Recombinant Erwinia carotovora subsp. atroseptica Protein AaeX (aaeX)

What is Erwinia carotovora subsp. atroseptica and what makes its proteins significant for research?

Erwinia carotovora subsp. atroseptica (now reclassified as Pectobacterium atrosepticum) is an enterobacterial phytopathogen that causes soft rot and blackleg diseases in potatoes. The significance of studying its proteins lies in understanding plant pathogenicity mechanisms within the Enterobacteriaceae family. According to genome sequencing studies, approximately 33% of its genes are not shared with sequenced enterobacterial human pathogens, suggesting unique metabolic traits such as nitrogen fixation and opine catabolism . Proteins from this organism provide insights into novel pathogenicity determinants and bacterial adaptation to plant hosts, making them valuable targets for agricultural research aimed at disease control and prevention.

How does the structure of recombinant proteins from E. carotovora subsp. atroseptica compare to native proteins?

Recombinant proteins like the ATP synthase subunit c (atpE) from E. carotovora subsp. atroseptica are typically expressed with fusion tags (such as His-tags) that facilitate purification while maintaining functional properties. The recombinant atpE protein (79 amino acids in length) retains its primary sequence integrity as evidenced by the amino acid sequence: MENLSVDLLYMAAALMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . When expressing such proteins, researchers should consider that N-terminal tags may affect protein folding or activity in some cases. For structural studies, it's advisable to compare both tagged and untagged versions of the protein to ensure the tag doesn't interfere with native conformation or function.

What expression systems are most effective for recombinant E. carotovora proteins?

E. coli expression systems are widely used for the recombinant production of E. carotovora proteins due to their genetic compatibility as fellow members of the Enterobacteriaceae family. Commercial recombinant proteins such as the ATP synthase subunit c (atpE) are successfully expressed in E. coli with high purity (>90% as determined by SDS-PAGE) . For optimal expression, researchers should consider codon optimization based on the expression host and use vectors with appropriate promoters. While E. coli is the most common host, alternative expression systems may be required for proteins that form inclusion bodies or require post-translational modifications. The choice of expression system should be guided by the specific properties of the target protein and the intended downstream applications.

What are the recommended protocols for transforming E. carotovora subsp. atroseptica with recombinant constructs?

For efficient transformation of E. carotovora subsp. atroseptica, researchers should employ a modified version of the Hanahan method. This approach yields transformation frequencies ranging from 1 × 10² to 4 × 10⁴ colonies per microgram of plasmid DNA, as demonstrated with plasmids pBR322, pBR325, and pAT153 . The methodology involves preparing competent cells in the presence of divalent cations, followed by a heat shock procedure.

For optimal results, researchers should:

• Use fresh overnight cultures diluted 1:100 and grown to early log phase (OD₆₀₀ 0.4-0.6)

• Harvest cells by gentle centrifugation and maintain at 4°C throughout the procedure

• Resuspend cells in ice-cold transformation buffer containing Ca²⁺ and Mg²⁺ ions

• Introduce plasmid DNA (50-500 ng) to the competent cells

• Apply heat shock at 42°C for 90 seconds followed by immediate cooling

• Allow for expression of antibiotic resistance genes before plating on selective media

Confirmation of successful transformants should be performed through plasmid isolation and restriction analysis to verify the integrity of the introduced construct .

How can researchers optimize the expression and purification of membrane proteins like ATP synthase subunits from E. carotovora?

Optimizing the expression and purification of membrane proteins such as ATP synthase subunits requires specialized approaches due to their hydrophobic nature. For the ATP synthase subunit c (atpE), which contains multiple transmembrane domains, researchers should consider the following protocol:

• Expression optimization:

  • Test multiple expression strains (BL21(DE3), C41(DE3), C43(DE3)) specifically designed for membrane proteins

  • Employ lower induction temperatures (16-25°C) to slow protein synthesis and improve folding

  • Use lower IPTG concentrations (0.1-0.5 mM) for induction

  • Include membrane-stabilizing agents such as glycerol (5-10%) in growth media

• Purification strategy:

  • Solubilize membrane fractions using mild detergents (DDM, LDAO, or Triton X-100)

  • Employ IMAC (Immobilized Metal Affinity Chromatography) for His-tagged proteins

  • Perform size exclusion chromatography as a polishing step

  • Store in buffer containing 6% trehalose at -20°C/-80°C to maintain stability

For highest purity (>90%), researchers should implement a multi-step purification process and analyze results using SDS-PAGE. The reconstitution of lyophilized protein should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol for long-term storage .

