Recombinant Escherichia coli Sensor protein CreC (creC)

What is the CreC sensor protein in Escherichia coli?

CreC is a sensor histidine kinase that functions as part of a two-component regulatory system in Escherichia coli. It works in conjunction with the response regulator CreB to control the expression of the cre regulon, a group of genes with functions that are still being fully characterized. CreC is also sometimes referred to as PhoM in scientific literature, indicating its potential involvement in phosphate regulation pathways. As a sensory histidine kinase, CreC detects specific environmental signals and, through phosphorylation, transfers this information to CreB, which then modulates gene expression accordingly. This signaling pathway plays a crucial role in bacterial adaptation to changing environmental conditions.

What is the structural composition of recombinant CreC protein?

The recombinant Escherichia coli sensor protein CreC is typically produced with a purity of at least 85% as determined by SDS-PAGE analysis. The protein is encoded by the creC gene (also known as phoM, ECK4391, or JW4362) and functions as a sensory histidine kinase in a two-component regulatory system. The structure includes characteristic domains common to histidine kinases: a sensor domain that detects environmental signals, a transmitter domain containing the histidine phosphorylation site, and in some cases, a receiver domain. When expressed recombinantly, CreC protein can be produced in various expression systems including E. coli, yeast, baculovirus, or mammalian cell systems, depending on the specific research requirements and desired post-translational modifications.

How does the CreC-CreB two-component system function in E. coli?

The CreC-CreB two-component regulatory system in Escherichia coli operates through a phosphorelay mechanism that allows the bacterium to respond to specific environmental cues. In this system, CreC functions as the sensor kinase that detects environmental stimuli, while CreB serves as the response regulator. When appropriate signals are detected, CreC undergoes autophosphorylation at a conserved histidine residue and subsequently transfers the phosphate group to an aspartate residue on CreB. This phosphorylation activates CreB, enabling it to bind to specific DNA sequences and regulate the expression of genes within the cre regulon. The system controls various cellular processes and has been implicated in responses to carbon source availability and antibiotic stress. Research has demonstrated that the CreC-CreB system can influence the expression of other regulatory proteins, including CreD, which is known to affect the cell's response to certain antibiotics.

What are the recommended methods for purifying recombinant CreC protein?

To purify recombinant Escherichia coli sensor protein CreC with high yield and activity, researchers should implement a multi-step purification protocol optimized for histidine kinases. Begin with an appropriate expression system, with cell-free expression systems showing particular promise for maintaining functional CreC protein. After cell lysis under non-denaturing conditions (typically using a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors), initial purification should employ affinity chromatography, typically using a nickel-NTA column if the recombinant protein contains a His-tag. This should be followed by size exclusion chromatography to separate the target protein from aggregates and other contaminants. Ion exchange chromatography can be implemented as an additional purification step when higher purity is required. Throughout the purification process, maintaining a temperature of 4°C and including stabilizing agents such as glycerol (10%) and DTT (1 mM) helps preserve protein activity. The final purified CreC protein should achieve ≥85% purity as verified by SDS-PAGE analysis, with functional verification through in vitro phosphorylation assays to confirm kinase activity.

How can researchers effectively detect CreC protein expression in experimental systems?

For effective detection of CreC protein expression in experimental systems, researchers should employ a multi-faceted approach combining immunological methods and activity assays. Western blotting represents the primary detection method, utilizing specific anti-CreC polyclonal antibodies (such as rabbit anti-E. coli CreC antibodies) at optimized dilutions (typically 1:1000 to 1:5000). For enhanced sensitivity in complex samples, immunoprecipitation followed by western blotting is recommended. Functional detection can be accomplished through in vitro phosphorylation assays that measure the kinase activity of CreC using radiolabeled ATP (γ-32P) and monitoring the transfer of phosphate to its cognate response regulator CreB. For in vivo studies, researchers can utilize reporter gene constructs where the expression of fluorescent proteins or enzymatic reporters is placed under the control of CreB-regulated promoters, providing indirect evidence of CreC activity. Mass spectrometry-based approaches offer the highest specificity for protein identification and can detect post-translational modifications that may affect CreC function. When designing experiments, researchers should include appropriate positive controls (purified recombinant CreC) and negative controls (samples from creC deletion strains) to validate detection methods.

