Recombinant Treponema denticola Ornithine carbamoyltransferase, catabolic (arcB)

What is the role of catabolic ornithine carbamoyltransferase (arcB) in the T. denticola arginine deiminase system?

The catabolic ornithine carbamoyltransferase (cOTC), encoded by the arcB gene, is a critical component of the arginine deiminase system (ADS) in T. denticola. This enzyme catalyzes the conversion of citrulline to ornithine and carbamoyl phosphate in the catabolic direction. In the ADS, arcB functions alongside arginine deiminase (AD) and carbamate kinase (CK) to metabolize arginine as an energy source.

The complete ADS in T. denticola consists of the following components:

• arcA - encoding arginine deiminase (AD)

• arcB - encoding catabolic ornithine carbamoyltransferase (cOTC)

• arcC - encoding carbamate kinase (CK)

• arcD - encoding the arginine-ornithine antiporter

• arcT - encoding a putative transporter

Transcriptional analysis has shown that these genes are cotranscribed in T. denticola, forming a polycistronic operon . The system allows T. denticola to utilize arginine as a nitrogen source, which is particularly important in the nutrient-limited environment of periodontal pockets.

How does the arcB gene and expression differ between T. denticola strains?

Significant strain-specific variations exist in the expression and functionality of the ADS components, including arcB, among different T. denticola strains. These differences have important implications for metabolism and virulence:

Research has demonstrated that strain TIGR4 actively takes up arginine and releases ornithine, indicating a functional ADS system. In contrast, strain D39 shows significantly lower arginine uptake (20-fold lower than TIGR4) and no detectable ornithine secretion, correlating with lower expression of AD enzymes .

The differences in ADS functionality appear to be linked to the presence of ArgR2, which functions as an activator of the arcABCDT operon in strain-specific patterns. These variations suggest evolutionary adaptations to different oral microenvironments.

What are the best methodological approaches for cloning and expressing recombinant T. denticola arcB in heterologous systems?

The successful expression of recombinant T. denticola arcB requires careful consideration of several methodological factors:

Expression Vector Selection

• Shuttle plasmids derived from the pTS1 cryptic plasmid have been successfully used for expressing T. denticola genes

• E. coli expression vectors like pET series plasmids (particularly pET28b and pET30b) have demonstrated success for recombinant expression of T. denticola proteins

Modification Strategies for Improved Expression

• Codon optimization: T. denticola has different codon usage preferences compared to E. coli. Research has shown that codon optimization can significantly improve expression yields.

• Promoter selection: For expression in T. denticola, the P-msp promoter drives approximately 7-fold higher expression than P-tap1 (normalized to plasmid copy number)

• Tag addition: C-terminal 6×His tags have been successful for purification without compromising enzymatic activity

• Start codon selection: Testing expression from different potential start codons (Met1, Met7, and Val38) may be necessary to determine optimal protein production

Transformation Considerations for T. denticola

When transforming T. denticola with recombinant constructs:

• Consider the restriction-modification systems present in the target strain

• Use SyngenicDNA shuttle plasmids resistant to T. denticola R-M systems to enhance transformation efficiency

• Methylation status of transforming plasmid DNA is critical for successful transformation

How can researchers confirm the enzymatic activity of recombinant T. denticola arcB?

Verification of enzymatic activity for recombinant arcB should employ multiple complementary approaches:

Biochemical Activity Assays

• Citrulline-to-ornithine conversion assay: Measure the phosphorolysis of citrulline, yielding ornithine and carbamoyl phosphate

• Coupled enzyme assay: Linking carbamoyl phosphate production to subsequent reactions that generate measurable products

• Spectrophotometric detection: Monitor the formation of products using appropriate chromogenic substrates

Comparative Analysis Methods

• Compare activity of recombinant protein with that of native protein from T. denticola extracts

• Include appropriate controls such as heat-inactivated enzyme, substrate-free reactions, and reactions with known inhibitors

• Test substrate specificity using structurally similar compounds to confirm reaction specificity

Functional Complementation

Researchers have successfully used E. coli strains deficient in related enzymes for functional complementation studies. For example, E. coli ME8359 (thyA pyrB::Tn5), which lacks aspartate carbamoyltransferase activity, has been used as a host to confirm functional expression of T. denticola carbamoyltransferase enzymes .

