Recombinant Bartonella quintana Translation initiation factor IF-2 (infB), partial

What is Translation Initiation Factor IF-2 and what is its role in Bartonella quintana?

Translation Initiation Factor IF-2 is a GTPase protein essential for bacterial protein synthesis. In Bartonella quintana, IF-2 serves multiple critical functions: promoting ribosomal subunit association, recruiting and binding formylmethionyl-transfer RNA (fMet-tRNA) to the ribosomal P-site, and facilitating initiation dipeptide formation. Unlike eukaryotic translation initiation systems which employ 13 factors, bacterial translation relies on only three initiation factors, with IF-2 combining several activities that are distributed across multiple factors in higher organisms . The IF-2 protein plays a crucial role in B. quintana survival during host-vector transitions, as protein synthesis regulation is vital for adapting to the different environmental conditions.

How do the forms of IF-2 encoded by the infB gene differ structurally?

The infB gene in bacteria codes for two distinct forms of translation initiation factor IF-2: IF-2 alpha (97,300 daltons) and IF-2 beta (79,700 daltons). These forms differ specifically at their N-terminal regions, as established through Edman degradation sequencing. The N-terminal amino acid sequences of these variants match perfectly with DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 base pairs downstream . The production of these two forms results from independent translation events rather than from proteolytic cleavage of a single precursor. This has been experimentally verified through gene fusion studies where the proximal half of infB was fused with the lacZ gene, resulting in the expression of two distinct fusion proteins corresponding to IF-2 alpha-beta-galactosidase and IF-2 beta-beta-galactosidase .

What are the key structural domains of bacterial IF-2 and their functions?

Bacterial IF-2 contains several functional domains with specific roles in translation initiation:

The G2 domain binds nucleotides (GTP or GDP) and contains the GTPase activity essential for IF-2 function. NMR studies have revealed that this domain undergoes significant structural rearrangements upon nucleotide binding . The C2 domain is responsible for binding fMet-tRNA. Interestingly, the C1 and C2 modules demonstrate completely independent mobility, indicating that the bacterial interdomain connector lacks the rigidity found in archaeal IF-2 homologs. This suggests that structural signals from the G2 domain upon GTP hydrolysis are unlikely to be mechanically forwarded to the fMet-tRNA binding domain .

What methodologies are most effective for expressing and purifying recombinant B. quintana IF-2?

Effective expression and purification of recombinant B. quintana IF-2 requires specialized techniques due to the protein's size and structural complexity. The recommended methodology includes:

• Gene cloning: PCR amplification of the infB gene from B. quintana genomic DNA using specifically designed primers with appropriate restriction enzyme sites. For complete functional analysis, it's advisable to clone both the full-length gene and individual domains.

• Vector selection: Utilizing the pET-28a(+) vector system for bacterial expression with an N-terminal 6×His tag facilitates subsequent purification . For the study of specific interactions, alternative tagging systems such as the maltose binding protein (MBP) fusion strategy may be employed.

• Expression conditions: Expression in Escherichia coli BL21(DE3) at reduced temperatures (16-20°C) after IPTG induction minimizes inclusion body formation. For B. quintana proteins, consider the organism's natural growth temperature range (28-37°C) when optimizing expression conditions .

• Purification protocol: A two-step purification approach is recommended:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Size exclusion chromatography to remove aggregates and obtain highly pure protein

The success of this methodology has been demonstrated in structural studies of bacterial IF-2, yielding protein preparations suitable for NMR analysis and functional assays .

How can researchers study the GTPase activity of recombinant B. quintana IF-2?

Studying the GTPase activity of recombinant B. quintana IF-2 requires multiple complementary approaches:

• Spectrophotometric GTPase assays: The release of inorganic phosphate can be measured using malachite green or MESG (2-amino-6-mercapto-7-methylpurine riboside) coupled with purine nucleoside phosphorylase. These assays allow for real-time monitoring of GTP hydrolysis.

• Structural analysis during GTP hydrolysis: NMR spectroscopy has proven valuable for analyzing structural changes in IF-2 domains upon nucleotide binding. Studies with isolated domains (particularly the G2 domain) reveal that GDP binding induces significant structural rearrangements in the G2 subdomain . These conformational changes may be critical for IF-2 function during translation initiation.

• Ribosome-stimulated GTPase assays: Since IF-2's GTPase activity is enhanced in the presence of ribosomes, assays incorporating purified 50S ribosomal subunits more accurately reflect the physiological activity. Evidence indicates that the isolated G2 domain of B. stearothermophilus IF-2 (highly similar to B. quintana) can bind the 50S ribosomal subunit and hydrolyze GTP .