What analytical techniques are most informative for characterizing structural features of E. carotovora proteins?

When characterizing structural features of E. carotovora proteins, researchers should employ a multi-technique approach:

• Primary structure analysis:

  • Mass spectrometry (MS) for molecular weight confirmation and post-translational modifications

  • N-terminal sequencing to verify protein identity and signal peptide cleavage

• Secondary structure determination:

  • Circular dichroism (CD) spectroscopy to quantify α-helix, β-sheet, and random coil content

  • FTIR spectroscopy as a complementary technique for secondary structure analysis

• Tertiary structure characterization:

  • X-ray crystallography for high-resolution structural information (resolution <2.5Å)

  • NMR spectroscopy for solution structure and dynamics (for proteins <30 kDa)

  • Cryo-EM for larger protein complexes or membrane proteins

• Quaternary structure assessment:

  • Size exclusion chromatography combined with multi-angle light scattering (SEC-MALS)

  • Analytical ultracentrifugation to determine oligomerization state

For membrane proteins like the ATP synthase subunit c, which has a distinctive amino acid sequence with hydrophobic regions (MENLSVDLLYMAAALMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA), structural predictions using computational tools can provide preliminary insights before experimental verification .

How should researchers design experiments to study the function of E. carotovora proteins in plant-pathogen interactions?

When designing experiments to study E. carotovora protein function in plant-pathogen interactions, researchers should implement a comprehensive multi-level approach:

• In vitro functional assays:

  • Enzymatic activity assays for relevant functions (e.g., pectate lyase, cellulase activity)

  • Protein-protein interaction studies using pull-down assays, yeast two-hybrid, or surface plasmon resonance

  • Substrate specificity determination through enzyme kinetics

• Cell-based assays:

  • Bacterial mutant construction through insertional mutagenesis or gene deletion

  • Complementation studies with wild-type and mutant protein variants

  • Heterologous expression in E. coli to confirm protein function

• Plant-pathogen interaction studies:

  • Plant bioassays with wild-type and mutant bacterial strains

  • Microscopy to visualize infection process

  • Quantification of virulence factors in planta

  • Transcriptome analysis during infection

• Controls and validation:

  • Include positive and negative controls in all assays

  • Validate protein expression using Western blotting or mass spectrometry

  • Perform statistical analysis of replicated experiments

This experimental framework has proven effective in investigating novel pathogenicity determinants in E. carotovora subsp. atroseptica, as demonstrated by studies using insertional mutants that showed reduced virulence in plant bioassays .

What storage and handling protocols ensure optimal stability of recombinant E. carotovora proteins?

For optimal stability of recombinant E. carotovora proteins, researchers should follow these evidence-based storage and handling procedures:

• Short-term storage (up to one week):

  • Store working aliquots at 4°C

  • Avoid repeated freeze-thaw cycles which can cause protein degradation

• Long-term storage:

  • Store lyophilized protein at -20°C/-80°C

  • For reconstituted proteins, add glycerol to a final concentration of 5-50% (50% is recommended)

  • Prepare small single-use aliquots to avoid repeated freeze-thaw cycles

• Reconstitution protocol:

  • Briefly centrifuge vials before opening to bring contents to the bottom

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • For ATP synthase subunit c, use Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Stability assessment:

  • Monitor protein integrity using SDS-PAGE before experiments

  • Check activity using functional assays periodically

  • Document storage duration and conditions for reproducibility

These protocols are based on established guidelines for recombinant proteins from E. carotovora, such as the ATP synthase subunit c, which maintains >90% purity when properly stored and handled .

How can researchers accurately measure and interpret the effects of protein modifications on E. carotovora protein function?