What structural features distinguish catabolic OTC (arcB) from anabolic OTC in oral spirochetes?

Catabolic and anabolic ornithine carbamoyltransferases (OTCs) have distinct structural features that reflect their different physiological roles:

Key Distinguishing Features

• 80s Loop Conformation: In catabolic OTCs, the 80s loop has a unique conformation that connects the carbamoyl phosphate binding site to the center of the trimer, influencing cooperativity. This feature is absent in anabolic OTCs .

• Substrate Binding Preferences: Thermal shift assays indicate different substrate preferences that align with their unidirectional functions:

  • cOTC shows higher stability with citrulline (catabolic direction)

  • aOTC shows higher stability with ornithine and carbamoyl phosphate (anabolic direction)

• Active Site Architecture: cOTC exhibits a partially blocked binding site for carbamoyl phosphate, consistent with its preferred reaction direction .

Structural Organization

Both enzymes typically form homotrimeric structures, but with distinct quaternary arrangements that impact substrate binding and catalytic activity. These structural differences explain the preferential direction of catalysis despite the theoretical reversibility of the reaction.

While most research on OTC structural differences has been conducted in other bacterial species, like Psychrobacter sp. PAMC 21119, similar structural principles likely apply to T. denticola arcB based on sequence homology and functional conservation across bacterial species.

How does arcB contribute to T. denticola virulence and inter-species interactions in periodontal disease?

The arcB gene product contributes to T. denticola virulence through multiple mechanisms:

Metabolic Adaptation and Survival

• ArcB enables T. denticola to utilize arginine as an alternative nitrogen source in the nutrient-limited periodontal pocket

• Mouse infection models have demonstrated that deletion of arcABCDT genes attenuates T. denticola TIGR4, confirming the importance of this system for in vivo fitness

Polymicrobial Interactions

T. denticola functions in a polymicrobial community where ornithine produced by ArcB can impact other species:

• Metabolic cross-feeding: Similar to S. gordonii, the ornithine exported by T. denticola ArcD (after ArcB activity) may support the growth of other periodontal pathogens like Fusobacterium nucleatum

• Community development: Studies with S. gordonii demonstrated that "sustained delivery of ornithine from accessory pathogens induce a state of dysbiosis, by sustaining the growth of the entire microbial community" . This principle likely applies to T. denticola's role as well.

• Synergistic virulence: T. denticola exhibits synergistic virulence with Porphyromonas gingivalis. This cooperation includes metabolic interactions where P. gingivalis provides glycine through proteolysis, which T. denticola uses as a major energy and carbon source .

Immune Evasion

Products of arginine metabolism may help T. denticola evade host immune responses through:

• Modulation of nitric oxide production, which depends on arginine availability

• Alteration of the local microenvironment pH, affecting neutrophil function

• Interference with arginine-dependent antimicrobial mechanisms

What methods are most effective for purifying recombinant T. denticola arcB?

Successful purification of recombinant T. denticola arcB requires careful consideration of protein properties and purification conditions:

Affinity Chromatography

• Immobilized Metal Affinity Chromatography (IMAC):

  • His-tagged recombinant arcB can be effectively purified using nickel affinity chromatography

  • PrtP has been shown to copurify with PrcB-6×His in nickel affinity chromatography, suggesting similar approaches may work for arcB-associated proteins

• Tag selection considerations:

  • C-terminal 6×His tags have been successfully used for T. denticola proteins

  • Tags should be positioned to avoid interfering with the active site

Protein Solubility Enhancement

T. denticola proteins can present solubility challenges when expressed in E. coli. Consider these approaches:

• Expression at lower temperatures (16-25°C)

• Use of solubility-enhancing fusion partners (MBP, SUMO, etc.)

• Addition of compatible solutes or mild detergents during purification

• Optimization of buffer conditions based on theoretical isoelectric point

Chromatographic Strategies

A multi-step purification approach is often required:

• Initial capture using affinity chromatography

• Intermediate purification using ion exchange chromatography

• Polishing using size exclusion chromatography to obtain homogeneous protein preparations

Activity Preservation

During purification, maintaining arcB activity requires careful attention to:

• Inclusion of reducing agents (2-mercaptoethanol or dithiothreitol) to preserve enzyme activity

• Avoiding serine protease inhibitors like TLCK that can inactivate the enzyme

• Protection from proteinase K, which has been shown to inactivate related enzymes

How is arcB expression regulated in T. denticola compared to other bacterial species?

