Recombinant Bartonella quintana Phosphoribosylformylglycinamidine synthase 1 (purQ)

What is Phosphoribosylformylglycinamidine synthase 1 (purQ) in Bartonella quintana?

Phosphoribosylformylglycinamidine synthase subunit PurQ in Bartonella quintana is a 227-amino acid protein (identified by accession number Q6FZJ0) that functions as part of the purine biosynthesis pathway . It is encoded by the purQ gene in the B. quintana genome and represents one of the essential components in the de novo purine nucleotide synthesis machinery. The protein likely participates in the conversion of formylglycinamide ribonucleotide (FGAR) to formylglycinamidine ribonucleotide (FGAM), which is a critical step in purine biosynthesis.

How does B. quintana PurQ differ from similar proteins in other bacterial species?

While the core functional domains of PurQ are conserved across bacterial species due to the essential nature of purine biosynthesis, B. quintana PurQ has evolved specific adaptations that may reflect the organism's unique lifecycle. B. quintana transitions between the hemin-restricted human bloodstream and the hemin-rich body louse vector, requiring specialized metabolic adaptations . The specific differences may include variations in amino acid sequence, protein structure, and regulatory mechanisms that allow B. quintana to optimize purine biosynthesis in its distinctive dual-host environment.

What is the genomic context of purQ in the Bartonella quintana genome?

In B. quintana (strain Toulouse), the purQ gene is found in proximity to other purine biosynthesis genes, suggesting an organized functional arrangement. The genome analysis indicates that purQ (coding for Q6FZJ0) is positioned near purL (coding for Q6FZI9, Phosphoribosylformylglycinamidine synthase subunit PurL) . This genomic organization likely enables coordinated expression of functionally related genes involved in the same biosynthetic pathway. The arrangement reflects the common prokaryotic strategy of organizing related metabolic genes in operons or gene clusters for efficient transcriptional regulation.

What are the optimal conditions for recombinant expression of B. quintana PurQ?

For recombinant expression of B. quintana PurQ, a prokaryotic expression system using E. coli is typically preferred. Based on successful approaches with similar proteins, the gene can be PCR-amplified from B. quintana genomic DNA using specific primers targeting the purQ gene sequence. The amplified gene can then be ligated into an expression vector such as pET-28a(+) to generate a recombinant protein with an N-terminal 6×His tag for purification purposes . Expression should be optimized at temperatures between 18-30°C, with IPTG induction at concentrations of 0.1-1.0 mM, with lower temperatures often yielding better soluble protein. Purification via nickel affinity chromatography followed by size exclusion chromatography typically produces the purest protein preparations.

How can researchers quantify expression levels of native purQ in B. quintana under different environmental conditions?

To quantify the expression levels of native purQ in B. quintana under varying environmental conditions, reverse transcriptase quantitative PCR (RT-qPCR) is the method of choice. Following the protocol described for other B. quintana genes, researchers should isolate total RNA from bacteria cultured under different conditions (such as varying temperature, hemin concentration, or pH), prepare cDNA through reverse transcription, and perform qPCR using specific primers for the purQ gene . The reaction mixture would typically include SYBR Fast qPCR master mix, ROX low, and purQ-specific primers at appropriate concentrations. Expression levels should be normalized to stable reference genes such as 16S rRNA or housekeeping genes to ensure accurate comparisons across different conditions.

What are the challenges in purifying active recombinant B. quintana PurQ?

Purifying active recombinant B. quintana PurQ presents several challenges. First, PurQ typically functions as part of a multi-subunit enzyme complex with PurL and potentially other proteins, making isolation of functionally active PurQ alone difficult. Second, maintaining protein solubility during expression and purification can be problematic, often requiring optimization of buffer conditions (pH 7.0-8.0, 100-300 mM NaCl, with potential additives such as glycerol or mild detergents). Third, ensuring proper folding of the recombinant protein may require co-expression with molecular chaperones or expression at lower temperatures. Finally, assessing enzymatic activity requires either reconstitution of the complete enzyme complex or development of specialized assays that can detect PurQ's glutaminase activity independent of the full complex.

How does the structure of B. quintana PurQ influence its function in the FGAR amidotransferase complex?