To accurately measure and interpret the effects of protein modifications on E. carotovora protein function, researchers should implement the following methodological approach:

• Systematic protein modification strategy:

  • Site-directed mutagenesis of conserved or putative functional residues

  • Truncation analysis to identify functional domains

  • Fusion protein construction to assess the impact of tags

  • Post-translational modification analysis using mass spectrometry

• Comparative functional assessment:

  Protein Variant	Activity Assay	Binding Affinity	Structural Stability	In Vivo Function
  Wild-type	Baseline	Baseline	Baseline	Baseline
  Modified	% of wild-type	Kd value	Tm value	% of wild-type

• Structural impact analysis:

  • Circular dichroism to assess secondary structure changes

  • Thermal shift assays to determine protein stability

  • Molecular dynamics simulations to predict conformational changes

• Biological significance verification:

  • Complementation studies in bacterial mutants

  • Plant infection assays with modified protein variants

  • Expression analysis in native conditions vs. experimental conditions

This approach has been successfully applied to regulatory proteins in E. carotovora, such as the activator of extracellular protein production (aepA) and aepH, which control the production of pectolytic enzymes, cellulase, and protease that are critical for virulence .

What are common challenges in expressing and purifying E. carotovora proteins and how can they be overcome?

Researchers working with E. carotovora proteins frequently encounter several challenges that can be addressed with specific strategies:

• Low expression levels:

  • Optimize codon usage for the expression host

  • Test different promoter systems (T7, tac, araBAD)

  • Vary induction conditions (temperature, inducer concentration, induction time)

  • Screen multiple E. coli strains (BL21, Rosetta, Arctic Express)

• Inclusion body formation:

  • Lower expression temperature to 16-20°C

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Fuse with solubility-enhancing tags (MBP, SUMO, Trx)

  • Optimize refolding protocols for proteins like ATP synthase subunits that may form inclusion bodies

• Protein instability:

  • Include protease inhibitors during purification

  • Add stabilizing agents like trehalose (6%) in buffer formulations

  • Maintain cold chain throughout purification

  • Determine optimal pH and ionic strength conditions

• Purification interference:

  • For His-tagged proteins, include low concentrations of imidazole in binding buffers

  • Perform multiple chromatography steps for >90% purity

  • Consider on-column refolding for difficult proteins

These strategies have been successfully applied to the expression and purification of E. carotovora proteins, resulting in high-purity preparations (>90% as determined by SDS-PAGE) suitable for functional and structural studies .

How can researchers effectively troubleshoot transformation efficiency issues when working with E. carotovora strains?

When troubleshooting transformation efficiency issues with E. carotovora strains, researchers should implement a systematic approach:

• Competent cell preparation optimization:

  • Use early log phase cultures (OD₆₀₀ 0.4-0.6)

  • Ensure all solutions and equipment are pre-chilled

  • Minimize exposure to room temperature

  • Test different buffer compositions with varying concentrations of divalent cations

• DNA quality and quantity assessment:

  • Use highly purified plasmid DNA (A₂₆₀/A₂₈₀ ratio >1.8)

  • Optimize DNA concentration (50-500 ng per transformation)

  • Verify plasmid integrity by gel electrophoresis

  • Consider plasmid size (smaller plasmids generally transform more efficiently)

• Transformation parameter optimization:

  Parameter	Standard Condition	Optimization Range	Effect on Efficiency
  Heat shock duration	90 seconds	30-120 seconds	Strain-dependent
  Heat shock temperature	42°C	37-45°C	Critical for membrane permeability
  Recovery medium	SOC	LB, 2XYT, SOB	Nutrient availability affects recovery
  Recovery time	1 hour	0.5-3 hours	Extended time for slow-growing strains

• Strain-specific considerations:

  • Different E. carotovora subspecies may require modified protocols

  • Consider using ColE1-based plasmids which have demonstrated utility as cloning vectors for E. carotovora

  • Test chemical transformation versus electroporation for recalcitrant strains

This systematic approach has been successful in achieving transformation frequencies ranging from 1 × 10² to 4 × 10⁴ colonies per microgram of plasmid DNA with E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica .

What approaches can address challenges in determining protein-protein interactions involving E. carotovora virulence factors?

