Recombinant Chromobacterium violaceum ATP synthase subunit beta (atpD)

What is Chromobacterium violaceum and why is its ATP synthase subunit beta (atpD) significant for research?

Chromobacterium violaceum is a Gram-negative betaproteobacterium that inhabits tropical and subtropical ecosystems, including freshwater and soil environments . It is characterized by production of a violet pigment called violacein that has antimicrobial and antitumoral properties . While C. violaceum rarely causes human infections, when it does, it leads to severe sepsis with mortality rates of 60-80% .

The ATP synthase subunit beta (atpD) is particularly significant because:

• It forms part of the catalytic core (F1) of ATP synthase, the enzyme responsible for ATP production via oxidative phosphorylation

• The gene is highly conserved, making it useful for phylogenetic studies and species identification

• Understanding atpD structure and function may provide insights into C. violaceum's adaptations to its ecological niche

• ATP synthase components represent potential antimicrobial targets, particularly relevant given C. violaceum's intrinsic resistance to many antibiotics

How does the genomic organization of C. violaceum inform our understanding of atpD expression and function?

The complete genome of C. violaceum ATCC 12472 has been sequenced by The Brazilian National Genome Project Consortium . While specific information on the ATP synthase operon organization is not detailed in the search results, the genomic analysis reveals important contextual information:

Understanding this genomic context provides valuable insights into how atpD expression might be regulated and coordinated with other cellular processes in C. violaceum.

What evolutionary insights can be gained from studying C. violaceum atpD compared to other bacterial species?

Studying C. violaceum atpD in comparison to other bacterial species can reveal:

• Evolutionary adaptations specific to tropical/subtropical environments

• Conservation of catalytic domains across diverse bacterial phyla

• Potential horizontal gene transfer events in ATP synthase evolution

• Sequence variations that correlate with unique physiological traits of C. violaceum

Methodological approach for evolutionary analysis:

• Perform multiple sequence alignment of atpD sequences from diverse bacteria

• Construct phylogenetic trees using maximum likelihood or Bayesian methods

• Calculate selection pressures (dN/dS ratios) to identify regions under positive selection

• Map sequence variations onto structural models to understand functional implications

What are the optimal expression systems and conditions for producing recombinant C. violaceum atpD?

The choice of expression system is critical for successful production of functional recombinant atpD:

Methodological recommendations:

• Clone the C. violaceum atpD gene into a vector with an inducible promoter (T7, tac)

• Include a removable affinity tag (His6, GST) for purification

• Express initially at small scale to optimize conditions:

  • Test multiple temperatures (16-37°C)

  • Vary inducer concentration (0.1-1.0 mM IPTG)

  • Test rich vs. minimal media

• Confirm expression by SDS-PAGE and Western blotting

• Assess solubility through fractionation experiments

How can the violacein pigment interference be managed during purification of recombinant C. violaceum proteins?

When working with C. violaceum directly (rather than heterologous expression), the violacein pigment can interfere with protein purification:

For recombinant atpD expressed in E. coli or other hosts, these concerns are largely avoided, but cross-contamination of equipment should be considered if working with both native and recombinant systems.

What are the most reliable methods for assessing the structural integrity and activity of purified recombinant atpD?

A multi-method approach ensures comprehensive characterization:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Thermal shift assays to evaluate stability

  • Limited proteolysis to assess proper folding

  • Size exclusion chromatography to evaluate oligomeric state

• Functional assessment:

  • ATPase activity assay (colorimetric phosphate release)

  • ATP synthesis assay (when reconstituted with other subunits)

  • Nucleotide binding assays (fluorescence-based or isothermal titration calorimetry)

• Interaction assessment:

  • Surface plasmon resonance for binding to other ATP synthase subunits

  • Pull-down assays to verify complex formation

  • Native gel electrophoresis to assess complex assembly

How might C. violaceum atpD contribute to the bacterium's adaptation to its ecological niche?

C. violaceum inhabits tropical and subtropical regions, often in aquatic environments with varying conditions. The atpD subunit may play key roles in adaptation:

• Temperature adaptation mechanisms:

  • Structural modifications that enhance stability at higher temperatures

  • Altered regulatory sites that respond to temperature fluctuations

  • Research approach: Compare thermal stability and activity profiles of C. violaceum atpD with homologs from temperate species

• pH and ion concentration responses:

  • Modified catalytic sites to maintain efficiency under varying pH conditions

  • Altered ion binding sites for functioning in low-nutrient environments

  • Methodology: Site-directed mutagenesis of conserved residues followed by activity assays under various pH and ion conditions

• Biofilm-related adaptations:

  • C. violaceum produces cellulose for biofilm formation

  • ATP synthase activity may be specifically regulated in biofilm conditions

  • Experimental approach: Compare atpD expression and activity between planktonic and biofilm growth states

What is the relationship between atpD function and antibiotic resistance in C. violaceum?

C. violaceum shows intrinsic resistance to several antibiotics while remaining susceptible to others :

Research approaches to investigate this relationship:

• Comparative proteomics:

  • Compare ATP synthase expression levels in resistant vs. susceptible strains

  • Methodology: Quantitative mass spectrometry with stable isotope labeling

• Energetic burden analysis:

  • Measure ATP production rates in the presence of sublethal antibiotic concentrations

  • Approach: Luciferase-based ATP quantification in real-time

• Membrane potential studies:

  • Investigate the relationship between membrane potential (maintained by ATP synthase) and antibiotic uptake

  • Method: Fluorescent dye-based membrane potential measurement combined with antibiotic accumulation assays

How does the violacein pigment interact with C. violaceum energy metabolism components?

