Recombinant Desulfitobacterium hafniense UPF0316 protein Dhaf_3052 (Dhaf_3052)

What is Dhaf_3052 protein and what is its origin?

Dhaf_3052 is a UPF0316 family protein from the bacterium Desulfitobacterium hafniense. It consists of 174 amino acids and is classified in the UniProt database with ID B8G014 . Desulfitobacterium hafniense is notable for its versatile metabolism, particularly its ability to use organohalogens as terminal electron acceptors via organohalide respiration (OHR) . While many Desulfitobacterium proteins have well-characterized roles in metabolic pathways, Dhaf_3052 belongs to a protein family (UPF0316) whose specific function remains to be fully elucidated .

What are the recommended storage and handling conditions for recombinant Dhaf_3052?

For optimal stability and activity, recombinant Dhaf_3052 should be stored at -20°C or -80°C upon receipt. Aliquoting is necessary to avoid repeated freeze-thaw cycles, which can compromise protein integrity . The protein is typically supplied as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

For reconstitution:

• Briefly centrifuge the vial before opening

• 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 commonly recommended)

• Store working aliquots at 4°C for up to one week

What expression systems are most effective for producing recombinant Dhaf_3052?

Based on the available commercial preparations, E. coli appears to be a suitable heterologous expression system for recombinant Dhaf_3052 . The protein has been successfully expressed with an N-terminal His-tag, facilitating purification via affinity chromatography .

When designing your own expression system, consider:

• Codon optimization for E. coli if expressing the native Desulfitobacterium sequence

• Inclusion of appropriate affinity tags (His-tag has been demonstrated to work)

• Selection of promoters suitable for regulated expression

• Optimization of induction conditions to maximize soluble protein yield

The purity achieved through established protocols exceeds 90% as determined by SDS-PAGE, making this approach suitable for most research applications .

How can researchers design experiments to investigate the potential function of Dhaf_3052?

Given the limited functional information about Dhaf_3052, a systematic approach to functional characterization would include:

• Sequence-based analysis: Use bioinformatics tools to identify conserved domains and predict potential functions based on homology to characterized proteins. The UPF0316 family designation suggests Dhaf_3052 belongs to a group of proteins with unknown function .

• Expression pattern analysis: Employ proteomics approaches similar to those described by Frontiers in Microbiology (2023) to determine under which metabolic conditions Dhaf_3052 is expressed . Tandem Mass Tag labeling proteomics has successfully identified nearly 60% of the predicted proteome of Desulfitobacterium hafniense strain DCB-2, making it a viable approach for studying Dhaf_3052 expression patterns .

• Protein interaction studies: Identify potential interaction partners through pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens to place Dhaf_3052 in a functional context .

• Gene knockout/knockdown studies: Create deletion mutants in Desulfitobacterium hafniense to observe phenotypic changes, particularly under different respiratory conditions.

• Heterologous expression impact: Express Dhaf_3052 in model organisms to observe phenotypic changes that might suggest function.

What statistical approaches are appropriate for analyzing experimental data involving Dhaf_3052?

When designing experiments involving Dhaf_3052, consider the following statistical approaches:

• For comparative proteomics studies: Use fold change analysis with appropriate thresholds (e.g., logFC >1 or <-1) and false discovery rate (FDR) controls (e.g., FDR <0.05) as demonstrated in the Frontiers study on Desulfitobacterium hafniense proteome adaptations .

• For experimental designs with nested factors: Software packages like NeDPy (Nested Design Python) can be valuable for analyzing experiments with hierarchical designs . This approach may be relevant when studying Dhaf_3052 expression across different strains, growth conditions, or timepoints.

• For general experimental analysis: Standard statistical methods for analyzing central tendency and variability are essential, including:

  • Measures of central tendency (mean, median, mode)

  • Measures of variability (range, variance, standard deviation)

  • Appropriate hypothesis testing (null vs. alternative hypotheses)

  • Consideration of Type I and Type II errors

• For complex multi-factor experiments: Use factorial designs and appropriate ANOVA techniques to distinguish between the effects of different experimental variables while controlling for random error .

