Recombinant Pelobacter carbinolicus UPF0316 protein Pcar_2434 (Pcar_2434)

What is Pelobacter carbinolicus and what makes its metabolism unique?

Pelobacter carbinolicus is a bacterium with versatile metabolic capabilities, able to grow through multiple mechanisms including fermentation, syntrophic hydrogen/formate transfer, and electron transfer to sulfur from short-chain alcohols, hydrogen, or formate. Unlike its Geobacter relatives, P. carbinolicus does not oxidize acetate and has not been observed to ferment sugars or grow autotrophically . Genome analysis has revealed that P. carbinolicus possesses unexpected metabolic pathways for catabolism of various substrates including 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline, and ethanol . This metabolic flexibility allows the organism to thrive in specialized ecological niches.

What is UPF0316 protein Pcar_2434 and why is it of research interest?

UPF0316 protein Pcar_2434 is a protein encoded in the genome of Pelobacter carbinolicus with currently uncharacterized function (UPF), as indicated by its classification in the UPF0316 family . This protein is of particular research interest because it may play a role in the unique metabolic capabilities of P. carbinolicus. Studying uncharacterized proteins like Pcar_2434 is crucial for fully understanding the organism's physiological features and metabolic versatility . As genomic analysis has revealed that P. carbinolicus possesses more metabolic capabilities than previously anticipated, characterizing proteins like Pcar_2434 may lead to discoveries of novel biochemical pathways or regulatory mechanisms.

How does recombinant production of Pcar_2434 differ across expression systems?

Recombinant production of Pcar_2434 can be accomplished using various expression systems including yeast, E. coli, baculovirus, and mammalian cells, each offering distinct advantages . The E. coli system typically provides high protein yields with relatively simple culture conditions, making it suitable for initial structural studies. The yeast expression system (CSB-YP662632PAAO1) offers eukaryotic post-translational modifications while maintaining moderate to high yields .

For more complex studies requiring mammalian-like glycosylation patterns, the mammalian cell expression system (CSB-MP662632PAAO1) is preferable despite its higher cost and lower yields. The baculovirus expression system (CSB-BP662632PAAO1) offers a middle ground, with insect cell post-translational modifications and relatively high yields . Methodologically, researchers should select the expression system based on their specific experimental requirements, considering factors such as required protein yield, post-translational modifications, and downstream applications.

What experimental approaches are most effective for determining the function of uncharacterized proteins like Pcar_2434?

For uncharacterized proteins like Pcar_2434, a multi-faceted experimental approach is most effective. Begin with in silico analysis including sequence homology searches, protein domain prediction, and structural modeling to generate initial functional hypotheses. Follow with biochemical characterization techniques including substrate binding assays, enzymatic activity screens, and protein-protein interaction studies.

A methodological workflow might include:

• Phylogenetic analysis to identify evolutionary relationships

• Expression and purification of recombinant Pcar_2434

• Structural determination via X-ray crystallography or NMR spectroscopy

• Protein-protein interaction studies using pull-down assays or yeast two-hybrid screens

• Knockout/knockdown studies in P. carbinolicus to observe phenotypic changes

• Metabolomic analysis comparing wild-type and Pcar_2434-deficient strains

This comprehensive approach allows for triangulation of function from multiple experimental angles. Given that P. carbinolicus exhibits unique metabolic capabilities including specialized electron transfer mechanisms , testing Pcar_2434 for involvement in these pathways should be prioritized.

How should researchers design experiments to investigate Pcar_2434's potential role in the TCA cycle of P. carbinolicus?

To investigate Pcar_2434's potential role in the TCA cycle of P. carbinolicus, researchers should employ a systematic experimental design approach:

• Isotope labeling studies: Use 13C-labeled substrates (such as acetate, pyruvate, or other TCA cycle intermediates) combined with mass spectrometry to track metabolic flux through the TCA cycle in wild-type versus Pcar_2434 knockout strains.