The violacein pigment is a distinctive feature of C. violaceum with potential implications for energy metabolism:

• Membrane localization effects:

  • Violacein is thought to incorporate into bacterial membranes

  • May affect the lipid environment surrounding membrane-embedded ATP synthase

  • Research approach: Compare ATP synthase activity in native membranes vs. violacein-depleted membranes

• Redox interactions:

  • Violacein has redox-active properties that might influence electron transport chain

  • Could indirectly affect the proton gradient that drives ATP synthase

  • Methodology: Measure proton motive force in violacein-producing vs. non-producing strains

• Regulatory relationships:

  • Production of violacein requires energy, creating feedback relationships

  • ATP levels may regulate violacein biosynthesis pathway

  • Experimental design: Controlled modulation of ATP levels followed by measurement of violacein production

How can researchers address solubility issues when expressing recombinant C. violaceum atpD?

Inclusion body formation is a common challenge when expressing membrane-associated proteins like ATP synthase components:

• Prevention strategies:

  • Reduce expression rate through lower inducer concentration and temperature

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Use solubility-enhancing fusion partners (MBP, SUMO, TrxA)

  • Express truncated constructs lacking hydrophobic regions

• Recovery methods if inclusion bodies form:

  • Develop refolding protocols using gradual dialysis

  • Screen various detergents and lipids for solubilization

  • Try mild solubilization using arginine or low concentrations of chaotropes

  • Implement on-column refolding during purification

• Analytical approaches to monitor improvements:

  • Use dynamic light scattering to assess aggregation state

  • Employ thermal shift assays to evaluate stability of refolded protein

  • Confirm secondary structure recovery by circular dichroism

  • Validate function through activity assays

What are the best approaches for analyzing atpD sequence variations across C. violaceum strains?

Comparative sequence analysis requires systematic methodologies:

• Dataset preparation:

  • Collect atpD sequences from multiple C. violaceum isolates

  • Include sequences from related species as outgroups

  • Ensure accurate annotation and sequence quality

• Sequence analysis workflow:

  • Perform multiple sequence alignment (MUSCLE, CLUSTAL)

  • Identify conserved domains and variable regions

  • Calculate nucleotide diversity and selection pressures

  • Map variations to structural models

• Correlation with phenotypic data:

  • Link sequence variations to differences in antibiotic resistance

  • Correlate with ecological origin of isolates

  • Associate with virulence or pathogenicity data

• Data interpretation framework:

  • Distinguish functionally significant from neutral variations

  • Identify potential adaptation signatures

  • Generate testable hypotheses about structure-function relationships

How can researchers differentiate between direct effects on atpD and indirect metabolic consequences in experimental studies?

When studying ATP synthase function, distinguishing direct from indirect effects requires careful experimental design:

• Genetic approach:

  • Create point mutations in atpD rather than gene deletions

  • Use complementation studies to verify phenotype rescue

  • Implement controllable expression systems for dose-dependent analysis

• Biochemical discrimination:

  • Perform in vitro reconstitution experiments with purified components

  • Use ATP synthase inhibitors with varying mechanisms of action

  • Develop assays specific for different steps in the catalytic cycle

• Systems biology framework:

  • Implement metabolic flux analysis to track energy flow

  • Use transcriptomics to identify compensatory responses

  • Develop mathematical models that integrate direct and indirect effects

• Controls and validations:

  • Include parallel studies on other ATP synthase subunits

  • Compare effects across multiple growth conditions

  • Validate key findings using complementary methodological approaches

What structural biology approaches would advance understanding of C. violaceum atpD?

Advanced structural biology techniques can reveal critical insights:

• Cryo-electron microscopy (cryo-EM):

  • Allows visualization of the entire ATP synthase complex

  • Can capture different conformational states during catalytic cycle

  • Methodology: Purify intact ATP synthase complexes in nanodiscs or detergent micelles for imaging

• X-ray crystallography:

  • Provides high-resolution structures of individual domains

  • Can reveal detailed binding interactions with ligands

  • Approach: Screen multiple crystallization conditions with various nucleotide analogs

• Hydrogen-deuterium exchange mass spectrometry:

  • Maps dynamic regions and conformational changes

  • Identifies regions involved in subunit interactions

  • Protocol: Compare exchange patterns in isolated atpD versus assembled complex

How might studying atpD contribute to understanding C. violaceum pathogenicity?

The virulence of C. violaceum (causing fatal sepsis with 60-80% mortality) may be linked to energy metabolism:

• Host-pathogen energetic interactions:

  • Investigation of ATP synthase regulation during infection

  • Analysis of energy requirements for virulence factor secretion

  • Method: Develop infection models with atpD reporter constructs

• Type III secretion system connections:

  • C. violaceum pathogenicity depends on Cpi-1/-1a-encoded TTSS

  • TTSS assembly and function require significant ATP

  • Research design: Measure ATP consumption rates during TTSS activation

• Stress adaptation during pathogenesis:

  • ATP synthase may be modified to function under host-induced stress

  • Important for survival within phagocytes or under oxidative stress

  • Approach: Compare atpD expression and modifications between free-living and infection states