The regulation of arcB expression in T. denticola shows both similarities and differences compared to other bacterial species:

denticola Regulation

In T. denticola, arcB is part of the arcABCDT operon, which research indicates is regulated by:

• ArgR-type regulators: T. denticola genomes contain three ArgR-type regulators (ArgR1, ArgR2, and AhrC) involved in regulating arginine metabolism

• ArgR2 as specific activator: In strain TIGR4, ArgR2 binds to promoter sequences of the arc operon and functions as an activator. EMSAs confirm that ArgR2 binds to both arcA and arcD promoter regions

• Strain-specific regulation: Unlike TIGR4, strain D39 lacks ArgR2 expression and constitutively expresses the ADS with a truncated nonfunctional AD

Comparison with Other Species

This comparison reveals that while many bacteria regulate ADS components in response to arginine availability, T. denticola has evolved strain-specific regulatory mechanisms that may reflect adaptation to different oral microenvironments.

How does the relationship between arcB and other ADS components impact experimental design for recombinant expression?

The functional relationship between arcB and other ADS components has important implications for experimental design when working with recombinant proteins:

Operon Structure and Co-expression Considerations

Northern blot analysis with probes specific to arcA, arcB, arcC, and arcDT mRNAs detected transcripts of 2.6 and 1.6 kb in wild-type TIGR4, indicating the genes form a regulatory unit . This co-transcription suggests:

• Potential for protein-protein interactions: ArcB may function optimally when co-expressed with other ADS components

• Expression stoichiometry: Natural expression levels of ADS components may be important for proper function, suggesting multi-protein expression systems may be valuable

Experimental Design Implications

When designing experiments with recombinant arcB:

• Co-expression strategies: Consider co-expressing arcB with other ADS components, particularly:

  • arcA (arginine deiminase) - supplies the substrate for arcB

  • arcC (carbamate kinase) - utilizes the product of arcB

• Functional assays: Design assays that can detect coupled enzymatic activities of the full ADS system:

  • Measure arginine consumption and ornithine production

  • Detect ATP generation through the complete pathway

• Reconstitution experiments: Combine individually purified components to reconstruct the functional pathway in vitro

• Protein interaction studies: Investigate whether physical interactions between ArcB and other ADS components enhance enzymatic activity

What unique aspects of arcB should researchers consider when designing site-directed mutagenesis experiments?

When planning site-directed mutagenesis of T. denticola arcB, researchers should consider these key aspects:

Essential Functional Residues

Based on homology with other ornithine carbamoyltransferases and studies of related enzymes:

• Carbamyl phosphate binding residues: These highly conserved residues are essential for substrate binding and should be primary targets for mutagenesis studies

• 80s loop region: This region plays a critical role in catalytic activity of catabolic OTCs, with a unique conformation that influences cooperativity by connecting the CP binding site and the center of the trimer

• Oligomerization interface: Residues at the trimerization interface are important for maintaining the functional quaternary structure

Strain-Specific Variation Hotspots

Comparing arcB sequences across T. denticola strains can identify:

• Regions of high sequence conservation (likely essential for function)

• Variable regions that may contribute to strain-specific functional differences

Functional Domain Architecture

When designing mutagenesis experiments, consider:

• Domain organization: Target mutations to specific functional domains rather than randomly throughout the protein

• Regulatory regions: Investigate potential regulatory sites that might affect enzyme activity or response to environmental factors

• Post-translational modification sites: Identify potential modification sites based on homology to other species (e.g., acetylation at Lys residues, similar to human OTC where acetylation at Lys-88 negatively regulates activity )

Contextual Considerations

• Genetic background: Effects of mutations may differ between T. denticola strains due to differences in genetic background and regulation

• Expression context: Mutations may have different effects when arcB is expressed alone versus in the context of the complete ADS operon

• Experimental validation: Plan for both in vitro enzymatic assays and in vivo functional studies to comprehensively characterize mutant phenotypes