Recombinant Clavibacter michiganensis subsp. michiganensis ATP synthase subunit c (atpE)

How is recombinant Cmm atpE protein typically produced for research?

Recombinant Cmm atpE protein is typically produced using E. coli expression systems. The full-length protein (1-77aa) is cloned with an N-terminal His-tag to facilitate purification. After expression in E. coli, the protein is purified, typically using affinity chromatography methods that leverage the His-tag . The purified protein is generally provided as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE. For research applications, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol added as a stabilizing agent .

What are the recommended storage conditions for recombinant Cmm atpE protein?

For optimal stability and activity of recombinant Cmm atpE protein, the following storage conditions are recommended:

Repeated freezing and thawing significantly reduce protein stability and should be avoided. It is advisable to make small working aliquots after reconstitution to minimize freeze-thaw cycles .

What molecular detection methods can be used to specifically identify Cmm atpE gene?

The atpE gene can serve as a specific molecular target for Cmm detection using quantitative real-time PCR (qPCR). While atpE sequences are conserved across members of the genus, they show sufficient variation compared to related bacterial genera, making them suitable for specific detection.

For designing primers and probes:

• Perform sequence alignment of atpE genes from Cmm and closely related species

• Identify regions of conservation within Cmm but divergence from other genera

• Design primers with melting temperatures between 58-62°C

• Select probes with at least 5°C higher melting temperature than primers

• Validate specificity using control samples from related genera such as Corynebacterium, Nocardia, and Rhodococcus

The benefit of targeting atpE is that it exists as a single copy in the genome, allowing for precise quantification of bacterial load, unlike multi-copy targets like rRNA genes .

How can PMAxx-qPCR be optimized for detecting viable Cmm cells in environmental samples?

PMAxx-qPCR is a powerful method for detecting viable Cmm cells while excluding dead cells from detection. The optimization process involves several critical parameters:

For environmental sample processing:

• Extract bacterial cells from the sample matrix

• Treat the suspension with 20 μM PMAxx in the dark for 10 minutes

• Expose to bright light to photoactivate PMAxx

• Extract DNA from the treated samples

• Perform qPCR using atpE-specific primers and probes

This method has shown high specificity and sensitivity for detecting Cmm in tomato seed samples with as few as 10 seeds per set .

How can researchers establish viable but nonculturable (VBNC) Cmm models for studying bacterial persistence?

The viable but nonculturable (VBNC) state of Cmm represents a significant challenge in disease management and detection. To establish a VBNC model:

• Prepare a suspension of Cmm cells at 10⁸ CFU mL⁻¹ in appropriate buffer

• Induce VBNC state by adding 50 μM copper sulfate to the suspension

• Incubate at room temperature for up to one month

• Monitor viability using:

  • PMAxx-qPCR (for membrane integrity)

  • Flow cytometry with appropriate viability dyes

  • Attempted culture recovery on standard and enriched media

• Validate VBNC state by confirming cells are metabolically active but non-culturable

For VBNC confirmation, researchers should observe:

• Positive detection by PMAxx-qPCR

• No growth on standard culture media

• Membrane integrity confirmed by flow cytometry

• Metabolic activity using respiratory indicators

This model can be valuable for studying bacterial persistence mechanisms, evaluating disinfection strategies, and developing detection methods for difficult-to-culture bacterial states .

What are the critical factors for developing a standard curve for absolute quantification of Cmm using atpE-targeted qPCR?

Developing a reliable standard curve for absolute quantification requires careful consideration of multiple factors:

• Plasmid standard preparation:

  • Clone the target atpE gene fragment into a plasmid vector

  • Verify sequence integrity through sequencing

  • Determine plasmid concentration using spectrophotometry

  • Calculate copy number based on plasmid size and DNA concentration

• Standard curve generation:

  • Create 10-fold serial dilutions ranging from 10² to 10⁸ copies/μL

  • Perform qPCR in triplicate for each dilution point

  • Plot Ct values against log-transformed copy numbers

  • Verify linearity (R² > 0.99) and efficiency (90-110%)

• Assay validation:

  • Include no-template controls to detect contamination

  • Use internal amplification controls to identify inhibition

  • Determine limit of detection (LOD) and limit of quantification (LOQ)

  • Assess inter-assay and intra-assay variation coefficients

When properly established, the standard curve should show a negative linear correlation between bacterial cell numbers and Ct values. An example relationship derived from experimental data shows that for viable Cmm cells in the range of 10³ to 10⁷ CFU mL⁻¹, plotting Ct values versus log-concentration yields a straight-line regression with R² = 0.996 .

How does the atpE gene in Cmm compare with other ATP synthase subunits in terms of conservation and potential as a molecular marker?