What experimental models are most appropriate for studying CreC function?

When studying CreC function, researchers should select experimental models based on specific research questions regarding this sensor kinase. For basic biochemical characterization, in vitro reconstitution systems using purified recombinant CreC and CreB proteins allow for controlled assessment of phosphotransfer kinetics and interaction dynamics. For cellular-level investigations, isogenic E. coli strains with wild-type creC, creC deletion mutants, and strains expressing constitutively active CreC variants provide valuable comparative models. The well-characterized E. coli K-12 strain serves as an excellent baseline model, while clinically relevant isolates should be incorporated when studying CreC's role in antibiotic responses or pathogenesis. To investigate gene regulation, reporter strains carrying transcriptional fusions between CreB-regulated promoters and reporter genes (such as lacZ, gfp, or luciferase) enable real-time monitoring of the CreC-CreB signaling pathway. Microfluidic single-cell analysis systems prove particularly valuable for observing heterogeneity in CreC-mediated responses within bacterial populations. For in vivo relevance, infection models using Galleria mellonella larvae or murine systems can assess the impact of CreC mutations on bacterial fitness during host colonization or infection. Each model system should be selected based on its ability to address specific aspects of CreC function, from molecular interactions to physiological roles in antibiotic responses.

How does CreC activation influence antibiotic susceptibility in E. coli?

CreC activation has been shown to significantly enhance Escherichia coli growth in the presence of certain antibiotics without altering the minimum inhibitory concentrations (MICs). Specifically, activatory mutations in the CreC sensor kinase improve bacterial growth when exposed to β-lactam antibiotics including cefoxitin, cefotaxime, and the carbapenem antibiotic meropenem. This phenomenon represents a distinct form of antibiotic tolerance rather than classical resistance, as the MIC values remain unchanged despite the enhanced growth capability. The mechanism appears to operate through CreC-mediated activation of the response regulator CreB, which subsequently upregulates expression of the cre regulon, particularly the inner-membrane protein CreD. The overproduction of CreD is essential for this enhanced growth phenotype, as deletion of creD abolishes the effect even in strains with activated CreC. Rather than directly degrading or modifying antibiotics, this pathway likely enhances cellular tolerance through alterations in membrane properties or stress response pathways. This CreC-mediated tolerance mechanism represents a clinically relevant adaptation strategy that may allow bacteria to persist during antibiotic treatment despite appearing susceptible by standard susceptibility testing methods.

What is the relationship between CreC and carbapenem resistance in clinical E. coli isolates?

The relationship between CreC and carbapenem resistance in clinical Escherichia coli isolates presents a nuanced interaction distinct from traditional resistance mechanisms. While CreC activation does not directly increase the minimum inhibitory concentrations (MICs) of carbapenems like meropenem, it significantly enhances bacterial growth capability in the presence of these antibiotics. This presents a potential complication in clinical settings where traditional susceptibility testing might categorize isolates as carbapenem-susceptible despite their enhanced ability to grow in the presence of these drugs. In contrast to the primary carbapenem resistance mechanisms in E. coli, which typically involve the production of carbapenemases such as KPC, NDM, or OXA-181 enzymes, CreC-mediated tolerance operates through alterations in cellular physiology rather than direct antibiotic degradation. The clinical significance of this phenotype warrants careful consideration, particularly in the context of the global emergence of carbapenem-resistant E. coli (CREC) clones, such as those from the ST410 lineage, which represent serious challenges for clinical management and public health. Research suggests that CreC-mediated tolerance may work in concert with other resistance mechanisms, potentially enhancing bacterial fitness during antibiotic therapy and contributing to treatment failure despite apparent susceptibility in standard testing.

What molecular mechanisms underlie CreC sensor activation in response to environmental signals?