How might Dhaf_3052 relate to the organohalide respiration (OHR) capabilities of Desulfitobacterium hafniense?

While direct evidence linking Dhaf_3052 to organohalide respiration is not presented in the provided references, researchers can investigate potential connections through:

• Comparative proteomics: Analyze whether Dhaf_3052 is differentially expressed when Desulfitobacterium hafniense is grown with organohalides as electron acceptors compared to other respiratory conditions . The proteomics approach used by Frontiers in Microbiology (2023) could serve as a model, which successfully identified proteins involved in specific metabolic pathways by comparing growth in different electron donor/acceptor combinations .

• Co-expression analysis: Determine if Dhaf_3052 is co-expressed with known components of the OHR machinery, such as reductive dehalogenases (rdh genes) .

• Localization studies: Investigate whether Dhaf_3052 is localized in the cell membrane, which would be consistent with a role in respiratory electron transport chains.

• Protein structure prediction: Analyze whether Dhaf_3052 contains structural features consistent with redox proteins involved in electron transport chains.

The versatile energy metabolism of Desulfitobacterium hafniense involves numerous redox enzyme families , and determining where Dhaf_3052 fits within this complex metabolic network requires systematic investigation across multiple respiratory conditions.

How can researchers investigate potential protein-protein interactions involving Dhaf_3052?

To elucidate the functional network of Dhaf_3052, consider these protein interaction study approaches:

• Affinity purification coupled with mass spectrometry (AP-MS): Use His-tagged Dhaf_3052 as bait to capture interacting proteins directly from Desulfitobacterium hafniense lysates.

• Bacterial two-hybrid system: Adapt yeast two-hybrid methodology for bacterial proteins to screen for potential interactors.

• Co-immunoprecipitation: Develop antibodies against Dhaf_3052 to pull down protein complexes from native cells.

• Crosslinking studies: Use chemical crosslinkers to stabilize transient protein-protein interactions before purification and identification.

• Surface plasmon resonance (SPR) or microscale thermophoresis (MST): Validate and quantify specific interactions identified through screening approaches.

When designing these experiments, it's crucial to include appropriate controls to distinguish specific from non-specific interactions. Given that Desulfitobacterium hafniense has a highly versatile energy metabolism with numerous redox enzyme families , interaction studies may reveal connections to specific metabolic pathways.

What approaches can be used to investigate the evolutionary conservation of UPF0316 family proteins like Dhaf_3052?

To understand the evolutionary context of Dhaf_3052:

• Comprehensive phylogenetic analysis: Compare Dhaf_3052 sequences across bacterial species to trace evolutionary relationships and identify conserved regions that might indicate functional importance.

• Synteny analysis: Examine the genomic context of Dhaf_3052 homologs across species to identify consistently co-located genes that might suggest functional relationships.

• Structural homology modeling: Use available crystal structures of related proteins to model Dhaf_3052 and identify structurally conserved features that might indicate functional sites.

• Selection pressure analysis: Calculate dN/dS ratios across the protein sequence to identify regions under purifying or diversifying selection, providing clues about functionally critical domains.

• Horizontal gene transfer analysis: Determine if the UPF0316 family shows evidence of horizontal gene transfer, which might explain its distribution across bacterial lineages.

This evolutionary perspective could provide valuable insights into the functional importance of Dhaf_3052 and guide experimental approaches to characterize its role in bacterial metabolism.

What are the recommended approaches for studying potential enzymatic activity of Dhaf_3052?

Without prior knowledge of Dhaf_3052's function, a systematic approach to enzymatic characterization includes:

• Bioinformatic prediction: Use tools like InterPro, PFAM, and structure prediction to identify potential catalytic residues or substrate binding pockets.

• High-throughput activity screening: Test purified Dhaf_3052 against panels of potential substrates covering major classes of enzymatic reactions (hydrolases, oxidoreductases, transferases, etc.).

• Metal ion dependency: Test activity in the presence of different metal cofactors, as many UPF (uncharacterized protein family) members require specific metal ions for activity.

• pH and temperature optimization: Determine optimal conditions for activity, which might provide clues about the cellular localization and physiological role.

• Coupled enzyme assays: Design assays where potential Dhaf_3052 activity is linked to a detectable enzymatic reaction.

