Recombinant Vibrio vulnificus UPF0207 protein VV1113 (VV1113)

What is UPF0207 protein VV1113 and how is it identified in Vibrio vulnificus genomes?

UPF0207 protein VV1113 belongs to a family of proteins with unknown function (UPF) found in Vibrio vulnificus. It is typically identified through whole genome sequencing (WGS) and subsequent bioinformatic analysis. Similar to how researchers identified genes in V. vulnificus VV2018, genome annotation tools can identify coding sequences (CDSs) and categorize them according to functional classifications . Comprehensive genomic analysis reveals that UPF0207 family proteins are commonly identified during annotation but lack experimentally verified functions. The identification process typically involves:

• Whole genome sequencing of V. vulnificus isolates

• Annotation of coding sequences using tools like RAST, PROKKA, or NCBI Prokaryotic Genome Annotation Pipeline

• Classification of proteins based on homology to known protein families

• Designation of unknown function proteins into UPF categories based on conserved domains

What are the predicted structural features of VV1113 and how might they inform functional studies?

Based on comparative genomic approaches similar to those used for other V. vulnificus proteins, VV1113 likely possesses characteristic structural features that can guide functional studies. Bioinformatic analysis would predict:

Structural predictions serve as the foundation for designing targeted experiments, similar to the approach used when characterizing virulence factors in V. vulnificus . The presence of specific structural motifs might suggest potential interactions with other cellular components or involvement in particular biochemical pathways.

How is recombinant VV1113 typically produced for research purposes?

Producing recombinant VV1113 follows methodologies similar to those used for other bacterial proteins. Drawing from techniques used in V. vulnificus elastase studies , the typical workflow involves:

• PCR amplification of the VV1113 gene from V. vulnificus genomic DNA

• Cloning into an appropriate expression vector (e.g., pET series for E. coli)

• Transformation into a suitable expression host (commonly E. coli BL21(DE3))

• Optimization of expression conditions (temperature, induction time, inducer concentration)

• Protein purification via affinity chromatography (if tagged) or conventional chromatographic methods

• Verification of purity by SDS-PAGE and confirmation of identity by mass spectrometry

Researchers should optimize buffer conditions based on predicted protein properties to maintain stability and solubility throughout the purification process.

What gene knockout methods are most effective for studying VV1113 function in Vibrio vulnificus?

Creating precise gene knockouts allows for phenotypic evaluation to determine protein function. Based on successful approaches used for other V. vulnificus genes, such as vvpE , the following methods are recommended:

• Allelic exchange using suicide vectors (e.g., pNQ705) carrying truncated VV1113 fragments

• CRISPR-Cas9 based genome editing, which offers greater precision

• Transposon mutagenesis for large-scale screening, followed by specific targeting of VV1113

The methodology described for constructing vvpE knockout mutants provides an excellent template: "The desired transconjugants were selected by chloramphenicol resistance and screened for [specific phenotype]... Potential mutants were subsequently tested for lack of [specific] activity." This approach ensures both positive selection for the mutation and verification of the altered phenotype.

For VV1113, researchers should:

• Design constructs that avoid polar effects on adjacent genes

• Include complementation studies to confirm phenotypes are due to VV1113 deletion

• Perform whole genome sequencing to verify no additional mutations occurred

How can transcriptomic analysis reveal the regulatory network involving VV1113?

Transcriptomic studies can elucidate the regulatory context of VV1113, similar to approaches used in fish vibriosis research . A comprehensive approach would include:

Analysis should include both early and late phase responses, similar to the two-phase inflammatory response observed in V. vulnificus infections: "The early phase... and the late phase (detectable at 12 hpi) is characterized by the upregulation of genes for typical inflammatory cytokines..."

Differential gene expression can be validated using RT-qPCR for selected marker genes, providing a more quantitative assessment of expression changes.

What protein interaction studies are most informative for deciphering VV1113 function?

