Recombinant Cronobacter sakazakii ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Cronobacter sakazakii?

ATP synthase subunit c (atpE) in C. sakazakii is a transmembrane protein component of the F0 sector of ATP synthase. The protein consists of 79 amino acids with the sequence MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . This hydrophobic protein functions as part of the membrane-embedded F0 complex, forming the c-ring structure that rotates during ATP synthesis. The c-ring acts as a proton channel, allowing protons to pass through the membrane, which drives the conformational changes necessary for ATP synthesis in the F1 sector of the enzyme . As a lipid-binding protein, it plays a crucial role in energy production within C. sakazakii cells, converting the proton motive force across the membrane into chemical energy in the form of ATP .

How does ATP synthase function relate to C. sakazakii's pathogenicity and survival?

ATP synthase plays a vital role in C. sakazakii's energy metabolism, directly affecting its ability to survive in stressful environments. As an opportunistic pathogen associated with severe neonatal infections, C. sakazakii's ability to produce energy efficiently contributes to its persistence under various environmental conditions, including desiccation stress .

Energy production genes, including those encoding ATP synthase components, have been identified as important factors in C. sakazakii's biofilm formation , which enhances bacterial survival in unfavorable conditions and increases resistance to antimicrobial agents. The relationship between energy metabolism and stress response is particularly relevant for C. sakazakii, as this pathogen is known for its exceptional desiccation tolerance, allowing it to persist in low-water activity foods such as powdered infant formula, which increases the risk of infection, especially in immunocompromised neonates .

What expression systems are optimal for producing recombinant C. sakazakii atpE protein?

The optimal expression system for producing recombinant C. sakazakii ATP synthase subunit c is an in vitro E. coli expression system . This approach is particularly effective for this transmembrane protein due to several factors:

• E. coli provides the prokaryotic cellular machinery that closely resembles the native environment of C. sakazakii proteins

• The relatively small size of atpE (79 amino acids) makes it amenable to expression in E. coli

• The addition of an N-terminal 10xHis-tag facilitates subsequent purification steps without significant interference with protein structure or function

For optimal expression, researchers should consider using specialized E. coli strains designed for membrane protein expression, such as C41(DE3) or C43(DE3), which are engineered to tolerate potentially toxic membrane proteins. Expression should be conducted under controlled temperature conditions (typically 18-25°C after induction) to minimize inclusion body formation and protein misfolding that commonly occurs with membrane proteins .

What are the critical considerations for maintaining stability of purified atpE protein?

Maintaining stability of purified recombinant C. sakazakii ATP synthase subunit c requires careful attention to storage conditions and handling practices. The protein should be stored at -20°C, with extended storage preferably at -20°C or -80°C . Several critical considerations include:

• Buffer composition: The presence of appropriate detergents is essential for maintaining the solubility of this hydrophobic membrane protein

• Temperature management: Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and aggregation

• Working aliquots: It is recommended to store working aliquots at 4°C for no more than one week to maintain protein integrity

• Shelf life considerations: Liquid formulations typically have a shelf life of approximately 6 months at -20°C/-80°C, while lyophilized preparations can be stored for up to 12 months

These stability considerations are particularly important for experimental reproducibility, as protein degradation or aggregation can significantly impact functional assays and structural studies.

How can researchers effectively use recombinant atpE in studies of C. sakazakii energy metabolism?

Researchers can effectively use recombinant C. sakazakii ATP synthase subunit c in several experimental approaches to study energy metabolism:

• Reconstitution studies: Purified atpE can be reconstituted into liposomes with other ATP synthase subunits to study proton translocation and ATP synthesis activity in a controlled system

• Inhibitor screening: The recombinant protein can serve as a target for screening potential antimicrobial compounds that specifically inhibit C. sakazakii ATP synthase

• Structure-function analysis: Site-directed mutagenesis of key residues in recombinant atpE followed by functional assays can elucidate the specific amino acids critical for proton translocation

• Protein-protein interaction studies: Techniques such as cross-linking or pull-down assays using the His-tagged recombinant atpE can identify interactions with other ATP synthase components or regulatory proteins

These approaches are particularly valuable as energy production genes have been implicated in C. sakazakii biofilm formation and environmental persistence , making ATP synthase a potential target for controlling this pathogen in food production environments.

