Recombinant Burkholderia cepacia Anthranilate 1,2-dioxygenase regulatory protein (andR)

What is the andR regulatory protein in Burkholderia cepacia and what is its primary function?

The andR protein in Burkholderia cepacia is an AraC/XylS-type transcriptional regulator that positively controls the expression of genes encoding anthranilate 1,2-dioxygenase. This regulatory protein is indispensable for the stimulation of the anthranilate operon promoter (Pant) and plays a crucial role in the metabolism of aromatic compounds. In B. cepacia, andR specifically regulates the andAcAdAbAa gene cluster, which encodes the components of anthranilate 1,2-dioxygenase (AntDO-3C), a three-component Rieske-type [2Fe-2S] dioxygenase system . The enzyme system consists of a reductase (AndAa), a ferredoxin (AndAb), and a two-subunit oxygenase (AndAcAd), which collectively catalyze the conversion of anthranilate to catechol in the degradation pathway of aromatic compounds.

How does the structure of andR relate to its regulatory function?

AndR belongs to the AraC/XylS family of transcriptional regulators, which typically contain a DNA-binding domain with a helix-turn-helix motif in the C-terminal region and a ligand-binding domain in the N-terminal region. The protein's structure allows it to recognize specific DNA sequences in the promoter region of the anthranilate dioxygenase genes. Upon binding of anthranilate (the inducer molecule) to the N-terminal domain, andR undergoes a conformational change that enables it to bind to the promoter region and recruit RNA polymerase, thereby initiating transcription of the downstream genes . This structural feature explains why andR responds specifically to anthranilate but not to other related molecules like catechol, ensuring pathway-specific regulation.

What genomic organization is typical for the andR regulatory system in Burkholderia cepacia?

In B. cepacia, the andR gene is typically located upstream of the anthranilate dioxygenase gene cluster. Research has shown that in some strains, andR (also referred to as ORF23 in certain studies) is tandemly located with another regulatory gene (ORF22) approximately 3.2 kb upstream of the antA gene . The anthranilate dioxygenase genes in B. cepacia DBO1 are organized as andAcAdAbAa, which differs from the organization seen in other bacteria. This genomic arrangement facilitates coordinated regulation of the entire pathway, as andR can influence the expression of multiple genes involved in anthranilate metabolism from a single regulatory position.

What are the recommended methods for cloning and expressing recombinant andR protein?

For successful cloning and expression of recombinant andR from Burkholderia cepacia, researchers should consider the following methodological approach:

• PCR Amplification: Design primers targeting the complete andR coding sequence with appropriate restriction sites for subsequent cloning.

• Expression Vector Selection: Choose an E. coli expression system with an inducible promoter (such as T7 or tac) and appropriate tags for purification (His-tag or SUMO-tag systems have shown good results) .

• Expression Conditions: Optimize expression conditions with the following parameters:

  Parameter	Recommended Range	Notes
  Host strain	E. coli BL21(DE3), Rosetta	Strains with enhanced rare codon expression may improve yields
  Induction temperature	16-25°C	Lower temperatures reduce inclusion body formation
  IPTG concentration	0.1-0.5 mM	Lower concentrations often yield more soluble protein
  Induction time	4-16 hours	Longer times at lower temperatures are often beneficial

• Protein Purification: Implement a two-step purification strategy using affinity chromatography followed by size exclusion chromatography for highest purity.

• Functional Verification: Confirm DNA-binding activity using electrophoretic mobility shift assays (EMSA) with the Pant promoter region.

How can researchers effectively study the interaction between andR and its promoter region?

To study the interaction between andR and its target promoter, researchers should employ multiple complementary techniques:

• Electrophoretic Mobility Shift Assay (EMSA): Use purified recombinant andR protein with labeled DNA fragments containing the promoter region. Systematically test different fragments to identify the specific binding site. Include anthranilate in the reaction mixture to test inducer-dependent binding.

• DNase I Footprinting: This method can precisely identify the nucleotides protected by andR binding, providing base-pair resolution of the binding site.

• Reporter Gene Assays: Construct luciferase or β-galactosidase reporter systems containing the Pant promoter region and various truncations to identify the minimal region required for andR-dependent activation . This approach is particularly useful for defining the region up to at least 70 bp from the transcription start point that has been shown necessary for activation.

• ChIP-seq: For genome-wide binding studies, chromatin immunoprecipitation followed by sequencing can identify all andR binding sites across the genome.

• Surface Plasmon Resonance (SPR): This technique allows real-time measurement of binding kinetics between andR and its DNA targets, enabling determination of association and dissociation constants.

What approaches are most effective for studying andR-mediated gene regulation in vivo?