• Enzyme activity assays: Prepare purified recombinant Pcar_2434 and test its activity with various TCA cycle intermediates and cofactors. Monitor substrate consumption and product formation using spectrophotometric methods or HPLC.

• Protein-protein interaction studies: Perform co-immunoprecipitation experiments to identify if Pcar_2434 interacts with known TCA cycle enzymes. This is particularly important since genomic analysis has revealed that P. carbinolicus contains catabolic glutamate dehydrogenases, suggesting that the TCA cycle can function catabolically .

• Growth phenotype characterization: Compare growth rates of wild-type and Pcar_2434-deficient strains on various carbon sources that require TCA cycle function, particularly under conditions where P. carbinolicus is known to utilize syntrophic hydrogen/formate transfer or electron transfer to sulfur .

Each experiment should include appropriate controls and be repeated with biological triplicates to ensure statistical significance. The integration of results from these complementary approaches will provide robust evidence for Pcar_2434's role in TCA cycle function.

What are the critical considerations for designing protein-protein interaction studies involving Pcar_2434?

When designing protein-protein interaction studies for Pcar_2434, researchers must consider several critical factors:

These considerations ensure that protein-protein interaction studies with Pcar_2434 produce reliable and physiologically relevant results that can inform our understanding of its role in P. carbinolicus metabolism.

How can researchers determine if Pcar_2434 contributes to the electroconductive properties of P. carbinolicus?

To investigate Pcar_2434's potential contribution to electroconductive properties of P. carbinolicus, researchers should implement a multi-faceted experimental approach:

• Comparative genomic analysis: Analyze Pcar_2434's sequence and structural similarities to the three identified proteins in P. carbinolicus with similarity to geopilin of electroconductive nanowires . This computational approach can provide initial evidence for functional relationships.

• Protein localization studies: Use fluorescently tagged Pcar_2434 or immunolocalization techniques to determine if the protein is associated with cell surface appendages in P. carbinolicus. Co-localization with known conductive appendage proteins would support a role in conductivity.

• Knockout/complementation studies: Generate Pcar_2434 knockout strains and measure changes in electrical conductivity using techniques such as:

  • Scanning tunneling microscopy

  • Conductive atomic force microscopy

  • Four-point probe measurements of biofilms

• Purified protein conductivity measurements: Reconstitute purified Pcar_2434 into liposomes or solid-state devices and measure electron transfer capabilities under varying redox conditions.

• Heterologous expression: Express Pcar_2434 in model organisms lacking conductive appendages and assess if conductivity is conferred.

The results should be analyzed in the context of P. carbinolicus's known electron transfer mechanisms, particularly its ability to transfer electrons to sulfur from short-chain alcohols, hydrogen, or formate .

What methodological approaches can determine if Pcar_2434 plays a role in the novel asparaginyl-tRNA formation in P. carbinolicus?

To investigate Pcar_2434's potential role in novel asparaginyl-tRNA formation in P. carbinolicus, researchers should employ the following methodological approaches:

• Sequence and structural analysis: Compare Pcar_2434 with known tRNA synthetases and RNA-modifying enzymes to identify potential functional domains related to tRNA processing.

• In vitro tRNA binding assays: Using purified recombinant Pcar_2434 (preferably from E. coli expression system CSB-EP662632PAAO1 ), perform electrophoretic mobility shift assays (EMSAs) with labeled tRNA^Asn to detect direct interactions.

• tRNA aminoacylation assays: Develop an in vitro system containing purified Pcar_2434, asparagine, ATP, and tRNA^Asn to test for aminoacylation activity, measured by incorporation of radioactive asparagine into tRNA.

• Structural studies of Pcar_2434-tRNA complexes: Utilize X-ray crystallography or cryo-electron microscopy to determine the structure of Pcar_2434 in complex with tRNA^Asn, providing insights into the mechanism of interaction.

• Genetic complementation experiments: Express Pcar_2434A in organisms with known defects in asparaginyl-tRNA formation to test for functional complementation.