What methods can be used to study the interaction between atpE and other ATP synthase components?

To study interactions between ATP synthase subunit c (atpE) and other components of the ATP synthase complex, researchers can employ several complementary techniques:

• Co-immunoprecipitation (Co-IP): Using the N-terminal 10xHis-tag of recombinant atpE , researchers can perform pull-down assays to identify proteins that directly interact with atpE

• Surface Plasmon Resonance (SPR): This technique can quantitatively measure binding affinities between atpE and other purified ATP synthase subunits in real-time

• Cryo-electron microscopy: For structural studies of the entire ATP synthase complex with a focus on the arrangement of c-subunits in the c-ring

• Förster Resonance Energy Transfer (FRET): By labeling atpE and potential interaction partners with fluorophores, researchers can monitor protein-protein interactions in solution or in reconstituted membrane systems

• Cross-linking mass spectrometry: Chemical cross-linking followed by mass spectrometry analysis can identify specific contact points between atpE and other subunits in the assembled complex

These methods can provide valuable insights into how atpE functions within the ATP synthase complex and how it contributes to energy production in C. sakazakii, potentially revealing unique features that could be exploited for pathogen control.

How conserved is the atpE gene across different C. sakazakii strains and related Cronobacter species?

The atpE gene encoding ATP synthase subunit c shows relatively high conservation across Cronobacter species. While specific data for atpE variation is limited in the search results, we can infer its conservation pattern from related ATP synthase genes like atpD, which is used in multilocus sequence typing (MLST) of Cronobacter species .

The atpD gene (encoding ATP synthase β chain) demonstrates 10.8% polymorphic sites across different Cronobacter strains, which is the lowest polymorphism rate among the seven housekeeping genes used in MLST . Additionally, atpD has the lowest dN/dS ratio (0.006) among these genes, indicating strong purifying selection pressure . This suggests that ATP synthase components, including potentially atpE, are under functional constraints that limit sequence variation.

This conservation is biologically relevant as ATP synthase performs essential functions in cellular energy metabolism, and major changes in its structure would likely be deleterious. For researchers, this conservation pattern suggests that findings regarding atpE function in one C. sakazakii strain may be broadly applicable across the species and potentially to related Cronobacter species.

How can genomic data help predict functional variations in ATP synthase across different C. sakazakii isolates?

Genomic data analysis can provide valuable insights into potential functional variations in ATP synthase across different C. sakazakii isolates through several approaches:

• Comparative genomics: Analysis of complete genome sequences, like those available for C. sakazakii ATCC BAA-894 , allows identification of single nucleotide polymorphisms (SNPs) or small insertions/deletions in ATP synthase genes across isolates

• Gene neighborhood analysis: Examination of genomic regions surrounding ATP synthase genes can reveal differences in gene organization or regulatory elements that might affect expression

• Selection pressure analysis: Calculating dN/dS ratios for ATP synthase genes can identify specific codons under positive selection that might confer functional adaptations

• Structural prediction: Using genomic sequence data to predict protein structures can help identify amino acid changes that might alter protein-protein interactions or enzymatic function

Such analyses could potentially correlate genetic variations with phenotypic differences in growth rates, stress tolerance, or virulence across isolates. For instance, given the role of energy metabolism in stress responses, variations in ATP synthase genes might contribute to differences in desiccation tolerance or biofilm formation between strains.

How does ATP synthase activity correlate with C. sakazakii's desiccation tolerance?

Energy production is fundamental to stress response mechanisms, and ATP synthase is the primary enzyme responsible for ATP generation through oxidative phosphorylation. Studies on C. sakazakii's desiccation tolerance have revealed that metabolic pathways and energy production are crucial for survival under drying stress conditions .

For example, research on glutathione transport-related genes in C. sakazakii has shown that disruption of the glutathione transport system leads to decreased desiccation tolerance . The underlying mechanism involves altered potassium ion homeostasis, inhibited proline synthesis, and increased oxidative stress, all of which require energy to counteract . ATP synthase, as the cell's primary ATP producer, would be essential in providing the energy needed for these protective responses.