Given that Desulfitobacterium hafniense displays a highly versatile energy metabolism with capabilities for using different electron donors and acceptors , Dhaf_3052 might play a role in redox reactions, potentially involved in the organohalide respiration pathway or alternative energy conservation mechanisms.

How can proteomics approaches be applied to study Dhaf_3052 expression patterns?

Based on the successful proteomics analysis of Desulfitobacterium hafniense described in reference , researchers can employ similar approaches:

• Tandem Mass Tag (TMT) labeling: This method allowed researchers to identify and quantify almost 60% of the predicted proteome of Desulfitobacterium hafniense strain DCB-2 across six different growth conditions .

• Comparative growth conditions: Design experiments comparing multiple metabolic states, such as:

  • Fermentative vs. respiratory metabolism

  • Different electron donors (e.g., lactate vs. H₂)

  • Different electron acceptors (e.g., fumarate vs. organohalides)

• Data analysis: Apply statistical methods to identify significantly regulated proteins:

  • Use log fold change thresholds (e.g., logFC >1 or <-1)

  • Apply appropriate false discovery rate controls (e.g., FDR <0.05)

  • Perform clustering analysis to group proteins with similar expression patterns

• Validation: Confirm proteomics findings through targeted methods such as:

  • Western blotting with specific antibodies

  • RT-qPCR to correlate protein levels with transcript abundance

  • Targeted proteomics approaches like selected reaction monitoring (SRM)

This approach would allow researchers to determine under which metabolic conditions Dhaf_3052 is most abundantly expressed, providing valuable clues about its functional role in Desulfitobacterium hafniense metabolism.

What experimental design would be optimal for investigating Dhaf_3052's potential role in sulfite reduction pathways?

Building on findings that Desulfitobacterium hafniense utilizes dissimilatory sulfite reduction pathways , researchers could design experiments to investigate Dhaf_3052's potential involvement:

• Experimental design structure:

  • Independent variable: Growth conditions (with/without sulfite or sulfide)

  • Dependent variables: Dhaf_3052 expression levels, growth rates, metabolite production

  • Controls: Wild-type vs. Dhaf_3052 knockout strains

• Specific experimental approaches:

  • Create Dhaf_3052 knockout strains and assess growth with sulfite as an electron acceptor

  • Perform comparative proteomics of wild-type and knockout strains

  • Measure transcription of Dhaf_3052 after sulfide addition using methods similar to those described in reference

  • Analyze growth curves with and without sodium sulfide supplementation

• Data analysis considerations:

  • Apply nested design statistics if multiple factors are involved

  • Use measures of central tendency and variability to assess significance

  • Consider the importance of variability in detecting treatment effects

This experimental design would help determine whether Dhaf_3052 plays a role in the dissimilatory sulfite reduction pathway that was shown to be upregulated in certain growth conditions of Desulfitobacterium hafniense .

How can researchers integrate Dhaf_3052 studies with broader investigations of Desulfitobacterium hafniense metabolism?

To position Dhaf_3052 research within the broader context of Desulfitobacterium hafniense metabolism:

• Multi-omics integration approaches:

  • Combine proteomics data on Dhaf_3052 expression with transcriptomics and metabolomics data

  • Use systems biology approaches to model metabolic networks and predict Dhaf_3052's role

  • Correlate Dhaf_3052 expression patterns with specific metabolic states

• Comparative studies across growth conditions:

  • Design experiments similar to those in reference , comparing proteomes across different respiratory conditions

  • Focus on conditions where Dhaf_3052 shows significant up- or down-regulation

  • Correlate Dhaf_3052 expression with known metabolic pathways

• Integration with genetic mobility studies:

  • Investigate whether Dhaf_3052 is located on mobile genetic elements, as observed for some rdh genes in Desulfitobacterium

  • Assess whether genetic rearrangements affect Dhaf_3052 expression or function

By integrating Dhaf_3052 research with broader studies of Desulfitobacterium hafniense metabolism, researchers can better understand how this protein contributes to the remarkable metabolic versatility of this organism, particularly its ability to use diverse electron donors and acceptors and to grow fermentatively .