Recombinant Kocuria rhizophila Elongation factor Tu (tuf)

What is Elongation factor Tu (tuf) and what is its primary role in bacterial translation?

Elongation factor Tu (Ef-Tu) is one of the most abundant proteins in bacteria, functioning as an essential and universally conserved GTPase that ensures translational accuracy. It catalyzes the critical reaction that adds the correct amino acid to the growing nascent polypeptide chain during protein synthesis . After the incoming aminoacyl-tRNA docks with the mRNA, GTPase activity induces a conformational change releasing Ef-Tu from the ribosome .

The protein consists of three functional domains:

• Domain I (amino acids 1–200): Forms a helix structure with Rossmann fold topology, a structural motif found in proteins that bind nucleotides; houses the GTP/GDP binding domains

• Domain II (amino acids 209–299): Largely comprised of beta sheets

• Domain III (amino acids 301–393): Largely comprised of beta sheets

When studying this protein, researchers should consider its structural integrity across all three domains, as this is essential for its proper function in translation.

How should recombinant Kocuria rhizophila Elongation factor Tu be stored to maintain optimal activity?

For optimal stability and activity preservation of recombinant Kocuria rhizophila Elongation factor Tu:

• Short-term storage: Store at -20°C

• Extended storage: Conserve at -20°C or -80°C

• Working aliquots: Can be stored at 4°C for up to one week

• Avoid repeated freezing and thawing cycles as this significantly reduces protein activity

When reconstituting the lyophilized protein:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot for long-term storage at -20°C/-80°C

The shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form maintains stability for up to 12 months at -20°C/-80°C .

What moonlighting functions does Elongation factor Tu perform beyond its role in translation?

Elongation factor Tu exhibits remarkable moonlighting capabilities beyond its canonical role in translation:

• Surface Expression: Ef-Tu moonlights on the surface of human pathogens including Staphylococcus aureus and Mycoplasma pneumoniae, as well as the porcine pathogen Mycoplasma hyopneumoniae .

• Host Molecule Binding: Recombinant Ef-Tu (particularly from M. pneumoniae) binds strongly to a diverse range of host molecules .

• Plasminogen Activation: When bound to plasminogen, Ef-Tu can convert plasminogen to plasmin in the presence of plasminogen activators, potentially contributing to bacterial virulence .

• Fragment Functionality: Even fragments of Ef-Tu retain binding capabilities to host proteins, suggesting functional domains within the protein that maintain activity independently .

These moonlighting functions appear to be facilitated by:

• Accumulation of positively charged amino acids in short linear motifs (SLiMs)

• Protein processing events that occur on the cell surface

• Codon bias engendered by an A+T rich genome that may influence how positively-charged residues accumulate in SLiMs

When investigating these secondary functions, researchers should consider both the whole protein and its processed fragments.

How can researchers effectively detect and quantify Elongation factor Tu in bacterial samples?

Methodological Approach for Ef-Tu Detection and Quantification:

• Immunological Methods:

  • Western blotting using anti-Ef-Tu antibodies

  • ELISA for quantitative measurement

  • Immunofluorescence microscopy for localization studies

• Mass Spectrometry-Based Approaches:

  • Selected reaction monitoring (SRM) MS for targeted quantification

  • Data-dependent acquisition (DDA) for discovery-based analysis

  • Data-independent acquisition (DIA) for comprehensive detection

• PCR-Based Detection:

  • qRT-PCR targeting the tuf gene with specific primers

  • Digital PCR for absolute quantification

• 16S rRNA Gene Sequencing:

  • Can be used alongside tuf gene analysis for bacterial identification

  • Note that 16S rRNA gene sequencing has limitations in discriminating certain bacterial genera/species

For optimal results, researchers should combine multiple approaches, particularly when studying Ef-Tu's dual roles in translation and as a moonlighting protein.

What experimental conditions optimize the expression of recombinant Kocuria rhizophila Elongation factor Tu?

When expressing recombinant Kocuria rhizophila Elongation factor Tu, consider the following optimization parameters:

Expression System Selection:

• Yeast expression systems have been successfully used for producing recombinant K. rhizophila Ef-Tu

• K. rhizophila DC2201 itself can be utilized as an organic solvent-resistant expression host

Expression Vector Design:

• Shuttle vectors like pKITE301 and its derivatives can be constructed for K. rhizophila expression systems

• Include appropriate promoters, selection markers, and fusion tags based on downstream applications

Culture Conditions Matrix:

Purification Strategy:

• Cell lysis under native conditions

• Affinity chromatography using appropriate tags (His-tag commonly used)

• Size exclusion chromatography for higher purity

• Quality control by SDS-PAGE (expect >85% purity)

How can Kocuria rhizophila Elongation factor Tu be used in structural biology studies?