Further investigations specifically targeting the role of ATP synthase in desiccation tolerance could involve creating conditional mutants with regulated atpE expression to observe how varying levels of ATP synthase activity affect survival under drying conditions.

What role might ATP synthase inhibition play in controlling C. sakazakii contamination in food production environments?

ATP synthase inhibition represents a promising approach for controlling C. sakazakii contamination in food production environments, particularly for products like powdered infant formula where this pathogen poses significant health risks. The potential of this approach is based on several factors:

• Essential function: ATP synthase performs a vital role in cellular energy metabolism, making it an excellent target for antimicrobial development

• Biofilm prevention: Energy production genes, including those related to ATP synthesis, have been implicated in C. sakazakii biofilm formation , which contributes significantly to its persistence in food processing environments

• Structural distinctiveness: Differences between bacterial and human ATP synthases could potentially allow for selective targeting of the bacterial enzyme

• Connection to stress tolerance: ATP generation is linked to stress response mechanisms, including desiccation tolerance , which is crucial for C. sakazakii persistence in dry foods

A methodological approach to developing ATP synthase inhibitors would include:

• Screening for compounds that specifically bind to recombinant C. sakazakii atpE

• Evaluating inhibitor effects on ATP synthesis in membrane vesicles

• Testing promising candidates for efficacy against biofilm formation

• Assessing effects on bacterial survival under desiccation conditions relevant to food production

The goal would be to develop targeted interventions that reduce bacterial persistence without introducing harmful residues into food products.

How might post-translational modifications of atpE affect ATP synthase function in C. sakazakii?

Post-translational modifications (PTMs) of ATP synthase subunit c (atpE) could significantly influence ATP synthase function in C. sakazakii, although specific data on PTMs of this protein are not provided in the search results. Based on knowledge of ATP synthases in other bacteria, several potential PTMs and their functional implications can be hypothesized:

• Phosphorylation: Phosphorylation of specific residues could modulate c-ring rotation or interaction with other subunits, potentially serving as a regulatory mechanism to adjust ATP synthesis rates in response to environmental conditions

• Acetylation: Lysine acetylation might affect the proton-binding capacity of key residues involved in proton translocation

• Methylation: Methylation of specific amino acids could alter protein-protein interactions within the ATP synthase complex

• Lipid modifications: Given atpE's role as a lipid-binding protein , specific lipid attachments might influence its membrane insertion or interaction with membrane lipids

Methodological approaches to investigate these PTMs could include:

• Mass spectrometry-based proteomics to identify and map PTMs on atpE isolated from C. sakazakii grown under different conditions

• Site-directed mutagenesis of potential modification sites to create non-modifiable variants

• Comparative analyses of ATP synthase activity between wild-type and mutant proteins

• Analysis of how stress conditions (particularly desiccation) affect the PTM patterns of atpE

Understanding these modifications could provide insights into how C. sakazakii regulates energy production under different environmental conditions, potentially revealing new targets for pathogen control.

How does C. sakazakii atpE compare structurally and functionally with homologs in other pathogenic bacteria?

Comparative analysis of C. sakazakii ATP synthase subunit c (atpE) with homologs in other pathogenic bacteria reveals both conservation of essential features and potential species-specific adaptations:

• Differences in proton-binding affinity affecting ATP synthesis efficiency under various pH conditions

• Variations in c-ring size or structure potentially influencing the ATP yield per proton

• Species-specific interactions with other ATP synthase components that might affect complex stability under stress conditions

Understanding these comparative aspects can provide insights into potential selective targeting of C. sakazakii ATP synthase for antimicrobial development and help elucidate evolutionary adaptations in energy metabolism across pathogenic bacteria.

What insights can be gained from comparative genomics of ATP synthase genes across Cronobacter species?

Comparative genomics of ATP synthase genes across Cronobacter species can provide valuable insights into evolutionary relationships, functional adaptations, and potential species-specific features. While direct comparative data for atpE is limited in the search results, we can infer patterns based on related ATP synthase genes and general Cronobacter genomics:

• Phylogenetic relationships: Analysis of ATP synthase genes, like atpD which is used in multilocus sequence typing (MLST), helps establish evolutionary relationships among Cronobacter species and strains . The atpD gene shows 10.8% polymorphic sites across different strains , suggesting a relatively high conservation that reflects its essential function.