The structure of B. quintana PurQ is likely organized into domains that facilitate its specific function within the FGAR amidotransferase complex. While no crystal structure is currently available specifically for B. quintana PurQ , structural predictions based on homologous proteins suggest it contains a glutaminase domain responsible for hydrolyzing glutamine to produce ammonia. This ammonia is then channeled to the PurL subunit where it's incorporated into FGAR to form FGAM. The protein-protein interaction interfaces between PurQ and PurL are critical for this ammonia channeling mechanism, which prevents the premature release of reactive ammonia intermediates. The structural arrangement likely includes conserved catalytic residues forming the glutamine binding pocket and hydrolysis center, as well as interface residues that mediate precise docking with PurL to ensure efficient substrate channeling.

How do mutations in purQ affect B. quintana virulence and metabolism?

Mutations in purQ would likely have significant impacts on B. quintana virulence and metabolism, particularly under conditions where de novo purine synthesis is essential. Loss-of-function mutations would likely create purine auxotrophy, rendering the bacteria dependent on environmental purine sources. This would severely compromise bacterial survival in purine-limited niches within the host. Specific point mutations affecting catalytic activity or protein-protein interactions with PurL could result in reduced efficiency of purine biosynthesis without complete elimination, potentially creating metabolic bottlenecks that slow growth and reduce virulence. Additionally, mutations affecting regulation of purQ expression could disrupt the bacteria's ability to adapt to changing environments during host transition. The relationship between purQ function and virulence underscores the potential of targeting purine biosynthesis for antimicrobial development against B. quintana infections.

What techniques are most effective for studying the interaction between PurQ and other proteins in the purine biosynthesis pathway?

Several techniques are particularly effective for studying interactions between PurQ and other proteins in the purine biosynthesis pathway. Bacterial two-hybrid systems can identify binary interactions between PurQ and potential partner proteins. Co-immunoprecipitation using anti-PurQ antibodies followed by mass spectrometry can identify proteins that physically associate with PurQ in vivo. Surface plasmon resonance or isothermal titration calorimetry provides quantitative binding parameters such as association/dissociation constants between purified PurQ and partner proteins. For structural studies of complexes, cryo-electron microscopy is increasingly valuable for visualizing multi-protein assemblies without crystallization. Additionally, protein crosslinking coupled with mass spectrometry can map specific interaction interfaces. FRET (Förster Resonance Energy Transfer) analysis using fluorescently labeled proteins can confirm interactions in live bacteria and provide spatial information. These complementary approaches together provide a comprehensive picture of PurQ's interaction network within the purine biosynthesis machinery.

How can researchers effectively design mutagenesis studies to identify critical residues in B. quintana PurQ?

Effective mutagenesis studies for identifying critical residues in B. quintana PurQ should begin with computational analysis to predict potentially important amino acids based on sequence conservation across species, structural modeling, and identification of putative catalytic or binding sites. Site-directed mutagenesis should target these candidate residues, particularly focusing on predicted catalytic residues, substrate binding sites, and residues at the interface with PurL. Alanine scanning mutagenesis, where selected residues are systematically replaced with alanine, can identify amino acids essential for function. Conservative and non-conservative substitutions can provide insights into the specific chemical properties required at each position. Each mutant should be characterized for protein stability, solubility, complex formation with PurL, and enzymatic activity. Complementation studies in purQ knockout strains can assess the functional consequences of mutations in vivo. This systematic approach will map the relationship between PurQ structure and function at the molecular level.

What are the best approaches for developing inhibitors targeting B. quintana PurQ as potential antimicrobials?

Developing inhibitors targeting B. quintana PurQ requires a multifaceted approach. Structure-based drug design, leveraging homology models or experimentally determined structures, can identify potential binding pockets for small molecule inhibitors. Virtual screening of compound libraries against these pockets can identify candidate inhibitors for experimental testing. High-throughput screening assays should be developed that measure either the glutaminase activity of PurQ or the formation of FGAM in the reconstituted enzyme complex. Fragment-based drug discovery can identify small chemical fragments that bind to PurQ, which can then be elaborated into more potent inhibitors. Lead compounds should be evaluated for their ability to inhibit purified recombinant PurQ, their effect on bacterial growth in culture, and their selectivity over human enzymes. Medicinal chemistry optimization can improve potency, selectivity, and pharmacokinetic properties. Finally, animal models of B. quintana infection would be needed to validate the efficacy of promising inhibitors in vivo.