Recombinant Kocuria rhizophila Elongation factor Tu offers several advantages for structural biology investigations:

X-ray Crystallography Approach:

• Purify protein to >95% homogeneity using multi-step chromatography

• Screen crystallization conditions with commercial kits (Hampton Research, Molecular Dimensions)

• Optimize promising conditions by varying:

  • Protein concentration (5-15 mg/mL)

  • Precipitant type and concentration

  • pH (5.0-8.0)

  • Temperature (4°C, 16°C, 20°C)

• For co-crystallization with GTP/GDP:

  • Include 1-5 mM nucleotide in crystallization buffer

  • Consider adding 5-10 mM MgCl₂ to stabilize nucleotide binding

Cryo-EM Studies:

• Prepare grids with protein at 0.5-5 mg/mL

• Consider GraFix method for complex stabilization

• Use nucleotide-locked states (GTP analogues like GMPPNP)

NMR Spectroscopy:

• For domain-specific studies (Domains I, II, or III)

• Isotopic labeling (¹⁵N, ¹³C) required for detailed structural analysis

The Rossmann fold in Domain I and beta sheet structures in Domains II and III provide distinct structural features that can be targeted in these studies . When designing structural biology experiments, researchers should account for the nucleotide binding state (GTP vs. GDP), as this significantly affects protein conformation.

What approaches can be used to study the GTPase activity of Kocuria rhizophila Elongation factor Tu?

Methodological Framework for GTPase Activity Analysis:

• Spectrophotometric Coupled Enzyme Assays:

  • Measure inorganic phosphate release using malachite green

  • NADH-coupled system monitoring absorbance at 340 nm

  • EnzChek® Phosphate Assay for continuous measurement

• Radioactive Assays:

  • [γ-³²P]GTP hydrolysis tracking

  • Thin-layer chromatography separation

  • Quantification by scintillation counting

• Fluorescence-Based Methods:

  • FRET-based GTPase sensors

  • Fluorescent GTP analogues (BODIPY-GTP, Mant-GTP)

  • Stop-flow kinetics for rapid reaction analysis

Kinetic Parameter Determination Protocol:

• Prepare reaction buffer (typically 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 100 mM KCl)

• Add purified Ef-Tu (0.1-1 μM)

• Initiate reaction with varying GTP concentrations (1-500 μM)

• Measure initial reaction rates at physiologically relevant temperature (30-37°C)

• Calculate Km and kcat using Michaelis-Menten equation

• Compare intrinsic vs. ribosome-stimulated GTPase activity

When studying the GTPase activity of Kocuria rhizophila Ef-Tu, researchers should include appropriate controls and consider the influence of ribosomal components on activity rates.

How does Elongation factor Tu interact with the ribosome and aminoacyl-tRNAs during translation?

The interaction between Elongation factor Tu and its translation partners follows a precise sequence:

Interaction Mechanism:

• Ef-Tu binds GTP, forming an activated Ef-Tu·GTP complex

• This complex binds aminoacyl-tRNA, creating a ternary complex (Ef-Tu·GTP·aa-tRNA)

• The ternary complex delivers the aminoacyl-tRNA to the A-site of the ribosome

• Correct codon-anticodon pairing triggers GTP hydrolysis by Ef-Tu

• GDP-bound Ef-Tu undergoes conformational change and dissociates from the ribosome

• Ef-Tu is recycled through GDP-GTP exchange by Elongation factor Ts (EF-Ts)

Key Interaction Regions:

• Domain I interacts with GTP/GDP and contains the GTPase center

• Domains I and II form the aminoacyl-tRNA binding interface

• All three domains contribute to ribosome interaction

• The switch I and switch II regions undergo significant conformational changes upon GTP hydrolysis

Experimental Approaches to Study These Interactions:

• Cryo-EM of ribosome-Ef-Tu complexes in different states

• Fluorescence-based binding assays with labeled components

• Chemical cross-linking followed by mass spectrometry

• Mutagenesis of key residues and functional testing

These interactions should be studied in the context of the complete translation cycle to understand the dynamic nature of Ef-Tu's role.

What is the significance of Kocuria rhizophila as an organic solvent-resistant bacterium in biotechnology applications?