• Selection pressures: The low dN/dS ratio (0.006) observed for atpD indicates strong purifying selection, suggesting that ATP synthase components are under functional constraints that limit sequence variation. This pattern likely extends to atpE as well.

• Niche adaptations: Variations in ATP synthase genes might reflect adaptations to different environmental niches. For instance, differences between C. sakazakii and C. malonaticus in ATP synthase components could potentially correlate with their different prevalence in food production environments versus clinical settings.

• Regulatory differences: Comparative genomics can reveal variations in regulatory regions controlling ATP synthase expression, potentially explaining differences in stress response or virulence between species.

A methodological approach to such comparative analysis would include:

• Whole genome sequence alignment of multiple Cronobacter species

• Extraction and detailed comparison of ATP synthase operons

• Correlation of genetic variations with phenotypic differences in growth, stress tolerance, or virulence

• Experimental validation of predicted functional differences through gene replacement studies

What are the most promising directions for therapeutic targeting of C. sakazakii ATP synthase?

Several promising directions exist for therapeutic targeting of C. sakazakii ATP synthase, particularly in the context of preventing neonatal infections and controlling contamination in food production environments:

• Structure-based inhibitor design: With the recombinant atpE protein available , structural studies could guide the development of small-molecule inhibitors that specifically bind to unique features of C. sakazakii ATP synthase

• Peptide-based inhibitors: Designed peptides that mimic natural interaction partners of atpE could disrupt ATP synthase assembly or function without affecting human ATP synthases

• Targeting regulatory mechanisms: Approaches that disrupt the regulation of ATP synthase expression or assembly, potentially through RNA-based therapeutics, could provide selective inhibition

• Combination approaches: Developing strategies that target ATP synthase in combination with other stress-response mechanisms, such as the glutathione transport system , could be particularly effective against C. sakazakii

• Biofilm prevention: Given the connection between energy production genes and biofilm formation , ATP synthase inhibitors could potentially serve as biofilm prevention agents in food production settings

The most methodologically sound approach would involve:

• Initial screening of compound libraries against purified recombinant atpE

• Secondary screening in cellular systems to confirm target engagement

• Evaluation of effects on C. sakazakii survival under relevant stress conditions

• Assessment of specificity by testing against human ATP synthase

• In vivo safety and efficacy testing in appropriate animal models

How might systems biology approaches integrate ATP synthase function with broader C. sakazakii metabolism and stress responses?

Systems biology approaches offer powerful frameworks for integrating ATP synthase function with broader C. sakazakii metabolism and stress responses, potentially revealing novel insights into this pathogen's remarkable environmental persistence:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data from C. sakazakii under various stress conditions (particularly desiccation) could map how ATP synthase expression and activity correlate with global metabolic shifts

• Flux balance analysis: Mathematical modeling of C. sakazakii metabolism with varying ATP synthase activity could predict how energy production constraints affect various cellular processes and stress responses

• Protein-protein interaction networks: Mapping the interaction partners of ATP synthase components could reveal unexpected connections to stress response pathways, potentially explaining the link between energy metabolism and stress tolerance

• Comparative systems analysis: Comparing systems-level responses between wild-type C. sakazakii and ATP synthase mutants under stress conditions could identify key dependency relationships

• Metabolic control analysis: Determining how control of metabolic flux is distributed across different enzymes and pathways could reveal the relative importance of ATP synthase in different stress scenarios

A methodological framework for such approaches would include:

• Time-course sampling during exposure to relevant stresses (desiccation, osmotic stress, etc.)

• Parallel multi-omics data collection (RNA-seq, proteomics, metabolomics)

• Computational integration of datasets to identify correlated changes

• Network analysis to identify key regulatory nodes

• Experimental validation of predicted relationships through targeted gene manipulations

These systems approaches could potentially identify novel intervention points for controlling C. sakazakii in food production environments and reducing the risk of neonatal infections.