Understanding protein-protein interactions is crucial for elucidating function. Several complementary approaches should be employed:

• Co-immunoprecipitation with tagged VV1113 followed by mass spectrometry

• Bacterial two-hybrid or yeast two-hybrid screening

• Proximity-dependent biotin labeling (BioID)

• Cross-linking mass spectrometry for transient interactions

• Pull-down assays with recombinant VV1113 and V. vulnificus lysates

Results should be validated using independent methods, and interaction networks should be mapped in relation to known virulence pathways. Potential interactions with virulence systems, such as the RTX toxin or type II secretion systems identified in V. vulnificus strains , would be particularly valuable to investigate.

How conserved is VV1113 across different Vibrio vulnificus strains and what does this suggest about its function?

Conservation analysis provides insights into evolutionary importance and functional constraints. Using approaches similar to the comparative genomic analysis performed on VV2018 , researchers should:

• Extract VV1113 sequences from available V. vulnificus genomes

• Perform multiple sequence alignment to identify conserved residues

• Calculate sequence identity and similarity percentages

• Construct phylogenetic trees to visualize evolutionary relationships

• Compare conservation patterns between clinical and environmental isolates

Data from such analysis might appear as:

High conservation would suggest essential cellular functions, while variability might indicate adaptation to different ecological niches or host interactions.

What can pan-genome analysis reveal about the distribution and evolution of VV1113 among Vibrio species?

Pan-genome analysis, similar to that conducted for VV2018 , would determine whether VV1113 belongs to the core genome (present in all strains) or the accessory genome (variably present). This approach would include:

• Construction of a pan-genome from multiple Vibrio genomes

• Classification of VV1113 as core, soft core, shell, or cloud gene

• Analysis of genetic context and synteny around the VV1113 locus

• Identification of horizontal gene transfer signatures

The research on VV2018 provides a model: "VV2018 shared a total of 3,016 core genes (≥99% presence), including 115 core virulence factors (VFs) and 5 core antibiotic resistance-related genes, and 309 soft core genes (≥95 and <99% presence) with 25 other V. vulnificus strains." Determining whether VV1113 falls within the core or accessory genome provides crucial evolutionary context.

How can single nucleotide polymorphism (SNP) analysis of VV1113 contribute to molecular epidemiology studies?

SNP analysis of VV1113 could serve as a molecular marker for epidemiological tracking, using methods similar to those described for V. vulnificus strain typing: "The phylogenetic tree of single nucleotide polymorphisms (SNPs) using 26 representative genomes revealed that VV2108 grouped with two other V. vulnificus strains isolated from humans."

A comprehensive approach would include:

• Identification of SNPs within the VV1113 gene across multiple isolates

• Correlation of specific SNPs with isolation source (clinical vs. environmental)

• Temporal analysis to track evolutionary changes

• Geographic distribution mapping to identify regional variants

• Association of specific SNPs with virulence or host adaptation

What phenotypic changes might be observed in a VV1113 knockout mutant?

Phenotypic evaluation of VV1113 knockout mutants would follow approaches similar to those used for elastase mutants . Researchers should examine:

• Growth kinetics in various media (rich, minimal, iron-limited)

• Stress response (oxidative, osmotic, temperature)

• Biofilm formation capacity

• Motility and chemotaxis

• Virulence in cell culture and animal models

• Resistance to environmental challenges

Unlike the elastase study which found "inactivation of the V. vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage," VV1113 might show phenotypes in different assays depending on its function.

How might post-translational modifications affect VV1113 function?

Post-translational modifications (PTMs) often regulate protein function. Investigation should include:

• Mass spectrometry analysis to identify PTMs (phosphorylation, glycosylation)

• Site-directed mutagenesis of modified residues

• Comparison of modifications under different growth conditions

• Functional assays comparing native and recombinant protein activities

The discovery of glycosylation (pgl) genes in V. vulnificus VV2018 suggests potential glycosylation machinery that might modify VV1113: "The glycosylation (pgl) like genes were found in VV2018 compared with Pgl-related proteins in Neisseria that might affect the adherence of the strain in hosts."

What approaches can determine if VV1113 contributes to Vibrio vulnificus virulence?

Determining virulence contributions requires multiple complementary approaches:

The time course of infection should be carefully monitored, as V. vulnificus infections show distinct phases: "The early phase... and the late phase (detectable at 12 hpi)..." Both acute and later-stage effects should be examined.