The molecular mechanisms underlying CreC sensor activation involve a sophisticated interplay of structural changes and signal transduction events. CreC, as a histidine kinase sensor, contains a periplasmic sensing domain that detects specific environmental cues, though the precise signals remain incompletely characterized. Current evidence suggests that CreC may respond to alterations in carbon source availability, changes in cell envelope integrity, or perturbations in central metabolic pathways. Upon signal detection, the periplasmic domain undergoes conformational changes that are transmitted across the membrane to the cytoplasmic catalytic domain. This conformational shift activates the histidine kinase activity, leading to autophosphorylation at a conserved histidine residue within the dimerization and histidine phosphotransfer (DHp) domain. This phosphoryl group is subsequently transferred to an aspartate residue in the receiver domain of the cognate response regulator CreB. Activatory mutations in CreC typically affect the signal perception or transmission regions of the protein, leading to constitutive kinase activity independent of environmental stimuli. While the precise molecular details of CreC activation remain under investigation, research has demonstrated that its activation significantly impacts cellular processes including growth in antibiotic-containing environments through downstream effects on the cre regulon, particularly through overproduction of the inner-membrane protein CreD.

How do genetic variations in creC affect E. coli fitness across different environmental conditions?

Genetic variations in the creC gene exert profound effects on Escherichia coli fitness across diverse environmental conditions, reflecting the central role of this sensor kinase in bacterial adaptation. Activatory mutations in CreC, particularly those affecting the periplasmic sensing domain or signal transduction regions, have been demonstrated to enhance bacterial growth in the presence of β-lactam antibiotics including cefoxitin, cefotaxime, and meropenem, without altering minimum inhibitory concentrations. This enhanced growth phenotype represents a significant fitness advantage in antibiotic-containing environments and is dependent on CreC-mediated upregulation of the inner-membrane protein CreD. Beyond antibiotic responses, CreC variants likely influence bacterial adaptation to various carbon sources and metabolic stresses, though these relationships require further characterization. The fitness impacts of creC variations appear to be environment-specific, potentially conferring advantages under certain conditions while imposing fitness costs in others. For example, constitutively active CreC mutants might demonstrate enhanced growth under antibiotic stress but reduced competitive fitness in antibiotic-free environments due to the metabolic burden of continuously expressing the cre regulon. These complex fitness landscapes highlight the evolutionary trade-offs associated with creC mutations and underscore the importance of this regulatory system in bacterial adaptation to changing environmental conditions.

What techniques are recommended for studying CreC-CreB phosphotransfer dynamics in vitro?

For studying CreC-CreB phosphotransfer dynamics in vitro, researchers should implement a comprehensive technical approach that combines radioisotope-based assays with advanced biophysical methods. The gold standard technique involves radiolabeled phosphotransfer assays using purified recombinant CreC and CreB proteins. In this method, CreC is autophosphorylated with [γ-32P]ATP, followed by addition of purified CreB and monitoring the transfer of the phosphoryl group over time using SDS-PAGE and autoradiography or phosphorimaging. This allows quantification of phosphotransfer kinetics, including rate constants and phosphorylation half-lives.

For real-time measurement of phosphotransfer, researchers should consider fluorescence-based approaches using CreB proteins labeled with environment-sensitive fluorophores that exhibit measurable changes upon phosphorylation. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) provide valuable complementary data on CreC-CreB binding kinetics and thermodynamics, respectively. To assess the structural basis of phosphotransfer, hydrogen-deuterium exchange mass spectrometry (HDX-MS) reveals conformational changes associated with phosphorylation states.

The experimental conditions should be carefully optimized with the following buffer composition as a starting point: 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, and 5% glycerol. ATP concentrations should typically range from 0.1-1 mM, with protein concentrations adjusted to allow detection while maintaining physiologically relevant ratios (typically 1:1 to 1:4 CreC:CreB). Temperature and pH conditions should mirror physiological parameters (37°C, pH 7.4-8.0) to obtain biologically relevant kinetic data.

How does the CreC-CreB system interact with other regulatory networks in E. coli?

The CreC-CreB two-component regulatory system in Escherichia coli demonstrates significant cross-talk and integration with several other key regulatory networks, forming a complex web of cellular control mechanisms. Most notably, the CreC-CreB system shows functional overlap with the PhoR-PhoB phosphate-sensing system, evidenced by CreC's alternative designation as PhoM and its ability to cross-phosphorylate PhoB under certain conditions. This interconnection suggests coordination between carbon metabolism and phosphate utilization pathways. The cre regulon controlled by CreC-CreB includes genes involved in central carbon metabolism and stress responses, indicating potential interactions with global regulatory networks such as CRP-cAMP and the RpoS-mediated general stress response. Experimental evidence suggests that CreC activation influences the expression of genes beyond the canonical cre regulon, potentially through indirect effects on other transcriptional regulators or through modulation of cellular metabolic states. In the context of antibiotic tolerance, the CreC-CreB system's regulation of CreD connects it to cell envelope integrity pathways and potentially to other antibiotic response mechanisms. The complex interaction network surrounding CreC-CreB enables integration of multiple environmental signals and coordinated cellular responses, though many of these interconnections remain incompletely characterized and represent fertile ground for future research.

