Recombinant Xylella fastidiosa GTPase Era (era)

What is the basic function of GTPase Era in Xylella fastidiosa?

GTPase Era is a highly conserved bacterial protein that plays a significant role in ribosome biogenesis, particularly in 16S rRNA processing and 70S ribosome assembly. Based on studies in related bacterial systems, Era likely binds to the 30S ribosomal subunit and participates in the maturation of 16S rRNA . Its GTPase activity is essential for proper ribosome assembly and function, making it a critical factor in bacterial growth and survival .

How does Era GTPase differ structurally and functionally from other bacterial GTPases?

Era GTPase belongs to the Era/Obg family of bacterial GTPases but has distinct structural features compared to other GTPases like Der. While both Era and Der contain GTP-binding domains, Era typically has a KH (K homology) RNA-binding domain in its C-terminus that allows it to specifically interact with 16S rRNA . This specialized domain organization facilitates Era's role in ribosome assembly. In contrast, Der (as seen in the product sheet) has a different domain architecture that supports its distinct cellular functions .

What conservation patterns are observed in Era GTPase across different X. fastidiosa subspecies?

Analysis of genomic data from the 72 X. fastidiosa genomes suggests that core genome genes like Era GTPase are typically highly conserved across subspecies including X. fastidiosa subsp. fastidiosa, multiplex, and pauca . The core genome evolves at an estimated rate of 7.62 × 10^-7 substitutions per site per year within the X. fastidiosa subsp. pauca subclade . While subspecies-specific variations may exist, the functional domains of Era are likely preserved due to their essential role in bacterial viability.

What are the optimal expression systems for producing recombinant X. fastidiosa GTPase Era?

For recombinant expression of X. fastidiosa GTPase Era, E. coli-based expression systems (particularly BL21(DE3) or Rosetta strains) are recommended due to their efficiency in producing bacterial proteins. Expression should be conducted at lower temperatures (16-20°C) after IPTG induction to ensure proper protein folding. Based on similar GTPase purification protocols, using a construct with an N-terminal His-tag allows for efficient purification while minimizing interference with the C-terminal RNA-binding domain function .

What purification strategy yields the highest activity for X. fastidiosa GTPase Era?

A multi-step purification protocol is recommended:

• Initial capture using Ni-NTA affinity chromatography with imidazole gradient elution

• Intermediate purification via ion exchange chromatography (typically Q-Sepharose)

• Final polishing step using size exclusion chromatography (Superdex 75/200)

This approach typically yields >90% pure protein compared to the >85% purity reported for similar GTPases . Buffer optimization is critical, with typical final storage conditions being 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 10% glycerol to maintain GTPase activity.

How can researchers verify the functional activity of purified recombinant Era GTPase?

Functional verification should include:

These assays collectively confirm both catalytic function and RNA-binding capabilities essential for biological activity .

How can Era GTPase overexpression be used to study ribosome assembly defects in X. fastidiosa?

Building on knowledge from E. coli studies, Era GTPase overexpression can serve as a valuable tool to investigate ribosome assembly pathways in X. fastidiosa. Researchers can generate X. fastidiosa strains with controlled Era expression and analyze impacts on 16S rRNA processing using Northern blotting and ribosome profile analysis. Studies in E. coli have shown that elevated levels of Era GTPase can improve 16S rRNA processing and 70S ribosome assembly in strains lacking YbeY endoribonuclease . Similar approaches in X. fastidiosa could reveal subspecies-specific aspects of ribosome biogenesis and potentially identify novel intervention points for controlling this plant pathogen.

What role might Era GTPase play in stress response and virulence of X. fastidiosa?

Era GTPase likely functions as a regulatory checkpoint connecting ribosome biogenesis to stress response in X. fastidiosa. Research approaches to investigate this connection should include:

• Stress exposure experiments (temperature, nutrient limitation, oxidative stress) with Era expression monitoring

• Virulence assays in planta comparing wild-type and Era-modified strains

• Transcriptome analysis of Era-overexpressing strains to identify affected pathways

Given X. fastidiosa's status as an economically important plant pathogen affecting various crops , understanding Era's role in stress adaptation could provide insights into bacterial persistence mechanisms during plant infection.

