Recombinant Bacillus subtilis Elongation factor Tu (tuf)

What is the genetic organization of the tuf gene in Bacillus subtilis?

The tuf gene in B. subtilis is located within the str operon, a highly conserved five-gene transcriptional unit arranged as 5′-ybxF-rpsL-rpsG-fus-tuf-3′. This gene organization includes ybxF (unknown function with homology to eukaryotic ribosomal protein L30), rpsL (encoding ribosomal protein S12), rpsG (encoding ribosomal protein S7), fus (encoding elongation factor G), and tuf (encoding elongation factor Tu) . Unlike Escherichia coli, which contains two copies of the tuf gene (tufA and tufB), B. subtilis possesses only a single tuf gene, similar to the genetic arrangement found in Bacillus stearothermophilus .

How is the expression of the tuf gene regulated in B. subtilis?

The expression of the tuf gene in B. subtilis is regulated by two distinct promoters. The first is the main str operon promoter (strp) located upstream of the ybxF gene, which drives the transcription of the entire operon. The second is the tuf-specific promoter (tufp) situated in the fus-tuf intergenic region, which specifically drives the expression of the tuf gene . Interestingly, the tufp promoter has approximately 20-fold higher transcriptional activity compared to the strp promoter, as determined by chloramphenicol acetyltransferase assays . This differential expression is attributed to the presence of oppositely acting cis elements: an inhibitory region upstream of strp and a stimulatory A/T-rich block upstream of tufp . In B. stearothermophilus, the ratio between transcription products from these two promoters is approximately 10:1 in favor of the tuf gene transcript .

What are the basic structural and functional characteristics of B. subtilis EF-Tu?

B. subtilis EF-Tu is a G protein that catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome during protein translation . It constitutes one of the most abundant proteins in bacterial cells. The primary function of EF-Tu is to shuttle aminoacylated tRNAs to the ribosome, ensuring that the correct amino acid is added to the growing protein chain through a codon-anticodon recognition system . This process consumes GTP, with EF-Tu being released as an EF-Tu/GDP complex after GTP hydrolysis. The recharging of this complex in bacteria is executed by the elongation factor Ts (EF-Ts), in contrast to eukaryotes which use elongation factor 1B (eEF1B) .

What are the optimal expression systems for producing recombinant B. subtilis EF-Tu?

Several expression systems have been evaluated for the recombinant production of B. subtilis EF-Tu. The most successful approach reported is the glutathione-S-transferase (GST) fusion system in E. coli . Other attempted systems, including constitutive expression systems and inducible expression systems with His6 tags at either N-terminus or C-terminus in both E. coli and B. subtilis, were unsuccessful at the plasmid construction stage . The GST/EF-Tu fusion proteins can be highly expressed upon IPTG induction, resulting in both soluble and insoluble forms of the protein .

For researchers seeking to express recombinant proteins in B. subtilis itself, plasmid pHT254 has been developed as a typical expression vector, which can be used for introducing target genes into B. subtilis . This system utilizes promoters such as Pgrac and Pgrac100 to drive the expression of recombinant proteins .

What purification methods are most effective for recombinant B. subtilis EF-Tu?

One-step purification using glutathione-sepharose affinity column chromatography has proven highly effective for purifying recombinant B. subtilis EF-Tu when expressed as a GST fusion protein . Following affinity purification, treatment with factor Xa to cleave the GST tag results in near-homogeneous EF-Tu preparation . The purified EF-Tu demonstrates high GDP binding activity, confirming its functional integrity . This method avoids multiple chromatographic steps, reducing protein loss and degradation during the purification process.

How can researchers verify the functional activity of purified recombinant B. subtilis EF-Tu?

The functionality of purified recombinant B. subtilis EF-Tu can be assessed through several assays:

• GDP binding activity: Measuring the ability of purified EF-Tu to bind GDP is a primary indicator of functional integrity .

• Poly(U)-directed poly(Phe) synthesis: In cell-free systems, functional EF-Tu promotes the synthesis of polyphenylalanine when directed by polyuridylic acid .

• Aminoacyl-tRNA binding assay: The capacity of EF-Tu to form a ternary complex with GTP and aminoacyl-tRNA can be measured using techniques such as filter binding assays or fluorescence-based methods.

• GTPase activity: The intrinsic and ribosome-stimulated GTPase activity of EF-Tu provides another measure of its functional competence.

What post-translational modifications occur in B. subtilis EF-Tu and how do they affect function?

B. subtilis EF-Tu undergoes a specific post-translational modification that distinguishes it from Gram-negative bacterial EF-P. Research has identified a 5-aminopentanol moiety attached to Lys32 of B. subtilis EF-P that is required for swarming motility . This modification is distinct from the two phylogenetically distinct EF-P modification pathways described in Gram-negative bacteria .