• Mutagenesis studies: Site-directed mutagenesis of key residues in the G2 domain allows for correlation between structural features and enzymatic activity. Mutations in the conserved GTP-binding motifs would be particularly informative for understanding the catalytic mechanism.

What impact does temperature have on B. quintana IF-2 function and how can this be studied experimentally?

Temperature effects on B. quintana IF-2 function are particularly relevant given the pathogen's lifecycle between human hosts (37°C) and body louse vectors (28°C). Experimental approaches to study temperature-dependent effects include:

What techniques can be used to study IF-2 interactions with ribosomes and fMet-tRNA?

Investigating IF-2 interactions with ribosomes and fMet-tRNA requires sophisticated methodologies:

• Cryo-electron microscopy (cryo-EM): This technique provides high-resolution structural information about IF-2 in complex with ribosomes and fMet-tRNA. Sample preparation involves:

  • Purification of B. quintana ribosomes using sucrose gradient ultracentrifugation

  • Formation of initiation complexes with recombinant IF-2 and fMet-tRNA

  • Vitrification and imaging with transmission electron microscopy

• Fluorescence-based binding assays: Using fluorescently labeled components:

  • fMet-tRNA labeled with fluorophores like Cy3

  • FRET pairs to measure distances between IF-2 domains and tRNA

  • Fluorescence anisotropy to quantify binding affinities

• Biochemical crosslinking coupled with mass spectrometry: This approach identifies specific contact points between IF-2, ribosomes, and fMet-tRNA:

  • UV-induced or chemical crosslinking of complexes

  • Enzymatic digestion of crosslinked samples

  • Mass spectrometric analysis to identify crosslinked peptides

• NMR spectroscopy: While challenging for full complexes, NMR has successfully revealed structural dynamics of individual IF-2 domains. Studies show that the G2 domain undergoes significant conformational changes upon GDP binding, but these changes are not mechanically transmitted to the fMet-tRNA binding C2 domain due to the flexibility of the connecting regions .

How can researchers design experiments to compare the functions of IF-2 alpha and IF-2 beta forms?

A comprehensive experimental design to compare IF-2 alpha and IF-2 beta functions should include:

• Differential expression analysis:

  • Construct separate expression vectors for IF-2 alpha and IF-2 beta

  • Create precise deletions of the 5'-non-translated region to selectively express each form

  • Quantify expression levels under various conditions using western blotting with form-specific antibodies

• In vitro translation assays:

  • Reconstitute translation initiation complexes with purified components

  • Compare the efficiency of 30S binding, 50S joining, and dipeptide formation

  • Measure fMet-tRNA binding kinetics using filter binding assays

  • Assess GTPase activity using the methodologies described earlier

• Structure-function analysis:

  • Perform domain swapping between alpha and beta forms

  • Create chimeric proteins with fluorescent tags for localization studies

  • Use the dipeptide synthesis assay containing fMet-tRNA and labeled aminoacyl-tRNAs to measure initiation activity

• Complementation studies:

  • Generate B. quintana strains with selective expression of either IF-2 alpha or IF-2 beta

  • Evaluate growth characteristics under various conditions (temperature, hemin concentration)

  • Assess ribosome profiles to determine translation efficiency

What approaches can be used to investigate the relationship between GTP hydrolysis and fMet-tRNA positioning by IF-2?

The relationship between GTP hydrolysis and fMet-tRNA positioning remains a central question in translation initiation. To investigate this relationship:

• GTPase-deficient mutants:

  • Generate site-directed mutations in the G2 domain that reduce or eliminate GTPase activity

  • Assess the ability of these mutants to position fMet-tRNA correctly

  • Compare the structural dynamics of wild-type and mutant IF-2 using NMR spectroscopy

• Domain flexibility analysis:

  • Use NMR relaxation measurements to quantify the mobility of different IF-2 domains

  • Compare the flexibility of interdomain connectors in bacterial IF-2 with archaeal homologs

  • Current evidence suggests that bacterial IF-2 domains show independent mobility, indicating that GTP hydrolysis signals may not be mechanically transmitted to the fMet-tRNA binding domain

• Time-resolved structural studies:

  • Employ time-resolved cryo-EM to capture structural intermediates during GTP hydrolysis

  • Use rapid-mixing techniques coupled with chemical footprinting to identify conformational changes

  • Correlate structural rearrangements with functional outcomes in translation initiation

• Single-molecule studies:

  • Develop fluorescence-based assays to monitor IF-2 structural dynamics and fMet-tRNA positioning simultaneously

  • Use optical tweezers or other single-molecule techniques to measure forces generated during translation initiation

How should researchers interpret conflicting data on IF-2 structural dynamics?