These approaches are particularly relevant given that genomic analysis has revealed the absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes, suggesting P. carbinolicus may make asparaginyl-tRNA in a novel way .

How can researchers determine whether Pcar_2434 functions in the phosphotransferase system for sugar uptake in P. carbinolicus?

To investigate Pcar_2434's potential role in the phosphotransferase system (PTS) for sugar uptake in P. carbinolicus, researchers should implement the following methodological approach:

• Domain analysis and structural prediction: Compare Pcar_2434's sequence and predicted structure with known PTS components (EI, HPr, EIIA, EIIB, EIIC) to identify potential functional domains or structural similarities.

• Phosphorylation studies:

  • Perform in vitro phosphorylation assays with radiolabeled ATP or phosphoenolpyruvate (PEP) to determine if Pcar_2434 can be phosphorylated

  • Test if Pcar_2434 can transfer phosphate groups to other proteins in the PTS cascade

• Sugar transport assays:

  • Create Pcar_2434 knockout strains and measure uptake of radiolabeled sugars

  • Reconstitute purified Pcar_2434 into liposomes and assess sugar transport capabilities

• Protein-protein interaction studies:

  • Use pull-down assays with biotinylated Pcar_2434 (utilizing the Avi-tag Biotinylated version, CSB-EP662632PAAO1-B ) to identify interactions with other PTS components

  • Perform bacterial two-hybrid screening to map the PTS interactome in P. carbinolicus

• Comparative metabolomics:

  • Compare intracellular and extracellular sugar concentrations in wild-type versus Pcar_2434-deficient strains

This systematic approach will provide evidence for or against Pcar_2434's involvement in the phosphotransferase system for sugar uptake, which was discovered through genomic analysis of P. carbinolicus .

What are the most effective approaches for determining the three-dimensional structure of Pcar_2434?

For determining the three-dimensional structure of Pcar_2434, researchers should consider a hierarchical approach utilizing complementary techniques:

The selection of method should consider Pcar_2434's properties, including stability, solubility, and tendency to form crystals. Regardless of the approach, researchers should verify structural models through biochemical validation experiments.

How should researchers approach comparative analysis between Pcar_2434 and homologous proteins in related species?

For comparative analysis between Pcar_2434 and homologous proteins in related species, researchers should implement a systematic approach combining computational and experimental methods:

• Sequence-based analysis:

  • Perform multiple sequence alignment of UPF0316 family proteins across bacterial species

  • Identify conserved residues and motifs that may indicate functional importance

  • Calculate evolutionary rates across different regions to identify selection pressures

  • Generate phylogenetic trees to understand evolutionary relationships

• Structure-based comparisons:

  • Superimpose Pcar_2434 structure (experimental or predicted) with homologous proteins

  • Analyze conservation patterns in the context of three-dimensional structure

  • Identify structural motifs that may indicate shared functions

• Genomic context analysis:

  • Compare gene neighborhoods of Pcar_2434 and homologs across species

  • Identify conserved gene clusters that may indicate functional associations

  • Analyze co-evolution patterns with interacting partners

• Functional comparison:

  • Design experiments to test whether homologs can complement Pcar_2434 deletion

  • Compare biochemical properties (substrate specificity, kinetic parameters)

  • Analyze expression patterns across different conditions

This approach is particularly relevant given that P. carbinolicus shows metabolic differences from its Geobacter relatives . By comparing Pcar_2434 with homologs in both closely and distantly related species, researchers can gain insights into its specialized function in P. carbinolicus and identify conserved mechanisms across bacterial species.

What experimental designs can effectively test structure-function relationships in Pcar_2434?