Kocuria rhizophila, particularly strain DC2201, has emerged as a valuable organism for biotechnology applications due to its organic solvent resistance . This characteristic makes it particularly suitable for:

Biotechnological Applications:

• Biocatalysis in Non-Aqueous Media:

  • Enzymatic transformations in the presence of organic solvents

  • Production of chiral compounds that may have limited water solubility

  • Enhanced reaction rates for certain hydrophobic substrates

• Expression System for Industrial Enzymes:

  • The development of shuttle vector pKITE301 and its derivatives enables genetic manipulation

  • Successfully used for expression of various enzymes:

    • Styrene oxidase

    • Alcohol dehydrogenase

    • Flavonoid methylation enzymes

• Whole-Cell Biocatalysts:

  • Two-phase reaction systems (aqueous/organic)

  • In situ product extraction during biocatalysis

  • Reduced product inhibition in biphasic systems

Practical Considerations for K. rhizophila-Based Bioprocesses:

Researchers can exploit this organism's unique properties for developing sustainable biocatalytic processes, particularly for reactions involving hydrophobic substrates or products that benefit from organic solvent presence.

What are common challenges in purifying recombinant Kocuria rhizophila Elongation factor Tu and how can they be addressed?

Researchers frequently encounter several challenges when purifying recombinant K. rhizophila Elongation factor Tu:

Challenge 1: Protein Solubility Issues

• Solution: Optimize lysis conditions (buffer composition, detergents, salt concentration)

• Approach: Test various lysis buffers (50 mM Tris-HCl pH 7.5-8.0, 100-300 mM NaCl, 0-10% glycerol)

• Alternative: Consider fusion tags that enhance solubility (MBP, SUMO, TrxA)

Challenge 2: Nucleotide Binding Affecting Purification

• Solution: Include GDP/GTP in purification buffers (0.1-1 mM)

• Approach: Add 5-10 mM MgCl₂ to stabilize nucleotide binding

• Alternative: Create nucleotide-free preparations using EDTA treatment followed by size exclusion

Challenge 3: Protein Degradation

• Solution: Add protease inhibitors to all buffers

• Approach: Work at 4°C throughout purification

• Alternative: Consider shorter purification protocols or automated systems

Challenge 4: Co-purifying Contaminants

• Solution: Multi-step purification strategy

• Approach: Combine affinity chromatography with ion exchange and size exclusion

• Alternative: Try different affinity tags or tag positions (N-terminal vs. C-terminal)

Recommended Purification Protocol:

• Affinity chromatography (Ni-NTA for His-tagged protein)

• Tag cleavage (if required)

• Ion exchange chromatography (Resource Q/S)

• Size exclusion chromatography (Superdex 75/200)

• Quality control by SDS-PAGE (target >85% purity)

How can researchers verify the functional activity of purified recombinant Kocuria rhizophila Elongation factor Tu?

Multiple complementary assays should be used to verify the functional activity of purified recombinant K. rhizophila Elongation factor Tu:

1. GTPase Activity Assays:

• Malachite green phosphate detection assay

• Measure intrinsic and ribosome-stimulated GTPase activity

• Expected values: intrinsic activity ~0.5-5 min⁻¹, ribosome-stimulated ~50-100 min⁻¹

2. Nucleotide Binding Assays:

• Fluorescent nucleotide analogs (mant-GTP/GDP)

• Isothermal titration calorimetry (ITC)

• Expected Kd for GTP: 10⁻⁷-10⁻⁸ M; for GDP: 10⁻⁸-10⁻⁹ M

3. Aminoacyl-tRNA Binding Assays:

• Filter binding assays with radioactively labeled aa-tRNAs

• Fluorescence anisotropy with fluorescently labeled aa-tRNAs

• Expected Kd: 10⁻⁷-10⁻⁸ M for cognate aa-tRNAs

4. Translation Activity Assays:

• In vitro translation systems with purified components

• Measurement of polypeptide synthesis using radiolabeled amino acids

• Comparison with commercially available translation factors

5. Structural Integrity Verification:

• Circular dichroism (CD) spectroscopy

• Differential scanning fluorimetry (thermal shift assay)

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

Functional Verification Decision Tree:

What are the key differences between Kocuria rhizophila Elongation factor Tu and Ef-Tu from other bacterial species?

Understanding the similarities and differences between K. rhizophila Ef-Tu and other bacterial Ef-Tu proteins is crucial for research design:

Conserved Features Across Bacterial Ef-Tu:

Distinctive Features of K. rhizophila Ef-Tu:

Implications for Research:

• When designing experiments using K. rhizophila Ef-Tu as a model, consider these differences

• For heterologous expression, codon optimization may be necessary

• When studying moonlighting functions, species-specific differences may be significant

• In structural studies, focus on both conserved catalytic regions and variable surface features

Understanding these differences is particularly important when extrapolating findings from K. rhizophila to other bacterial species or when using Ef-Tu as a phylogenetic marker.