When faced with conflicting data on IF-2 structural dynamics, researchers should:

• Consider methodological differences:

  • Different structural techniques (X-ray crystallography, NMR, cryo-EM) may capture different conformational states

  • Solution conditions (pH, salt, temperature) can significantly affect protein dynamics

  • The study of isolated domains versus full-length protein may yield different results

• Evaluate the experimental context:

  • Free IF-2 versus ribosome-bound IF-2 may exhibit different behaviors

  • The presence of nucleotides (GTP, GDP, non-hydrolyzable analogs) alters conformational states

  • Current research indicates that GDP-induced rearrangements in the G2 domain are not transmitted to the fMet-tRNA binding C2 subdomain, suggesting functional independence

• Apply integrative structural biology approaches:

  • Combine multiple techniques to build a comprehensive model

  • Use molecular dynamics simulations to bridge experimental data

  • Develop testable hypotheses to resolve contradictions

• Consider species-specific differences:

  • Compare B. quintana IF-2 with well-studied homologs from E. coli or B. stearothermophilus

  • Note that bacterial IF-2 shows functional differences from archaeal and eukaryotic homologs

What are common challenges in working with recombinant B. quintana proteins and how can they be addressed?

Research with recombinant B. quintana proteins presents several challenges:

• Growth conditions and cultivation:

  • B. quintana is fastidious and slow-growing, requiring specialized media and growth conditions

  • For isolation, use confluent shell vials inoculated with blood or tissue samples, centrifuged at 700 × g for 1 h at 22°C, then incubated at 37°C under 5% CO₂

  • Detection can be performed using immunofluorescence with anti-Bartonella antibodies

• Protein expression optimization:

  • Codon optimization for expression hosts is crucial due to GC content differences

  • Lower expression temperatures (16-20°C) often improve solubility

  • Consider fusion partners like MBP that enhance solubility

• Hemin requirements and toxicity:

  • B. quintana has an extraordinarily high hemin requirement compared to other bacterial pathogens

  • When working with hemin-binding proteins, carefully optimize hemin concentrations to balance requirement versus toxicity

  • The use of hemin-agarose affinity chromatography may be beneficial for purifying hemin-binding proteins

• Functional assays:

  • Develop assays that function at both host (37°C) and vector (28°C) temperatures

  • Consider the impact of hemin concentration on protein function and stability

  • When studying adaptations between environments, recreate relevant conditions (temperature, pH, hemin concentration)

What novel approaches could advance our understanding of B. quintana IF-2 in pathogenesis?

Several innovative approaches could significantly advance our understanding of B. quintana IF-2's role in pathogenesis:

• CRISPR interference systems:

  • Develop inducible CRISPRi systems to modulate IF-2 expression levels in vivo

  • Create partial knockdowns to study dose-dependent effects on bacterial survival

  • Target specific IF-2 domains to disrupt selected functions while preserving others

• Host-pathogen interaction models:

  • Develop cell culture systems that mimic both human host and louse vector environments

  • Create microfluidic devices that allow for rapid environmental transitions

  • Use transcriptomics and proteomics to identify IF-2-dependent changes during host-vector transitions

• Structural biology approaches:

  • Apply hydrogen-deuterium exchange mass spectrometry to map dynamic regions of IF-2

  • Utilize AlphaFold2 or similar AI-based structural prediction tools to model full-length IF-2

  • Develop nanobodies or other crystallization chaperones to facilitate structural studies

• Systems biology integration:

  • Combine translation efficiency measurements, ribosome profiling, and proteomics

  • Develop mathematical models of translation initiation incorporating IF-2 dynamics

  • Study the broader impact of translation regulation on B. quintana adaptation

How might research on B. quintana IF-2 contribute to understanding bacterial adaptation mechanisms?

Research on B. quintana IF-2 offers several opportunities to enhance our understanding of bacterial adaptation mechanisms:

• Translation regulation as an adaptive strategy:

  • Explore how modulation of translation initiation contributes to rapid adaptation

  • Compare translation efficiency at different temperatures and hemin concentrations

  • Investigate how IF-2 variants might optimize protein synthesis under specific conditions

• Comparative analysis across vectors and hosts:

  • Extend studies to other vector-borne pathogens with similar transmission cycles

  • Compare translation initiation factors across Bartonella species with different host ranges

  • Identify conserved and divergent features of translation regulation during host switching

• Integration with stress response pathways:

  • Investigate potential crosstalk between IF-2 and the RpoE-NepR-PhyR regulatory system

  • Determine how translation initiation responds to environmental stresses

  • Explore potential coordinated regulation of hemin-binding proteins and translation factors

• Therapeutic targeting potential:

  • Evaluate IF-2 as a potential target for novel antimicrobials

  • Explore whether disrupting environment-specific translation regulation could reduce bacterial adaptation

  • Develop small-molecule inhibitors specific to bacterial IF-2 forms not found in eukaryotes