For studying andR-mediated regulation in living bacterial cells, the following methodological approaches are recommended:

• Gene Knockout and Complementation: Generate andR deletion mutants and complement with wild-type or mutated versions to establish causality in regulatory phenomena. This reveals the specific genes and phenotypes controlled by andR.

• RT-PCR and Northern Blotting: These techniques can verify operon structure and quantify transcript levels under different conditions . They're particularly useful for confirming that antABC forms a single transcriptional unit and for measuring the induction of gene expression by anthranilate.

• RNA-seq: This provides a comprehensive view of all transcriptional changes influenced by andR, revealing the complete regulon.

• Promoter-Reporter Fusions: Construct fusions between the Pant promoter and reporter genes (luciferase, GFP) to monitor promoter activity in real-time under various conditions and in different genetic backgrounds .

• Time-Course Experiments: Monitor changes in gene expression following anthranilate addition to cultures to understand the kinetics of andR-mediated regulation.

How does anthranilate induce andR-dependent gene expression?

Anthranilate functions as a specific inducer for andR-dependent gene expression through the following mechanism:

• In the absence of anthranilate, andR has limited or no affinity for the promoter region of the anthranilate dioxygenase genes, resulting in minimal transcription.

• When anthranilate is present, it binds to the ligand-binding domain of andR, triggering a conformational change that enhances the protein's affinity for specific DNA sequences in the promoter region.

• The anthranilate-bound andR recognizes and binds to the Pant promoter region, which has been shown to require at least 70 bp upstream of the transcription start site for activation .

• Upon binding to the promoter, andR recruits RNA polymerase, facilitating transcription initiation of the downstream anthranilate dioxygenase genes.

• The resulting increased expression of anthranilate dioxygenase enables the bacterial cell to metabolize anthranilate more efficiently, converting it to catechol for further degradation.

Notably, research has demonstrated that catechol does not induce andR-dependent gene expression, highlighting the specificity of the regulatory mechanism . This specificity ensures that the degradation pathway is only activated when the appropriate substrate is available.

How is the andR regulatory system integrated with other metabolic pathways in Burkholderia cepacia?

The andR regulatory system is strategically integrated with multiple metabolic pathways in B. cepacia:

• Carbazole Degradation Pathway: In Pseudomonas resinovorans strain CA10 (which has similar pathway organization), the andR regulatory system coordinates with the carbazole degradation pathway. The plasmid pCAR1 contains both the car gene cluster (carAaAaBaBbCAcAdDFE) for converting carbazole to anthranilate and the ant gene cluster (antABC) for converting anthranilate to catechol . Both operons appear to be regulated by the same AntR/AndR protein in the presence of anthranilate.

• Tryptophan Catabolism: Anthranilate is an important intermediate in tryptophan metabolism, connecting amino acid catabolism to aromatic compound degradation. The andR-regulated pathway provides a mechanism for utilizing tryptophan-derived anthranilate as a carbon and energy source .

• Quorum Sensing Systems: Burkholderia cepacia complex strains utilize N-acylhomoserine lactone (AHL) quorum-sensing systems that regulate various functions including protease production, swarming motility, biofilm formation, and pathogenicity . There may be cross-talk between these systems and the andR regulatory system, particularly in response to environmental stresses.

This integrated network allows B. cepacia to respond efficiently to changing environmental conditions and available carbon sources, optimizing resource utilization and metabolic flexibility.

What structural features of andR determine its specificity for anthranilate versus other aromatic compounds?

This advanced research question focuses on the molecular basis of andR's ligand specificity. Researchers investigating this question should consider:

• Protein Crystallography: Determine the three-dimensional structure of andR both with and without bound anthranilate to identify specific amino acid residues involved in ligand recognition.

• Site-Directed Mutagenesis: Based on structural data or homology modeling, systematically mutate potential ligand-binding residues and assess changes in binding affinity and transcriptional activation. Design an experimental matrix:

  Mutated Residue	Predicted Function	Anthranilate Binding	Promoter Activation
  Residue X	Direct ligand contact	Measure by SPR/ITC	Measure by reporter assay
  Residue Y	Conformational change	Measure by SPR/ITC	Measure by reporter assay

• Ligand Competition Assays: Test structurally similar compounds (e.g., benzoate, salicylate, 2-chlorobenzoate) for their ability to compete with anthranilate for andR binding, providing insights into the structural requirements for recognition.

• Molecular Dynamics Simulations: Perform computational analyses to model the dynamics of ligand binding and consequent conformational changes, identifying key interaction networks within the protein.

Understanding these specificity determinants could enable the engineering of andR variants with altered substrate specificity for biotechnological applications.