How is purQ expression regulated in response to changing environmental conditions?

The expression of purQ in B. quintana is likely regulated in response to changing environmental conditions through multiple mechanisms. Similar to other bacteria, purine availability probably serves as a key regulatory signal, with purQ expression being repressed when purines are abundant and induced when purines are scarce. Temperature changes, which B. quintana experiences during transition between human host (37°C) and louse vector (lower temperature), may also influence purQ expression . The regulation likely involves both transcriptional control through promoter elements and regulatory proteins, and post-transcriptional mechanisms affecting mRNA stability. The B. quintana RpoE (σE) transcription factor, known to respond to environmental stresses including temperature changes, might play a role in regulating purQ expression during host adaptation . Additionally, small RNAs and riboswitches responsive to purine levels could contribute to fine-tuning purQ expression to match cellular metabolic requirements under different growth conditions.

What methods can be used to characterize the promoter region and transcriptional regulation of the purQ gene?

To characterize the promoter region and transcriptional regulation of the purQ gene, researchers should begin with in silico analysis to identify putative promoter elements, transcription factor binding sites, and regulatory motifs. Primer extension or 5' RACE (Rapid Amplification of cDNA Ends) can precisely map the transcription start site. Reporter gene assays using constructs with varying lengths of the purQ upstream region fused to a reporter gene (such as lacZ or luciferase) can define the minimal promoter region and identify important regulatory elements. Electrophoretic mobility shift assays (EMSAs) can detect protein-DNA interactions between purified transcription factors and the purQ promoter region. DNase I footprinting can precisely map the binding sites of regulatory proteins. Chromatin immunoprecipitation (ChIP) can identify proteins that bind to the purQ promoter in vivo. Finally, transcriptome analysis under various growth conditions, using RT-qPCR or RNA-seq, can reveal patterns of purQ expression and potential co-regulated genes, providing insights into the regulatory networks controlling purQ expression .

How has the purQ gene evolved in different Bartonella species, and what implications does this have for host adaptation?

The purQ gene has likely undergone adaptive evolution across different Bartonella species as they specialized for different mammalian hosts. Comparative genomic analysis would reveal sequence conservation patterns, with functional domains likely showing higher conservation while interface regions may exhibit host-specific adaptations. Phylogenetic analysis of purQ sequences from different Bartonella species would illuminate the evolutionary relationships and potential horizontal gene transfer events. Selection pressure analysis (dN/dS ratios) could identify amino acid positions under positive selection, potentially indicating residues important for host-specific adaptation. The evolution of purQ in Bartonella species probably reflects both the core metabolic requirements for purine biosynthesis and specific adaptations to different host environments. Species-specific variations might influence enzyme efficiency, regulatory responsiveness, or protein-protein interactions, all potentially contributing to host adaptation. Understanding these evolutionary patterns could provide insights into how Bartonella species have optimized their purine metabolism to thrive in diverse host environments.

How does the purine biosynthesis pathway in B. quintana compare with other bacterial pathogens, and what unique features might be exploited for targeted interventions?

The purine biosynthesis pathway in B. quintana likely shares core similarities with other bacterial pathogens due to the essential nature of purine metabolism, yet may possess unique features reflecting its specialized lifestyle. Compared to other bacterial pathogens, B. quintana has a relatively small genome (1.58 Mb) resulting from reductive evolution, which might impact pathway organization and regulation. A distinguishing feature of B. quintana is its adaptation to dramatically different environments: the hemin-restricted human bloodstream and the hemin-rich body louse vector . This transition may require unique regulatory mechanisms for purine biosynthesis genes, including purQ. Potential targets for intervention include unique structural features of the PurQ protein, distinctive regulatory mechanisms controlling purQ expression, or B. quintana-specific protein-protein interactions within the purine biosynthesis machinery. Targeting these distinctive features could provide selective inhibition of B. quintana while minimizing effects on host cells or beneficial microbiota. Comparative analysis with human purine biosynthesis enzymes would be essential to identify bacterial-specific features amenable to selective targeting.