To effectively test structure-function relationships in Pcar_2434, researchers should implement a systematic site-directed mutagenesis approach combined with functional assays:

• Rational design of mutations:

  • Target conserved residues identified through sequence alignment of UPF0316 family proteins

  • Focus on predicted active sites or binding interfaces based on structural analysis

  • Create a library of point mutations, deletions, and domain swaps

• Expression and purification of mutant proteins:

  • Use the E. coli expression system (CSB-EP662632PAAO1) to produce wild-type and mutant Pcar_2434 variants

  • Verify protein integrity through circular dichroism spectroscopy and thermal stability assays

  • Confirm purity (>85% by SDS-PAGE) for all variants

• Functional characterization:

  • Develop quantitative assays based on hypothesized functions (e.g., enzymatic activity, protein binding, nucleic acid interaction)

  • Compare kinetic parameters (Km, kcat) between wild-type and mutant proteins

  • Measure binding affinities using isothermal titration calorimetry or surface plasmon resonance

• Structural analysis of mutants:

  • Determine structures of functionally significant mutants

  • Analyze structural perturbations caused by mutations

  • Correlate structural changes with functional alterations

• In vivo validation:

  • Test the ability of mutant variants to complement Pcar_2434 deletion in P. carbinolicus

  • Analyze phenotypic effects under various growth conditions that highlight P. carbinolicus' unique metabolic capabilities

This approach allows researchers to establish causal relationships between specific structural elements and functional properties of Pcar_2434, providing mechanistic insights into its role in P. carbinolicus metabolism.

How can researchers integrate Pcar_2434 functional studies with broader metabolic pathway analysis in P. carbinolicus?

To integrate Pcar_2434 functional studies with broader metabolic pathway analysis in P. carbinolicus, researchers should implement a systems biology approach:

• Metabolic flux analysis:

  • Cultivate wild-type and Pcar_2434-knockout strains with 13C-labeled substrates

  • Measure isotope distribution patterns in metabolic intermediates using LC-MS or GC-MS

  • Calculate flux distributions through central carbon metabolism

  • Compare fluxes during growth on various substrates (2,3-butanediol, acetoin, glycerol, ethanolamine, choline, ethanol)

• Transcriptomics and proteomics integration:

  • Perform RNA-Seq and quantitative proteomics on wild-type and Pcar_2434-deficient strains

  • Identify differentially expressed genes and proteins in metabolic pathways

  • Construct co-expression networks to identify metabolic modules associated with Pcar_2434

• Metabolic modeling:

  • Develop or refine genome-scale metabolic models of P. carbinolicus

  • Simulate metabolic phenotypes with and without Pcar_2434 function

  • Identify potential metabolic bottlenecks and alternative pathways

• Integration with electron transfer mechanisms:

  • Investigate how Pcar_2434 may interact with the electron transfer pathways for syntrophic growth or sulfur reduction

  • Measure redox potentials and electron transfer rates in different genetic backgrounds

  • Analyze potential connections to the TCA cycle functioning catabolically

This integrated approach will place Pcar_2434 function within the context of P. carbinolicus' unique metabolic capabilities, particularly its versatile growth strategies through fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur .

What experimental designs can test whether Pcar_2434 is involved in the pyruvate:ferredoxin/flavodoxin oxidoreductase pathway?

To test whether Pcar_2434 is involved in the pyruvate:ferredoxin/flavodoxin oxidoreductase pathway, which has been identified as a potential metabolic bottleneck in P. carbinolicus , researchers should implement the following experimental design:

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation experiments with Pcar_2434 and pyruvate:ferredoxin/flavodoxin oxidoreductase

  • Use the biotinylated version of Pcar_2434 (CSB-EP662632PAAO1-B) for pull-down assays

  • Confirm interactions with orthogonal methods such as bacterial two-hybrid or FRET analysis

• Enzymatic activity measurements:

  • Reconstitute the pyruvate:ferredoxin/flavodoxin oxidoreductase system in vitro

  • Add purified Pcar_2434 and measure changes in activity

  • Monitor electron transfer using spectrophotometric methods with artificial electron acceptors

  • Test activity under various redox conditions relevant to P. carbinolicus metabolism

• Metabolomics analysis:

  • Compare metabolite levels in wild-type and Pcar_2434 knockout strains

  • Focus on pyruvate, acetyl-CoA, and TCA cycle intermediates

  • Measure oxaloacetate levels specifically, since pyruvate:ferredoxin/flavodoxin oxidoreductase was identified as a bottleneck in oxaloacetate supply