The atpE gene presents specific advantages as a molecular marker compared to other ATP synthase subunits:

The atpE gene is particularly valuable because it shows sufficient sequence conservation within the genus to serve as a reliable marker, while exhibiting enough sequence divergence from related genera to avoid false positive results . Additionally, its small size makes it amenable to efficient amplification in PCR assays, and its single-copy nature in the genome ensures that quantification directly correlates with bacterial numbers.

What are the common pitfalls in detecting Cmm in complex environmental samples, and how can they be addressed?

Detection of Cmm in environmental samples presents several challenges:

• Inhibitory compounds in samples:

  • Problem: Humic acids, polyphenols, and other compounds can inhibit PCR

  • Solution: Use specialized DNA extraction kits with inhibitor removal technologies; include internal amplification controls to detect inhibition

• Low bacterial concentrations:

  • Problem: Environmental samples often contain low pathogen levels

  • Solution: Implement sample concentration steps (filtration, centrifugation); consider sample enrichment when appropriate

• Interfering background microflora:

  • Problem: Non-target bacteria can interfere with detection

  • Solution: Use highly specific primers and probes; incorporate selective enrichment steps

• Sample turbidity affecting PMAxx treatment:

  • Problem: Turbid samples prevent effective light penetration for PMAxx activation

  • Solution: Dilute samples at least 20× before PMAxx treatment; optimize light exposure time and intensity

• VBNC state detection:

  • Problem: Standard culture methods miss VBNC cells

  • Solution: Implement PMAxx-qPCR for viable cell detection; validate with flow cytometry when possible

A systematic approach combining proper sample preparation, inhibitor removal, and validated molecular detection can overcome most of these challenges.

What are the key considerations when comparing the effectiveness of different DNA-binding dyes (EMA, PMA, PMAxx) for viable Cmm detection?

When selecting DNA-binding dyes for viable cell detection, researchers should consider several factors:

Experimental validation should include:

• Side-by-side comparison using the same bacterial cultures

• Testing a range of concentrations for each dye

• Evaluating dye efficiency with both live and heat-killed cells

• Determining the detection range for each dye

• Assessing the impact of the sample matrix on dye performance

Based on research findings, PMAxx has demonstrated superior discrimination between viable and dead Cmm cells compared to PMA, and both PMA and PMAxx outperform EMA in terms of selectivity for membrane-compromised cells .

How might the atpE gene in Cmm be utilized for advanced molecular epidemiology and population genetics studies?

The atpE gene offers several opportunities for advancing molecular epidemiology and population genetics of Cmm:

• Single nucleotide polymorphism (SNP) analysis:

  • Identify strain-specific variations within the atpE gene

  • Develop SNP-based typing schemes for tracking outbreak strains

  • Correlate specific SNP patterns with virulence or environmental adaptation

• Population structure analysis:

  • Compare atpE sequences across geographic regions to assess population diversity

  • Identify potential founder effects or genetic bottlenecks in pathogen populations

  • Combine with other housekeeping genes for multi-locus sequence typing (MLST)

• Evolutionary studies:

  • Analyze selection pressures on the atpE gene using dN/dS ratios

  • Compare evolutionary rates with other ATP synthase subunits

  • Use atpE sequence data to construct time-calibrated phylogenies

• Biogeography applications:

  • Correlate atpE sequence variants with ecological niches or host range

  • Map the geographic distribution of specific variants

  • Identify potential sources of new pathogen introductions

As genomic databases expand, the utility of atpE as a marker for epidemiological studies will likely increase, particularly when integrated with whole-genome approaches .

What potential applications exist for studying atpE inhibitors as novel antimicrobial agents against Cmm?

The ATP synthase complex represents a promising antimicrobial target, with atpE being particularly interesting due to its essential role in energy metabolism. Research opportunities include:

• Identification of potential inhibitors:

  • High-throughput screening of chemical libraries against recombinant atpE

  • Rational design of inhibitors based on the protein structure

  • Repurposing existing ATP synthase inhibitors from other bacterial systems

• Mechanism of action studies:

  • Investigate binding modes of potential inhibitors

  • Determine the impact on proton translocation and ATP synthesis

  • Analyze the effect on membrane potential and cellular energy status

• Evaluation of antimicrobial efficacy:

  • Test inhibitor effects on bacterial growth in vitro

  • Assess activity against VBNC state bacteria

  • Evaluate potential for resistance development

• Structure-activity relationships:

  • Modify lead compounds to improve specificity for Cmm atpE

  • Optimize pharmacokinetic properties for agricultural applications

  • Develop formulations suitable for plant application

The recombinant Cmm atpE protein, with its well-characterized sequence and availability as a research reagent, provides an excellent starting point for such investigations, potentially leading to novel control strategies for bacterial canker disease in tomatoes .