How can Kocuria rhizophila Elongation factor Tu be used as a model for studying protein moonlighting?

K. rhizophila Ef-Tu represents an excellent model system for investigating protein moonlighting phenomena:

Research Framework for Moonlighting Studies:

• Structural Basis of Moonlighting:

  • Map molecular determinants of canonical vs. moonlighting functions

  • Identify SLiMs (Short Linear Motifs) enriched in positively charged residues

  • Create domain-specific mutants to dissect functional regions

• Surface Localization Mechanisms:

  • Investigate non-classical secretion pathways

  • Study cell surface retention mechanisms

  • Characterize processing events on the cell surface using N-terminomics

• Host-Pathogen Interaction Studies:

  • Characterize binding to host molecules (ECM proteins, plasminogen)

  • Assess the impact of Ef-Tu surface exposure on bacterial adhesion

  • Evaluate the role in virulence using in vitro and in vivo models

• Evolutionary Aspects:

  • Analyze how A+T rich genomes influence the accumulation of positively charged residues

  • Compare moonlighting capabilities across bacterial species with varying GC content

  • Study the evolution of moonlighting functions in relation to bacterial adaptation

Methodological Approaches:

• Site-directed mutagenesis of key residues

• Protein fragment analysis for functional mapping

• Heterologous expression of Ef-Tu variants

• Surface plasmon resonance for interaction studies

• Advanced imaging techniques (super-resolution microscopy)

Using K. rhizophila Ef-Tu as a model system provides insights applicable to the broader field of protein moonlighting in prokaryotes.

What are the implications of Elongation factor Tu's interaction with plasminogen for bacterial pathogenesis?

The ability of Elongation factor Tu to interact with and activate plasminogen has significant implications for bacterial pathogenesis:

Mechanistic Pathway:

• Surface-exposed Ef-Tu binds plasminogen on the bacterial surface

• In the presence of host plasminogen activators (tPA, uPA), plasminogen is converted to plasmin

• Bacterial-bound plasmin can degrade extracellular matrix (ECM) components and fibrin clots

• This degradation facilitates bacterial invasion and dissemination within host tissues

Pathogenesis Implications:

Therapeutic Potential:

• Targeting Ef-Tu-plasminogen interaction could limit bacterial dissemination

• Ef-Tu-derived peptides might serve as competitive inhibitors

• Antibodies against surface-exposed Ef-Tu epitopes could block this interaction

• Small molecule inhibitors of this interaction represent novel antimicrobial strategies

These findings suggest that seemingly housekeeping proteins like Ef-Tu can play unexpected roles in bacterial pathogenesis through their moonlighting functions. Researching these interactions provides new avenues for understanding and potentially treating bacterial infections.

How might recombinant Kocuria rhizophila Elongation factor Tu be applied in biotechnology beyond protein synthesis studies?

Recombinant K. rhizophila Ef-Tu has potential applications in biotechnology that extend far beyond its role in protein synthesis:

Biocatalysis Applications:

• Organic Solvent-Compatible Enzymatic Processes:

  • Utilize K. rhizophila's organic solvent resistance for two-phase reaction systems

  • Express Ef-Tu along with target enzymes for enhanced stability in non-aqueous media

  • Develop K. rhizophila expression systems using shuttle vectors like pKITE301

• Chiral Compound Production:

  • Leverage the organic solvent tolerance for synthesis of pharmaceutical intermediates

  • Express enzymes like styrene oxidase and alcohol dehydrogenase in K. rhizophila

  • Design cascade reactions combining multiple enzymatic steps

Biomedical Applications:

• Diagnostic Tools:

  • Develop Ef-Tu-based diagnostic markers for bacterial identification

  • Create biosensors using Ef-Tu's binding properties

  • Use as a carrier protein for antigen presentation

• Therapeutic Approaches:

  • Exploit Ef-Tu's moonlighting functions for novel antimicrobials

  • Develop Ef-Tu-derived peptides that interfere with bacterial pathogenesis

  • Create vaccine components targeting conserved Ef-Tu epitopes

Biosensing and Environmental Applications:

• GTP/GDP-Sensing Systems:

  • Utilize Ef-Tu's nucleotide binding properties for analytical applications

  • Develop fluorescence-based sensors for nucleotide detection

  • Create environmental monitoring tools based on conformational changes

• Protein Engineering Platform:

  • Use as a scaffold for designing novel protein functionalities

  • Exploit its domain architecture for creating chimeric proteins

  • Develop stress-resistant protein variants based on K. rhizophila adaptations

These diverse applications highlight the potential of this protein beyond its canonical role in translation, with particular promise in organic solvent-compatible bioprocesses and medical applications.