What are the major challenges in expressing and purifying sufficient quantities of functional recombinant B. quintana PurQ for structural studies?

Expressing and purifying sufficient quantities of functional recombinant B. quintana PurQ for structural studies presents several significant challenges. First, B. quintana proteins often have codon usage patterns that differ from common expression hosts like E. coli, potentially requiring codon optimization or specialized strains. Second, PurQ may require co-expression with PurL or other interacting partners to achieve proper folding and stability. Third, the protein may form inclusion bodies when overexpressed, necessitating refolding procedures or alternative expression conditions (lower temperature, reduced inducer concentration). Fourth, PurQ might require specific cofactors or post-translational modifications for stability. Fifth, the protein may be prone to aggregation during concentration steps required for structural studies. Finally, obtaining diffraction-quality crystals for X-ray crystallography can be particularly challenging for proteins like PurQ that naturally function as part of multi-protein complexes. Alternative approaches such as cryo-electron microscopy might be more suitable for structural characterization, particularly when studying PurQ in complex with its physiological partners.

How can researchers overcome the difficulties in creating genetic modifications in B. quintana to study purQ function?

Researchers face significant challenges in creating genetic modifications in B. quintana due to its slow growth, fastidious nature, and limited genetic tools. To overcome these difficulties, several approaches can be employed. First, optimizing transformation protocols specifically for B. quintana, including electroporation parameters and recovery conditions, can improve transformation efficiency. Second, developing shuttle vectors with appropriate origins of replication and selectable markers that function effectively in B. quintana is essential. Third, implementing CRISPR-Cas9 systems adapted for B. quintana could enable precise genome editing. Fourth, conditional knockdown systems (such as CRISPRi or antisense RNA) may be more feasible than complete knockouts if purQ is essential. Fifth, complementation studies can be performed by introducing modified versions of purQ on plasmids into B. quintana strains. Sixth, heterologous expression systems can evaluate B. quintana purQ function in more genetically tractable relatives. Finally, collaborating with specialized laboratories experienced in Bartonella genetics can provide access to established protocols and resources. These combined approaches can help overcome the technical barriers to genetic manipulation of B. quintana for purQ functional studies.

What is the relationship between purQ function and B. quintana pathogenesis in human infections?

The relationship between purQ function and B. quintana pathogenesis likely centers on the bacterium's ability to sustain purine biosynthesis during infection. Purines are essential for bacterial DNA/RNA synthesis and energy metabolism, making de novo purine biosynthesis via PurQ potentially critical when B. quintana cannot scavenge sufficient purines from the host environment. During the early stages of infection, B. quintana must adapt to the hemin-restricted human bloodstream environment , where purine availability may be limited. The ability to synthesize purines through the pathway involving PurQ could provide a significant growth advantage. Additionally, changes in purQ expression may be part of the broader metabolic remodeling that occurs during host adaptation. If purQ is essential for B. quintana survival in vivo, it represents a potentially valuable target for therapeutic intervention. Understanding the relationship between purQ function and pathogenesis requires further investigation using approaches such as conditional knockdown systems or specific inhibitors targeting PurQ function, combined with infection models to assess the impact on bacterial survival and virulence.

How might targeting PurQ functionality impact B. quintana infections, and what considerations are important for developing such interventions?

Targeting PurQ functionality could significantly impact B. quintana infections by disrupting de novo purine biosynthesis, potentially leading to growth inhibition or bacterial clearance. Several considerations are important for developing such interventions. First, the essentiality of purQ in different infection microenvironments must be established, as the bacterium might be able to scavenge purines in some host compartments but require de novo synthesis in others. Second, inhibitor specificity is crucial to avoid off-target effects on human purine metabolism enzymes. Third, drug delivery considerations must account for B. quintana's intracellular phases during infection. Fourth, the potential for resistance development through mutations in purQ or upregulation of purine salvage pathways should be evaluated. Fifth, combination therapies targeting multiple steps in purine biosynthesis or different essential pathways might improve efficacy and reduce resistance. Finally, PurQ inhibitors could potentially have broad-spectrum activity against multiple Bartonella species and possibly other bacterial pathogens that rely on de novo purine biosynthesis during infection, expanding their therapeutic potential beyond B. quintana infections.