Determining protein-protein interactions involving E. carotovora virulence factors presents unique challenges that can be addressed through complementary methodologies:

• In vitro interaction studies:

  • Pull-down assays using recombinant His-tagged proteins

  • Surface plasmon resonance (SPR) for quantitative binding parameters

  • Isothermal titration calorimetry (ITC) for thermodynamic characterization

  • Microscale thermophoresis (MST) for interactions in complex solutions

• Cell-based interaction methods:

  • Bacterial two-hybrid systems adapted for E. carotovora proteins

  • Förster resonance energy transfer (FRET) with fluorescent protein fusions

  • Bimolecular fluorescence complementation (BiFC) for visualizing interactions in situ

  • Co-immunoprecipitation from bacterial lysates with specific antibodies

• Proximity-based interaction mapping:

  • Crosslinking mass spectrometry (XL-MS) to capture transient interactions

  • BioID or APEX2 proximity labeling to identify interaction neighborhoods

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Validation strategies:

  • Mutagenesis of predicted interaction sites

  • Competitive binding assays with peptide mimics

  • Functional assays measuring virulence factor activity

  • In planta confirmation of interactions during infection

These approaches are particularly valuable for studying interactions between virulence regulators like aepA and aepH, which control the production of extracellular enzymes that are essential for E. carotovora pathogenicity .

What are promising research avenues for utilizing E. carotovora proteins in understanding bacterial pathogenicity mechanisms?

Several promising research avenues exist for utilizing E. carotovora proteins to enhance our understanding of bacterial pathogenicity mechanisms:

• Comparative genomics and proteomics:

  • Analyzing the unique 33% of E. carotovora genes not shared with human pathogens

  • Identifying novel virulence determinants through systematic protein characterization

  • Cross-species comparison of virulence factor homologs within Enterobacteriaceae

• Secretion system characterization:

  • Investigating type IV secretion systems identified through genomic analysis

  • Determining the substrate specificity of secretion machinery

  • Mapping protein-protein interactions within secretion complexes

• Regulatory network analysis:

  • Exploring the aepA and aepH regulatory system that controls virulence factor production

  • Identifying environmental signals that modulate protein expression

  • Constructing comprehensive models of virulence regulation

• Host-pathogen protein interactions:

  • Identifying plant proteins targeted by E. carotovora virulence factors

  • Characterizing molecular mechanisms of plant immunity suppression

  • Developing protein-based strategies for disease resistance

These research directions leverage the significant genomic and molecular tools available for E. carotovora subsp. atroseptica, including the complete genome sequence of strain SCRI1043 and established transformation protocols . The insights gained could lead to novel approaches for controlling bacterial plant diseases and provide broader understanding of pathogenicity mechanisms in the Enterobacteriaceae family.

How might new technologies enhance structural and functional studies of E. carotovora proteins?

Emerging technologies are poised to revolutionize structural and functional studies of E. carotovora proteins:

• Advanced structural biology approaches:

  • Cryo-electron microscopy for near-atomic resolution of membrane proteins without crystallization

  • Integrative structural biology combining multiple data sources (SAXS, XL-MS, cryo-EM)

  • Microcrystal electron diffraction (MicroED) for proteins resistant to traditional crystallization

  • AlphaFold2 and related AI methods for accurate structure prediction

• High-throughput functional genomics:

  • CRISPR-Cas9 genome engineering for rapid mutant generation

  • Transposon sequencing (Tn-Seq) to identify essential genes under infection conditions

  • RNA-Seq to map transcriptional responses to environmental stimuli

  • Ribosome profiling to monitor translation efficiency of virulence factors

• Advanced imaging technologies:

  • Super-resolution microscopy to visualize protein localization during infection

  • Correlative light and electron microscopy (CLEM) for structural context

  • Label-free imaging techniques to study native protein distributions

  • Multiplexed imaging to track multiple proteins simultaneously

• Systems biology integration:

  • Multi-omics data integration (genomics, transcriptomics, proteomics, metabolomics)

  • Network analysis to identify critical nodes in virulence pathways

  • Machine learning approaches to predict protein function from sequence and structure

  • Mathematical modeling of host-pathogen interactions

These technologies will provide unprecedented insights into the function of proteins like ATP synthase subunits and regulatory proteins such as aepA and aepH , advancing our understanding of E. carotovora pathogenicity mechanisms and potentially leading to novel disease control strategies.