Recombinant Vibrio vulnificus Elongation factor P-like protein (VV1229)

What is the VV1229 protein and how does it function in bacterial translation?

The VV1229 protein belongs to the family of Elongation Factor P-like proteins in Vibrio vulnificus. Similar to EF-P and its paralog EfpL (YeiP) in other bacteria, VV1229 likely plays a crucial role in overcoming ribosome stalling during translation, particularly at polyproline sequences. These proteins help ribosomes navigate challenging sequence motifs that would otherwise cause translation to stall . The protein functions by interacting with the ribosome and facilitating peptide bond formation at difficult sequences, thereby ensuring efficient protein synthesis.

How does VV1229 relate to other elongation factors in the bacterial kingdom?

VV1229 is part of the broader family of bacterial elongation factors that includes the well-characterized EF-P and its paralog EfpL. Research indicates that these proteins share structural similarities but may have evolved distinct functional specificities. In many bacterial species, the co-occurrence of EF-P and EfpL has been identified as an evolutionary driver for higher bacterial growth rates . VV1229 likely shares core functional mechanisms with these proteins while potentially possessing unique characteristics specific to Vibrio vulnificus.

What are the key structural features of VV1229 that enable its function?

Based on structural studies of related elongation factors, VV1229 likely possesses a three-domain architecture similar to other EF-P-like proteins. In EF-P, a modified lysine residue (K34) aligns with the tRNA trinucleotide backbone, while in EfpL, an unmodified arginine (R33) in the β3Ωβ4 loop can potentially reestablish interaction with the tRNA trinucleotide . The prolonged β3Ωβ4 loop and its central tip are critical structural features that may compensate for the lack of modified lysine in some EF-P paralogs. Molecular docking studies suggest these proteins mediate specific interactions with RNA as the central residue stacks between C-bases and makes polar interactions with the phosphate-sugar backbone .

How do post-translational modifications affect the functionality of VV1229?

Post-translational modifications are essential for the function of Elongation Factor P-like proteins. Similar to what has been observed with EfpL, VV1229 may undergo lysine acylation that allows it to sense the metabolic state of the cell . This modification likely affects its interaction with the ribosome and tRNA, ultimately influencing its ability to resolve translation stalling. The specific modifications of VV1229 would determine its efficacy in promoting translation at challenging sequence motifs.

What are the optimal conditions for expressing and purifying recombinant VV1229?

For optimal expression and purification of recombinant VV1229, researchers should consider the following methodological approach:

• Gene amplification using PCR with primers containing appropriate restriction sites

• Cloning into an expression vector with an inducible promoter (like the tac promoter)

• Expression in a suitable E. coli strain (such as DH5α)

• Purification using affinity chromatography, potentially with a FLAG-tag system

Based on successful approaches with other V. vulnificus proteins, researchers can clone the target gene into vectors like pBluescript II KS+ before subcloning into an expression vector like pMMB67EH.cam . Protein expression may be optimized using an inducible system with IPTG, and purification can utilize affinity tags such as the FLAG expression system that has been successfully employed for other V. vulnificus proteins .

How can researchers effectively detect protein-protein interactions involving VV1229?

To study protein-protein interactions involving VV1229, immunoprecipitation experiments have proven effective for other V. vulnificus proteins. Researchers can:

• Prepare clarified cell lysates from V. vulnificus cultures

• Pre-incubate protein A-coated magnetic beads with VV1229-specific antibodies

• Precipitate the protein-antibody complex from the cell lysate

• Analyze the precipitate using SDS-PAGE and Western blot with specific antibodies

This approach has successfully demonstrated the interaction between VvRsbR and VvRsbS proteins in V. vulnificus . For VV1229, similar techniques could reveal its interaction partners within the translation machinery or regulatory networks.

How does VV1229 contribute to resolving ribosome stalling at specific sequence motifs?

VV1229, like other EF-P family proteins, likely recognizes and resolves ribosome stalling at specific sequence motifs. Through ribosome profiling analysis, researchers have found that EF-P and EfpL can resolve stalling not only at canonical polyproline motifs but also at additional sequences . The precise mechanism involves the protein binding to the ribosome between the P and E sites, where it can interact with the tRNA and facilitate peptide bond formation at challenging sequences.

Table 1: Comparison of Sequence Motifs Resolved by EF-P Family Proteins

What computational approaches are most effective for predicting VV1229 function?

For predicting VV1229 function, researchers should employ a multi-faceted computational approach including:

• Sequence alignment and phylogenetic analysis to position VV1229 within the EF-P protein family

• Homology modeling based on known structures of EF-P and EfpL

• Molecular docking simulations to predict interactions with the ribosome and tRNA

• Molecular dynamics simulations to understand conformational changes during function

The molecular docking approach using HADDOCK, as employed for EfpL , would be particularly useful for predicting the interaction between VV1229 and its RNA targets. This would involve modeling the local geometry to determine how VV1229 might establish interactions with the tRNA trinucleotide.