The 5-aminopentanol modification plays a crucial role in modulating the synthesis of specific diprolyl motifs present in proteins required for swarming motility . Using a fluorescent in vivo B. subtilis reporter system, researchers identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P . Genomic analysis revealed that these EF-P-dependent peptide motifs are represented in flagellar genes, establishing a direct link between the post-translational modification of EF-P and swarming motility in B. subtilis .

How can researchers experimentally investigate the impact of EF-Tu modifications on B. subtilis physiology?

To investigate the physiological impact of EF-Tu modifications, researchers can employ several experimental approaches:

• Genetic Manipulation: Create mutants with alterations in putative modification enzymes or in the modification sites of EF-Tu.

• Motility Assays: Assess swarming and swimming motility on appropriate media to correlate EF-Tu modifications with motility phenotypes .

• Reporter Systems: Utilize fluorescent in vivo reporter systems to identify peptide motifs dependent on modified EF-Tu for efficient synthesis .

• Genomic Analysis: Examine the B. subtilis genome for the distribution of EF-Tu-dependent peptide motifs across different functional categories of genes .

• Mass Spectrometry: Apply high-resolution mass spectrometry to characterize the nature and site of post-translational modifications on purified EF-Tu.

• Structural Studies: Perform structural analyses to determine how modifications affect EF-Tu's interaction with tRNAs, ribosomes, and other factors.

What are the key considerations for designing a chassis strain of B. subtilis for recombinant EF-Tu expression?

When designing a B. subtilis chassis strain for recombinant EF-Tu expression, several factors should be considered:

• Autolysis Resistance: Knockout of autolysis genes such as lytC, sigD, pcfA, and flgD can increase biomass by 11-20% .

• Prophage Elimination: Removal of prophage-associated genes like xpf can increase biomass by approximately 10% .

• Sporulation Control: Manipulation of spore-associated genes can impact growth. While knockout of the global regulatory transcription factor spo0A reduced biomass by 20%, elimination of spore-associated autolysis enzyme genes skfA, sdpC, and spoIIE increased biomass by 8-14% .

• Promoter Selection: For EF-Tu expression, consider using the native tufp promoter, which shows significantly higher activity than the strp promoter .

• Lifespan Engineering: Applying chronological lifespan engineering strategies can create robust B. subtilis chassis cells that resist autolysis, tolerate toxic substrates, and achieve higher mass transfer efficiency .

What methodological approaches can be used to study the interaction between B. subtilis EF-Tu and antibiotics?

To study interactions between B. subtilis EF-Tu and antibiotics, researchers can employ several methodological approaches:

• Cell-free Protein Synthesis Systems: These can be used to directly assess the impact of antibiotics on EF-Tu function in a controlled environment .

• Resistance Profiling: Compare the sensitivity of wild-type and recombinant EF-Tu to various antibiotics, as demonstrated in studies with GE2270 and kirromycin .

• Binding Assays: Direct binding studies using techniques such as isothermal titration calorimetry, surface plasmon resonance, or fluorescence-based assays to quantify antibiotic-EF-Tu interactions.

• Structural Studies: X-ray crystallography or cryo-electron microscopy of EF-Tu in complex with antibiotics to determine binding sites and conformational changes.

• Recombinant Expression of Modified EF-Tu: Generate EF-Tu variants with specific mutations and assess their antibiotic sensitivity profiles.

• Poly(U)-directed Poly(Phe) Synthesis: This assay can be used to evaluate the impact of antibiotics on the functional activity of EF-Tu in vitro .

Beyond protein synthesis, what other cellular roles does B. subtilis EF-Tu play?

Although primarily known for its role in protein synthesis, EF-Tu has evolved to execute diverse functions beyond translation:

• Cell Surface Functions: EF-Tu can traffic to and be retained on cell surfaces where it can interact with membrane receptors and extracellular matrix components .

• Moonlighting Functions: Structural studies indicate that short linear motifs (SLiMs) in surface-exposed, non-conserved regions of EF-Tu may play key roles in its moonlighting functions .

• Pathogenesis-Related Functions: While primarily studied in pathogenic bacteria, EF-Tu's surface-exposed regions can contribute to various interactions with host cells .

• Swarming Motility: Through its interaction with specifically modified EF-P, EF-Tu contributes to the synthesis of proteins containing diprolyl motifs that are required for swarming motility in B. subtilis .

How can researchers investigate the non-canonical functions of B. subtilis EF-Tu?

To investigate non-canonical functions of B. subtilis EF-Tu, researchers can employ these methodological approaches:

• Subcellular Localization Studies: Immunofluorescence microscopy or fractionation studies to determine the distribution of EF-Tu within or on the surface of B. subtilis cells.

• Protein-Protein Interaction Assays: Techniques such as pull-down assays, co-immunoprecipitation, or yeast two-hybrid screening to identify novel interacting partners of EF-Tu.

• Mutational Analysis: Generate EF-Tu variants with mutations in regions suspected to be involved in non-canonical functions, particularly in surface-exposed short linear motifs (SLiMs) .