How does recombination across X. fastidiosa subspecies affect Era GTPase function?

Genomic studies have demonstrated that recombination is a major driver of diversity in X. fastidiosa, with nucleotides three times more likely to change due to recombination than point mutation . While Era likely remains functionally conserved due to its essential role, researchers should investigate whether subspecies-specific variations exist in regulatory regions or protein interaction domains. Comparative genomic analysis across the 72 genome dataset referenced in the literature could identify potential recombination events affecting Era regulation or function that might contribute to adaptation to different plant hosts .

What controls are necessary when studying Era GTPase mutants in X. fastidiosa?

When designing Era GTPase mutant studies, researchers should implement these essential controls:

These controls help distinguish Era-specific effects from experimental artifacts and provide mechanistic insights into observed phenotypes .

How can researchers efficiently generate site-directed mutations in X. fastidiosa Era GTPase for structure-function studies?

For comprehensive structure-function analysis of Era GTPase in X. fastidiosa, researchers should target conserved motifs identified through sequence alignment with characterized Era proteins. Key targets include:

• G1/P-loop motif (GxxxxGKT/S) – essential for GTP binding

• G3 motif (DxxG) – involved in GTP hydrolysis

• G4 motif (NKxD) – contributes to guanine specificity

• KH domain residues – critical for RNA recognition

Gibson Assembly or Q5 site-directed mutagenesis provides efficient generation of these mutations. Each mutant should be characterized for both GTPase activity and RNA binding to establish comprehensive structure-function relationships .

What challenges exist in studying Era-protein interactions in X. fastidiosa?

Investigating Era-protein interactions in X. fastidiosa presents unique challenges due to:

• Limited transformation efficiency of X. fastidiosa

• Slow growth rate complicating protein expression studies

• Potential toxicity of modified Era expression

• Subspecies-specific protein interaction networks

Researchers should employ complementary approaches including bacterial two-hybrid screening, co-immunoprecipitation followed by mass spectrometry, and in vitro reconstitution experiments with purified components to build a comprehensive interaction map.

How should researchers interpret conflicting data regarding Era GTPase function across different bacterial species?

When encountering conflicting reports on Era function between X. fastidiosa and model organisms like E. coli, researchers should:

• Examine methodology differences (in vivo vs. in vitro approaches)

• Consider evolutionary divergence between species (X. fastidiosa vs. E. coli)

• Evaluate experimental conditions (growth media, temperature, stress conditions)

• Analyze genetic background differences (presence/absence of complementary pathways)

For example, Era overexpression in E. coli partially suppresses defects in strains lacking YbeY endoribonuclease , but this relationship may differ in X. fastidiosa due to subspecies-specific genome content and selective pressures .

What bioinformatic approaches best predict Era GTPase substrate specificity in X. fastidiosa?

To predict Era GTPase substrate specificity in X. fastidiosa, researchers should employ a multi-faceted bioinformatic workflow:

• Structural homology modeling based on crystallized Era proteins

• RNA-binding site prediction focusing on the KH domain

• Molecular docking simulations with 16S rRNA fragments

• Conservation analysis across 72 X. fastidiosa genomes

• Comparative analysis with Era proteins from other bacterial species

This integrated approach can identify X. fastidiosa-specific interaction motifs and guide experimental validation of predicted Era-RNA interactions.

How can evolutionary rate analysis of Era GTPase inform understanding of X. fastidiosa subspecies divergence?

Evolutionary rate analysis of Era GTPase can provide insights into X. fastidiosa subspecies divergence, building on the established evolutionary rate of 7.62 × 10^-7 substitutions per site per year for core genome genes . Researchers should:

• Compare synonymous vs. non-synonymous substitution rates in Era across subspecies

• Identify potential signatures of selection using methods like PAML

• Map subspecies-specific variations onto structural models to predict functional impacts

• Correlate Era sequence changes with host-specificity patterns