• Growth phenotype characterization:

  • Test growth of wild-type and Pcar_2434-deficient strains under conditions requiring pyruvate:ferredoxin/flavodoxin oxidoreductase activity

  • Analyze growth on different carbon sources with varying requirements for this pathway

  • Perform growth competition experiments in mixed cultures

The results from these experiments would provide evidence for or against Pcar_2434's involvement in this critical metabolic pathway and potentially explain its role in the supply of oxaloacetate for the TCA cycle or the connection of glycolysis to ethanol production .

How can researchers utilize Pcar_2434 in synthetic biology applications for enhanced electron transfer systems?

To utilize Pcar_2434 in synthetic biology applications for enhanced electron transfer systems, researchers should follow this methodological framework:

• Functional characterization in heterologous hosts:

  • Express recombinant Pcar_2434 in model organisms such as E. coli or Shewanella oneidensis

  • Measure changes in electron transfer capabilities using electrochemical techniques

  • Quantify improvements in extracellular electron transfer using microbial fuel cell setups

  • Test performance under various electron donor and acceptor conditions

• Design of synthetic electron transfer modules:

  • Create fusion proteins linking Pcar_2434 with known electron transfer components

  • Design synthetic operons that co-express Pcar_2434 with complementary proteins

  • Incorporate regulatory elements for controlled expression

  • Test modular designs in different host organisms

• Optimization for specific applications:

  • For bioremediation: Engineer systems with Pcar_2434 for enhanced reduction of environmental contaminants

  • For bioelectrochemical systems: Optimize electrode interfaces with Pcar_2434-based electron conduits

  • For biosensors: Develop Pcar_2434-based systems that translate electron transfer to detectable signals

• Performance assessment in applied settings:

  • Evaluate long-term stability of engineered systems

  • Measure electron transfer rates and compare to natural systems

  • Quantify product formation in biotransformation applications

This approach leverages P. carbinolicus' natural capability for electron transfer to sulfur and the potential involvement of Pcar_2434 in electroconductive structures similar to the geopilin of electroconductive nanowires . The biotinylated version of Pcar_2434 (CSB-EP662632PAAO1-B) could be particularly valuable for creating oriented attachment to electrodes or other conductive surfaces.

What experimental designs can elucidate Pcar_2434's role in the adaptation of P. carbinolicus to different environmental conditions?

To elucidate Pcar_2434's role in P. carbinolicus' adaptation to different environmental conditions, researchers should implement a comprehensive experimental design that integrates multiple levels of analysis:

• Transcriptional regulation analysis:

  • Perform RNA-Seq under diverse growth conditions (varying electron donors/acceptors, nutrient limitations, stress conditions)

  • Quantify Pcar_2434 expression levels across conditions

  • Identify transcription factors regulating Pcar_2434 using ChIP-seq or DNA affinity purification

  • Characterize the promoter region through reporter gene assays

• Comparative fitness studies:

  • Create competition experiments between wild-type and Pcar_2434 knockout strains

  • Monitor population dynamics using strain-specific markers

  • Test relative fitness across environmental gradients (pH, temperature, redox potential)

  • Measure growth parameters in conditions mimicking natural habitats

• Stress response characterization:

  • Expose cultures to oxidative stress, nutrient limitation, or temperature shifts

  • Compare survival rates between wild-type and Pcar_2434-deficient strains

  • Measure biochemical indicators of stress response

  • Analyze protein stability and turnover under stress conditions

• In situ analysis:

  • Develop antibodies or fluorescent tags for detecting Pcar_2434 in environmental samples

  • Quantify expression levels in natural habitats

  • Correlate expression with environmental parameters

  • Track seasonal or geographical variations in expression

This multi-faceted approach will provide insights into how Pcar_2434 contributes to P. carbinolicus' remarkable metabolic versatility , particularly its ability to switch between different growth modes (fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur) in response to environmental conditions.