Recombinant Francisella tularensis subsp. novicida Malate dehydrogenase (mdh)

What is the genomic context of the mdh gene in Francisella tularensis subsp. novicida?

The mdh gene in F. tularensis subsp. novicida encodes malate dehydrogenase, a key enzyme in central metabolism. Like other Francisella species, F. novicida has a relatively small genome with limited regulatory systems compared to other bacteria. While F. tularensis possesses just two predicted orphan histidine kinases and two orphan response regulators, the regulation of metabolic genes like mdh is likely integrated into broader regulatory networks that respond to environmental conditions . The genomic neighborhood of mdh may contain other metabolic genes, though specific operon structures would need to be determined experimentally through methods similar to those used to confirm that FTT1236 and FTT1237 compose an operon .

How does F. novicida mdh differ structurally from other bacterial malate dehydrogenases?

While the search results don't provide specific structural information about F. novicida mdh, comparative analysis would typically involve:

• Sequence alignment with mdh from other bacterial species

• Homology modeling based on crystallized bacterial mdhs

• Analysis of active site residues and cofactor binding domains

Such analysis would require techniques similar to those used in proteomic comparisons between Francisella subspecies, as documented by Hubalek et al. (2004), who identified proteins unique to or upregulated in virulent subspecies of F. tularensis .

What expression systems are most effective for producing recombinant F. novicida mdh?

For recombinant expression of F. novicida mdh, researchers should consider:

• E. coli-based expression systems using vectors compatible with Francisella codon usage

• Native Francisella expression systems using shuttle vectors

The choice depends on research goals. For structural studies requiring high protein yields, E. coli systems may be preferable. For functional studies examining native regulation or post-translational modifications, Francisella-based expression might be more appropriate.

Several shuttle vectors have been developed for Francisella genetic manipulation, including those derived from pFNL10, pC194, and F. philomiragia plasmids pF242 and pF243 . When using non-Francisella expression systems, codon optimization may be necessary given the AT-rich genome of Francisella species.

What strategies enable successful cloning of the mdh gene from F. novicida?

F. novicida is the most genetically tractable Francisella subspecies, making it ideal for mdh cloning experiments . Researchers should consider:

• Direct PCR amplification from genomic DNA with primers containing appropriate restriction sites

• Synthetic gene synthesis based on the published F. novicida genome

• Homologous recombination approaches

An important consideration is that F. novicida contains restriction enzymes that can inhibit acquisition of foreign DNA. Gallagher et al. (2008) created strain MFN245 with four restriction enzymes disrupted, making it approximately 10,000-fold more efficient for transformation with foreign DNA . Using this strain as an intermediate host could significantly improve cloning efficiency.

For transformation into F. novicida, both chemical transformation and electroporation protocols have been established, with electroporation typically yielding higher efficiency.

How can researchers create targeted mdh mutants in F. novicida for functional studies?

Creating mdh mutants requires carefully designed strategies:

• For complete gene knockout: homologous recombination with a disruption cassette

• For point mutations: site-directed mutagenesis or allelic exchange

• For conditional expression: inducible promoter systems

F. novicida has a notable advantage over other Francisella subspecies in that it can be transformed with linear DNA fragments that integrate through homologous recombination . This property facilitates the creation of targeted mutations.

When designing experiments, researchers should consider:

For complementation studies to confirm phenotypes, shuttle vectors based on pFNL10 or other compatible plasmids can be used to reintroduce wild-type or modified mdh genes .

What purification methods yield the highest activity of recombinant F. novicida mdh?

Purification of recombinant mdh from F. novicida typically involves:

• Affinity chromatography (His-tag or other fusion tags)

• Ion exchange chromatography

• Size exclusion chromatography

Critical considerations include:

• Buffer optimization to maintain enzyme stability

• Addition of stabilizing agents (glycerol, reducing agents)

• Temperature control during purification

• Removal of bacterial endotoxins, especially important when working with Francisella-derived proteins

The purification approach should be validated by assessing enzyme activity, as improper purification can lead to inactive enzyme. Activity assays typically measure the conversion of malate to oxaloacetate by monitoring NADH production at 340 nm.

How do the kinetic properties of F. novicida mdh compare to homologs from other bacterial pathogens?

Comparative kinetic analysis should include:

• Determination of Km and Vmax for malate and NAD+

• pH and temperature optima

• Effects of potential inhibitors

• Allosteric regulation

These parameters would be evaluated through standard enzyme kinetic measurements under varying substrate concentrations, pH conditions, and temperatures. Comparing these values with mdh from other intracellular pathogens could provide insights into metabolic adaptations specific to Francisella's intracellular lifestyle.

How does mdh activity contribute to F. novicida's intracellular survival in macrophages?

