial compounds based on structural differences between bacterial and mammalian phosphoglycerate mutases

The methodology would involve iterative cycles of computational design, site-directed mutagenesis, and functional characterization to develop enzymes with desired properties.

What are the challenges and solutions for studying the in vivo dynamics of gpmA activity in P. luminescens during its lifecycle?

Investigating the in vivo dynamics of gpmA presents several challenges due to the complex lifecycle of P. luminescens involving both symbiotic and pathogenic stages . Methodological approaches to address these challenges include:

This research question addresses the technical difficulties in studying enzyme dynamics within a bacterium that transitions between different ecological niches and phenotypic states, requiring innovative experimental approaches.