• Functional Assays: Develop specific assays for phenotypes associated with non-canonical functions, such as motility, biofilm formation, or stress response.

• Comparative Genomics and Proteomics: Compare EF-Tu sequences and expression patterns across different bacterial species to identify conserved or divergent features that might relate to non-canonical functions.

How does B. subtilis EF-Tu differ from EF-Tu in other bacterial species?

B. subtilis EF-Tu exhibits several distinctive features compared to EF-Tu in other bacterial species:

• Gene Copy Number: B. subtilis possesses a single tuf gene, unlike E. coli which has two copies (tufA and tufB) .

• Operon Structure: The tuf gene in B. subtilis is part of a five-gene str operon (ybxF-rpsL-rpsG-fus-tuf), whereas in E. coli, the str operon consists of four genes (rpsL-rpsG-fus-tufA) .

• Transcriptional Regulation: B. subtilis utilizes a strong tuf-specific promoter (tufp) that produces approximately 10 times more tuf transcript than the main operon promoter, a regulatory mechanism not observed to the same extent in E. coli .

• Post-translational Modifications: The specific 5-aminopentanol modification on Lys32 of B. subtilis EF-P differs from the modifications observed in Gram-negative bacteria .

• Antibiotic Sensitivity: Different bacterial species show varying sensitivities to EF-Tu-targeting antibiotics. For example, Planobispora rosea EF-Tu is resistant to its own antibiotic GE2270 but is inhibited by kirromycin .

What methodological challenges exist when comparing recombinant EF-Tu from different bacterial species?

When comparing recombinant EF-Tu from different bacterial species, researchers face several methodological challenges:

• Expression System Compatibility: Expression systems optimized for one species' EF-Tu may not be equally effective for others. For example, the GST fusion system successful for B. subtilis EF-Tu may not work optimally for all bacterial EF-Tu proteins .

• Post-translational Modifications: Different bacterial species employ distinct modification pathways for EF-Tu, which may not be replicated in heterologous expression systems .

• Purification Strategy Differences: The physicochemical properties of EF-Tu can vary between species, necessitating adjustments to purification protocols.

• Functional Assay Standardization: Ensuring that functional assays (GDP binding, GTPase activity, etc.) are comparable across different species' EF-Tu requires careful standardization.

• Structural Analysis Interpretation: Subtle structural differences between EF-Tu proteins from different species can lead to significant functional divergences that may be challenging to interpret.

• Species-Specific Interacting Partners: EF-Tu may interact with species-specific partners, complicating the comparison of non-canonical functions across species.

What are common challenges in expressing recombinant B. subtilis EF-Tu and how can they be addressed?

Researchers commonly encounter several challenges when expressing recombinant B. subtilis EF-Tu:

• Plasmid Construction Difficulties: Many expression systems for B. subtilis EF-Tu fail at the plasmid construction stage . Solution: Use the GST fusion system in E. coli, which has proven successful for expressing B. subtilis EF-Tu .

• Protein Solubility Issues: Recombinant EF-Tu may form inclusion bodies. Solution: Optimize expression conditions (temperature, inducer concentration, expression duration) and consider fusion partners like GST that enhance solubility .

• Functional Activity Loss: Purified recombinant EF-Tu may lack full functional activity. Solution: Verify GDP binding activity as a measure of functional integrity and ensure proper folding during expression and purification .

• Low Expression Levels: Some expression systems may yield insufficient protein quantities. Solution: Utilize the strong tuf-specific promoter or optimize codon usage for the expression host .

• Post-translational Modification Absence: Heterologous expression systems may not reproduce essential modifications. Solution: Consider expressing in B. subtilis itself or engineer the expression host to include necessary modification enzymes .

What analytical methods are most effective for characterizing the structure and function of recombinant B. subtilis EF-Tu?

For comprehensive characterization of recombinant B. subtilis EF-Tu, researchers should consider these analytical methods:

• Mass Spectrometry: For precise molecular weight determination and identification of post-translational modifications, particularly the 5-aminopentanol moiety on Lys32 .

• Circular Dichroism Spectroscopy: To assess secondary structure content and thermal stability of the purified protein.

• X-ray Crystallography or Cryo-EM: For high-resolution structural determination, particularly in complex with GTP/GDP, aminoacyl-tRNAs, or antibiotics.

• GDP/GTP Binding Assays: Using either radioactive nucleotides or fluorescent analogs to quantify binding affinities and kinetics.

• In Vitro Translation Assays: Such as poly(U)-directed poly(Phe) synthesis to evaluate the functional activity in protein synthesis .

• Thermal Shift Assays: To assess protein stability under various conditions and in the presence of binding partners or inhibitors.

• Isothermal Titration Calorimetry: For thermodynamic characterization of interactions with nucleotides, aminoacyl-tRNAs, or antibiotics.