How do mutations in the andR gene affect the efficiency of anthranilate degradation in Burkholderia cepacia?

To address this research question comprehensively, researchers should design experiments that:

• Generate a Library of andR Mutants: Create both random and targeted mutations throughout the andR gene, focusing on:

  • DNA-binding domain mutations

  • Ligand-binding domain mutations

  • Linker region mutations

  • Promoter mutations affecting andR expression levels

• Functional Characterization:

  • Measure growth rates on anthranilate as sole carbon source

  • Quantify anthranilate consumption rates using HPLC

  • Determine enzyme activity levels of anthranilate 1,2-dioxygenase

  • Analyze transcription levels of andAcAdAbAa genes via RT-qPCR

• Phenotypic Analysis: Compare wild-type and mutant strains for:

  • Biofilm formation capacity

  • Stress tolerance

  • Competitive fitness in mixed cultures

  • Growth on alternative carbon sources

This comprehensive approach would reveal how different domains of andR contribute to regulatory efficiency and metabolic function, potentially identifying mutations that enhance anthranilate degradation for bioremediation applications.

What is the interplay between the andR regulatory system and quorum sensing in Burkholderia cepacia complex species?

This complex research question explores potential cross-regulation between two important signaling systems in B. cepacia. A methodical investigation would include:

• Dual Reporter Systems: Construct strains containing both andR-regulated promoters (Pant-luxCDABE) and QS-regulated promoters (PcepI-gfp) to simultaneously monitor both regulatory systems.

• Mutant Analyses: Generate and characterize:

  • andR deletion mutants - examine effects on QS-regulated phenotypes

  • QS system mutants (cepI/cepR) - examine effects on andR-regulated gene expression

  • Double mutants - determine epistatic relationships

• Temporal Expression Studies: Monitor the timing of andR and QS gene expression during growth phases to identify potential sequential activation.

• Protein-Protein Interaction Studies: Use techniques such as bacterial two-hybrid, co-immunoprecipitation, or FRET to detect potential physical interactions between andR and QS regulatory proteins.

Expected outcomes might include the identification of QS-regulated expression of andR, anthranilate-dependent modulation of QS signaling, or convergent regulation of downstream targets by both systems.

How does the andR-regulated anthranilate dioxygenase system in Burkholderia cepacia differ from analogous systems in other bacteria?

The anthranilate dioxygenase system in Burkholderia cepacia has several distinguishing features compared to similar systems in other bacteria:

• Component Structure: B. cepacia DBO1 contains a three-component anthranilate dioxygenase (AntDO-3C) composed of a reductase (AndAa), a ferredoxin (AndAb), and a two-subunit oxygenase (AndAcAd). This differs significantly from the two-component systems (an oxygenase and a reductase) found in Acinetobacter sp. strain ADP1, P. aeruginosa PAO1, and P. putida P111 .

• Evolutionary Relationships: The AntDO-3C system shows closer phylogenetic relationships to aromatic hydrocarbon dioxygenases from Novosphingobium aromaticivorans F199 and Sphingomonas yanoikuyae B1, as well as 2-chlorobenzoate dioxygenase from P. aeruginosa strains 142 and JB2. In contrast, anthranilate dioxygenases from other bacteria are more closely related to benzoate dioxygenases .

• Substrate Range: Comparative substrate specificity analysis reveals:

  Organism	Enzyme System	Anthranilate	Salicylate	2-Chlorobenzoate
  B. cepacia DBO1	AntDO-3C	+	+	-
  P. aeruginosa PAO1	AntDO	+	-	-
  P. putida P111	AntDO	+	-	-

• Regulatory Mechanism: The andR regulator in B. cepacia responds specifically to anthranilate, while related systems in other bacteria may have different inducer specificities .

These differences highlight the evolutionary divergence of aromatic compound degradation pathways across bacterial species, potentially reflecting adaptation to specific ecological niches.

What experimental design would best determine if the andR protein can regulate anthranilate metabolism genes when expressed in heterologous bacterial hosts?