The role of mdh in F. novicida's pathogenesis would require sophisticated experimental approaches:

• Creation of mdh knockdown or conditional mutants

• Intracellular growth assays in macrophages

• Metabolic profiling of wild-type versus mdh-deficient strains

• In vivo infection models

F. tularensis is known to replicate within macrophages and escape from phagosomes . Metabolic adaptations are likely crucial for this intracellular lifestyle. Studies could examine whether mdh activity changes during different stages of infection, similar to how researchers have analyzed other factors required for Francisella's lifecycle as an intracellular pathogen .

Microscopic analysis of macrophages infected with mdh mutants would reveal potential defects in intracellular replication or effects on host cell viability, similar to studies performed with mutants in FTT1236, FTT1237, and FTT1238 .

Could recombinant F. novicida mdh serve as a target for novel antimicrobial development?

Evaluating mdh as a drug target would require:

• Assessment of essentiality through gene knockout or silencing

• High-throughput screening of compound libraries against purified mdh

• Structure-based drug design using crystallographic data

• Validation in cell culture and animal models

The extreme virulence of F. tularensis and its classification as a potential bioweapon make the development of novel therapeutics particularly important . Metabolic enzymes like mdh often make attractive drug targets due to their essential nature and potential structural differences from host enzymes.

Researchers would need to demonstrate that:

• Inhibition of mdh activity correlates with reduced bacterial growth

• Potential inhibitors can access the target within infected cells

• Compounds show selectivity for bacterial versus human mdh

What role does mdh play in F. novicida's stress response and adaptation to host environments?

Investigating mdh's role in stress adaptation would involve:

• Expression analysis under various stress conditions (oxidative stress, nutrient limitation, etc.)

• Metabolic flux analysis using labeled substrates

• Comparison of wild-type and mdh mutant responses to stress

F. tularensis is known to modulate gene expression to adapt to varying conditions . The MglA/SspA regulatory system, which affects ~100 genes, could potentially influence mdh expression during infection . Additionally, the stringent response involving ppGpp signaling may impact metabolic gene regulation under stress conditions.

What crystallization conditions have proven successful for obtaining diffraction-quality crystals of F. novicida mdh?

Obtaining protein crystals suitable for X-ray diffraction typically requires:

• Highly pure (>95%) and homogeneous protein

• Screening of hundreds of crystallization conditions varying:

  • Precipitants (PEG, ammonium sulfate, etc.)

  • pH and buffer systems

  • Additives and cofactors

  • Temperature

• Optimization of promising conditions

For F. novicida mdh specifically, inclusion of the cofactor NAD+ or NADH often stabilizes the protein structure and improves crystallization. Additionally, surface entropy reduction through targeted mutagenesis of surface lysine and glutamate residues might enhance crystal packing.

How can researchers effectively apply molecular dynamics simulations to understand substrate binding in F. novicida mdh?

Molecular dynamics approaches for studying mdh would include:

• Building a homology model of F. novicida mdh based on crystallized bacterial mdhs

• Setting up simulation systems with appropriate force fields and solvent models

• Running equilibrium and substrate binding simulations

• Analyzing trajectories for binding pocket dynamics and substrate interactions

These computational studies complement experimental approaches and can provide insights into:

• Conformational changes upon substrate binding

• Identification of key residues for catalysis

• Differences between F. novicida mdh and potential drug targets

• Effects of mutations on enzyme function

Can recombinant F. novicida mdh serve as a biomarker for detecting Francisella infections?

Evaluating mdh as a biomarker would involve:

• Production of antibodies against purified recombinant mdh

• Development of immunoassays (ELISA, lateral flow, etc.)

• Testing specificity against other bacterial species

• Validation using clinical samples

The proteome of F. tularensis has been studied for its recognition by antibodies . Similar approaches could determine whether mdh is immunogenic during infection and whether anti-mdh antibodies could serve as diagnostic tools.

The comparison of proteomes between Francisella subspecies suggests that some proteins are uniquely expressed or upregulated in virulent strains . Understanding whether mdh expression differs between subspecies would be important for its potential as a biomarker.

How does mdh interact with other metabolic pathways during F. novicida infection?

Understanding mdh in the context of global metabolism requires:

• Metabolomics analysis of infected cells

• Flux balance analysis of metabolic networks

• Protein-protein interaction studies to identify potential regulatory partners

• Transcriptomics to identify co-regulated genes

F. tularensis adapts its metabolism to survive within macrophages . Studying how mdh activity coordinates with other pathways could reveal metabolic vulnerabilities that could be exploited for therapeutic intervention.

Interestingly, F. tularensis has been shown to activate the PI3K/Akt pathway during infection of macrophages . Investigating whether metabolic enzymes like mdh play a role in this process could reveal novel connections between bacterial metabolism and host cell signaling.