How is VV1229 expression regulated in response to environmental conditions?

The expression of VV1229 is likely regulated in response to changing environmental conditions, similar to other V. vulnificus proteins. Research on the V. vulnificus stressosome has shown that oxygen limitation triggers alterations in the proteome . VV1229 expression may similarly be regulated by oxygen availability, particularly given the importance of translation regulation during stress responses. Researchers have observed that starvation and oxygen-limitation lead to significant changes in protein abundance in V. vulnificus , suggesting that translation factors like VV1229 could be part of the adaptive response to these stressors.

What role might VV1229 play in V. vulnificus pathogenicity and stress adaptation?

VV1229 may contribute to V. vulnificus pathogenicity by enabling efficient translation of virulence factors, particularly those containing challenging sequence motifs. In bacterial pathogens, adaptation to host environments often involves rapid proteomic changes, and translation factors like VV1229 could be crucial for this response. V. vulnificus produces various virulence factors, including the elastase VvpE that is responsible for tissue necrosis and inflammation . The efficient translation of such virulence factors might depend on VV1229 function, especially if they contain sequences prone to ribosome stalling.

How can researchers address contradictory findings regarding VV1229 function?

To address contradictory findings in VV1229 research, researchers should apply systematic approaches to contradiction detection and resolution:

• Construct a corpus of potentially contradictory claims from the literature

• Normalize claims to facilitate literature-scale analysis

• Identify study characteristics that may explain contradictions

• Analyze methodological differences between studies

This approach, similar to what has been applied in cardiovascular research , involves presenting a specific research question and determining whether different studies support contradictory answers. For VV1229, researchers should carefully examine experimental conditions, protein preparations, and assay systems that might contribute to seemingly contradictory results.

What methodological challenges must be overcome when studying VV1229 in vivo?

Studying VV1229 in vivo presents several methodological challenges:

• Creating precise gene mutations without polar effects on downstream genes

• Developing specific antibodies for detecting native VV1229

• Distinguishing VV1229 function from other translation factors

• Establishing physiologically relevant conditions for functional studies

These challenges can be addressed through careful experimental design. For gene mutations, researchers can use approaches similar to what has been described for V. vulnificus pilA, where an Ω interposon was inserted and complementation was performed to confirm specificity . For protein detection, raising specific antibodies against recombinant VV1229 would allow for immunoprecipitation experiments similar to those performed for VvRsbR and VvRsbS .

How does VV1229 differ from EF-P and EfpL in other bacterial species?

VV1229 likely shares core functions with EF-P and EfpL while possessing unique features adapted to V. vulnificus physiology. Comparative analysis would reveal differences in:

• Structural features, particularly in the RNA-binding domains

• Post-translational modifications specific to Vibrio species

• Sequence motif recognition specificity

• Regulatory mechanisms responsive to marine environments

Research on EF-P and EfpL has shown that while they both resolve polyproline-induced stalling, they can also recognize different sequence motifs and even induce pauses at certain sequences . VV1229 may have evolved similar functional diversification specific to the translation needs of V. vulnificus.

What evolutionary advantages might the specialized function of VV1229 confer to V. vulnificus?

The specialized function of VV1229 likely confers evolutionary advantages to V. vulnificus, particularly in its natural marine habitat and during host infection. Research has shown that the co-occurrence of EF-P and EfpL in bacteria is an evolutionary driver for higher growth rates . For V. vulnificus, VV1229 might enable:

• More efficient translation of proteins required for environmental adaptation

• Rapid response to changing oxygen levels in marine environments

• Enhanced virulence through optimal production of pathogenicity factors

• Growth advantages in competitive microbial communities

These advantages would be particularly important for V. vulnificus as it transitions between marine environments and human hosts during infection.

What emerging technologies could advance our understanding of VV1229?

Several emerging technologies could significantly advance VV1229 research:

• Cryo-EM reconstruction of VV1229 bound to the ribosome, similar to what has been achieved for stressosome complexes

• Ribosome profiling to identify genome-wide VV1229-dependent translation events

• Mass spectrometry to characterize post-translational modifications

• CRISPR-based approaches for precise genome editing in V. vulnificus

These technologies would provide unprecedented insights into the structural basis of VV1229 function, its genome-wide impact on translation, and its regulation through post-translational modifications.

How might research on VV1229 contribute to developing novel antibacterial strategies?

Research on VV1229 could inform the development of novel antibacterial strategies through:

• Identification of VV1229-specific inhibitors that could disrupt V. vulnificus protein synthesis

• Understanding how VV1229 contributes to antibiotic tolerance during stress responses

• Targeting VV1229-dependent virulence factor production

• Rational design of antimicrobial peptides that exploit VV1229-dependent translation vulnerabilities

As V. vulnificus infections can be severe and life-threatening, developing targeted approaches that specifically inhibit this pathogen would be valuable for medical applications.