How can structural biology techniques be optimized for determining VV1113 three-dimensional structure?

Structural determination requires specialized approaches:

• X-ray crystallography

  • Optimize crystallization conditions (pH, temperature, precipitants)

  • Consider surface entropy reduction mutations to promote crystal packing

  • Use molecular replacement with homologous structures for phasing

• NMR spectroscopy

  • Produce 15N, 13C-labeled protein in minimal media

  • Optimize buffer conditions for long-term stability

  • Consider deuteration for larger constructs

• Cryo-electron microscopy

  • Particularly useful for protein complexes

  • Optimize grid preparation and vitrification conditions

  • Consider particle orientation issues

Structural information would significantly advance functional hypotheses for this protein of unknown function.

What are the optimal conditions for biochemical characterization of VV1113 enzymatic activity?

Without knowing the specific function of VV1113, a systematic approach is needed:

• Screen for common enzymatic activities (hydrolase, transferase, isomerase)

• Test activity under varying conditions:

  • pH range (5.0-9.0)

  • Temperature (4-42°C)

  • Salt concentration (0-500 mM NaCl)

  • Divalent cations (Mg2+, Mn2+, Ca2+, Zn2+)

  • Reducing agents (DTT, β-mercaptoethanol)

• Use substrate panels based on bioinformatic predictions

• Develop appropriate spectrophotometric or coupled assays for activity detection

This approach has been successful for characterizing other bacterial proteins of unknown function.

How can high-throughput screening approaches identify potential inhibitors or activators of VV1113?

Developing screening assays for VV1113 modulators would include:

• Developing a robust activity assay suitable for miniaturization

• Optimizing assay conditions for 96/384-well format

• Screening compound libraries:

  • Natural product extracts

  • Synthetic chemical libraries

  • Fragment-based screens

  • Repurposed drug libraries

• Validation of hits through:

  • Dose-response studies

  • Orthogonal assays

  • Target engagement studies

  • Structure-activity relationship analysis

• Testing effects on V. vulnificus growth and virulence

Chemical probes identified through such screens would serve as valuable research tools even before full functional characterization is achieved.

How might systems biology approaches integrate VV1113 into Vibrio vulnificus regulatory networks?

Systems biology integration would require:

• Multi-omics data collection:

  • Transcriptomics (RNA-seq) of VV1113 mutants

  • Proteomics (LC-MS/MS) to identify abundance changes

  • Metabolomics to identify pathway alterations

  • Interactomics from protein-protein interaction studies

• Network reconstruction using computational tools

• Identification of regulatory hubs connected to VV1113

• Perturbation studies to validate network predictions

This approach would place VV1113 in the broader context of cellular processes, potentially connecting it to known virulence mechanisms like the RTX toxin system or iron acquisition pathways identified in V. vulnificus .

What emerging technologies could accelerate functional characterization of VV1113?

Emerging technologies with potential application include:

• CRISPR interference (CRISPRi) for tunable gene repression

• Single-cell RNA-seq to capture heterogeneous responses

• Proximity labeling proteomics for in vivo interaction mapping

• Cryo-electron tomography for in situ structural studies

• AlphaFold2 and similar AI tools for structure prediction

• High-throughput phenotyping using automated imaging systems

These approaches offer advantages over traditional methods in terms of resolution, throughput, and contextual information.

How might studying VV1113 contribute to broader understanding of bacterial adaptation and pathogenesis?

Understanding VV1113 has implications beyond V. vulnificus biology:

• UPF0207 proteins are widely distributed across bacterial species, and functional insights could apply broadly

• Mechanisms of protein moonlighting (multiple functions) might be revealed

• Novel bacterial adaptation strategies to marine and host environments could be uncovered

• Potential new antimicrobial targets might emerge from functional studies

• Understanding the evolution of virulence might be enhanced by studying conserved proteins of unknown function

The comprehensive genomic and transcriptomic approaches used for V. vulnificus studies demonstrate the value of investigating uncharacterized proteins for understanding bacterial biology and pathogenesis.