To evaluate the functionality of andR in heterologous hosts, researchers should design a systematic experimental approach:

• Construct Expression Vectors:

  • Create vectors containing the andR gene under control of inducible promoters

  • Develop reporter constructs with the Pant promoter driving expression of easily quantifiable reporters (GFP, luciferase)

  • Design control vectors with mutated andR or promoter sequences

• Select Diverse Heterologous Hosts:

  • Closely related species (other Burkholderia)

  • Moderately related species (other Proteobacteria like Pseudomonas, Escherichia)

  • Distantly related species (Gram-positive bacteria)

• Test Functionality Under Various Conditions:

  • With/without anthranilate supplementation

  • Different growth phases and media compositions

  • Various expression levels of andR

• Measure Multiple Endpoints:

  • Reporter gene expression (fluorescence, luminescence)

  • Growth curves when anthranilate is provided as sole carbon source

  • RT-qPCR of reporter constructs and any endogenous genes with similar sequences

  • Protein production via Western blot

• Experimental Controls:

  • Express known functional regulators from the heterologous hosts

  • Test andR mutants with impaired function

  • Verify anthranilate uptake in each host

This comprehensive experimental design would evaluate both the sufficiency of andR alone for regulation and identify any host factors that might influence its function, contributing to our understanding of the transferability of bacterial regulatory systems.

What are the main technical challenges in studying andR-DNA interactions, and how can they be overcome?

Researchers studying andR-DNA interactions face several technical challenges:

• Protein Solubility Issues: AraC/XylS-type regulators like andR often show limited solubility when overexpressed.

  Solution: Use solubility-enhancing fusion tags such as SUMO or MBP, and optimize expression conditions with lower temperatures (16-20°C) and reduced inducer concentrations. Consider co-expression with molecular chaperones.

• Requirement for Anthranilate Co-factor: andR may require anthranilate binding for proper DNA interaction, complicating in vitro studies.

  Solution: Include anthranilate in purification and binding buffers at physiologically relevant concentrations. Perform parallel experiments with and without anthranilate to capture condition-dependent interactions.

• Non-specific DNA Binding: AraC/XylS family proteins may exhibit non-specific DNA binding that obscures specific interactions.

  Solution: Include competitor DNA (poly dI-dC) in binding reactions and optimize salt concentrations. Use techniques like EMSA with varying protein:DNA ratios to distinguish specific from non-specific interactions.

• Identification of Precise Binding Sites: The exact andR binding motif may be difficult to define.

  Solution: Employ a combination of techniques including DNase I footprinting, systematic evolution of ligands by exponential enrichment (SELEX), and high-throughput sequencing approaches like ChIP-seq to comprehensively map binding sites.

• Functional Relevance of Binding: Not all DNA binding events lead to transcriptional regulation.

  Solution: Correlate binding data with transcriptional activity using reporter gene assays and in vivo transcriptomics to distinguish functionally relevant binding events.

By addressing these technical challenges with appropriate methodological solutions, researchers can generate more reliable and physiologically relevant data on andR-DNA interactions.

How can researchers effectively design experiments to study the kinetics of andR-mediated gene regulation?

To effectively study the kinetics of andR-mediated gene regulation, researchers should implement time-resolved experimental approaches:

• Real-time Reporter Systems:

  • Construct strains containing fast-maturing fluorescent proteins (e.g., sfGFP) or luciferase under control of andR-regulated promoters

  • Design experiments with continuous monitoring capabilities:

  Time Point (min)	Fluorescence/Luminescence	RNA Levels	Protein Levels	Anthranilate Concentration
  0 (pre-induction)	Baseline measurement	RT-qPCR	Western blot	HPLC analysis
  5	Rapid monitoring	RT-qPCR	Western blot	HPLC analysis
  15	Rapid monitoring	RT-qPCR	Western blot	HPLC analysis
  30	Rapid monitoring	RT-qPCR	Western blot	HPLC analysis
  60	Rapid monitoring	RT-qPCR	Western blot	HPLC analysis
  120	Rapid monitoring	RT-qPCR	Western blot	HPLC analysis

• Pulse-Chase Experiments:

  • Expose cells to a pulse of anthranilate inducer

  • Remove the inducer and monitor the decay of transcriptional activity

  • Calculate half-lives of mRNA and proteins to understand system dynamics

• Single-Cell Analysis:

  • Use microfluidic devices combined with time-lapse microscopy to monitor gene expression in individual cells

  • Assess cell-to-cell variability in response timing and magnitude

  • Correlate with cell cycle stage and growth parameters

• Mathematical Modeling:

  • Develop differential equation models incorporating:

    • Anthranilate uptake rates

    • andR-anthranilate binding kinetics

    • andR-DNA association/dissociation rates

    • Transcription and translation rates

    • mRNA and protein degradation rates

  • Validate models with experimental data and use for predictive simulations

• Experimental Controls:

  • Include andR mutants with altered DNA-binding or anthranilate-binding properties

  • Test varying concentrations of anthranilate to establish dose-response relationships

  • Compare wild-type responses to those in strains with altered regulatory network components

This multi-faceted approach would provide comprehensive insights into the dynamics of andR-mediated gene regulation at both population and single-cell levels.