What roles does CreC play in E. coli metabolism and stress response pathways?

CreC plays multifaceted roles in Escherichia coli metabolism and stress response pathways, functioning as a critical sensor and regulator that integrates environmental cues with appropriate cellular adaptations. As a sensor histidine kinase in the CreC-CreB two-component system, it influences carbon metabolism through regulation of the cre regulon, which contains genes involved in various metabolic processes. While initially identified in relation to carbon source utilization, recent research has revealed its significant impact on antibiotic stress responses, particularly to β-lactams including cefoxitin, cefotaxime, and meropenem. Activatory mutations in CreC enhance bacterial growth in the presence of these antibiotics without altering minimum inhibitory concentrations, demonstrating its role in stress adaptation rather than direct resistance. This phenotype depends on CreC-mediated overproduction of the inner-membrane protein CreD, suggesting involvement in cell envelope integrity maintenance during antibiotic stress. The CreC sensor likely detects metabolic perturbations or envelope stress signals, translating these into appropriate transcriptional responses via CreB phosphorylation. Its alternative designation as PhoM and potential cross-talk with the PhoR-PhoB phosphate regulatory system further suggests roles in coordinating carbon and phosphate metabolism under varying environmental conditions. Together, these functions position CreC as an important component of E. coli's adaptive response network, integrating metabolic regulation with stress resistance mechanisms.

What emerging technologies are advancing our understanding of CreC function and regulation?

Recent technological advances are revolutionizing our understanding of CreC function and regulation in Escherichia coli. CRISPR-Cas9 genome editing has enabled precise modification of creC and associated genes, allowing researchers to create targeted mutations, domain swaps, and fluorescent protein fusions for in vivo studies. Advanced structural biology techniques, including cryo-electron microscopy and X-ray crystallography, are beginning to reveal the three-dimensional architecture of CreC in different activation states, providing unprecedented insights into conformational changes during signal transduction. Single-molecule techniques such as FRET (Förster Resonance Energy Transfer) are being applied to monitor CreC-CreB interactions and conformational dynamics in real-time, offering detailed kinetic information about phosphotransfer events. High-throughput phenotypic screening approaches using transposon insertion sequencing (Tn-Seq) have identified genetic interactions between creC and other cellular components, revealing previously unrecognized functional connections. Transcriptomics and proteomics studies employing RNA-seq and mass spectrometry are defining the complete regulon controlled by CreC-CreB under various conditions, expanding our understanding beyond previously characterized targets. Microfluidic systems coupled with time-lapse microscopy allow researchers to monitor CreC-mediated responses in single cells across changing environmental conditions, revealing important insights into population heterogeneity and the dynamics of CreC activation. These technological innovations are collectively driving rapid advances in our understanding of CreC biology and its roles in bacterial adaptation to various stresses, including antibiotic exposure.

How might targeting CreC be exploited in developing novel antimicrobial strategies?

Targeting the CreC sensor kinase presents a promising avenue for developing novel antimicrobial strategies that could address the growing challenge of antibiotic resistance. Since CreC activation enhances bacterial growth in the presence of certain antibiotics without changing minimum inhibitory concentrations, inhibiting CreC function could potentially sensitize bacteria to existing antibiotics, particularly β-lactams like cefoxitin, cefotaxime, and meropenem. Such CreC inhibitors would represent antibiotic adjuvants rather than direct antimicrobials, potentially extending the useful lifespan of existing antibiotics. The development of small molecule inhibitors targeting the ATP-binding domain of CreC or compounds that disrupt CreC-CreB interactions could prevent phosphotransfer signaling and downregulate the cre regulon, including CreD, which is essential for the enhanced growth phenotype in antibiotic environments. Another promising approach involves the design of peptide mimetics that interfere with the dimerization interface of CreC, preventing the conformational changes necessary for kinase activation. While targeting two-component systems presents challenges due to their conserved nature across bacterial species, the periplasmic sensing domain of CreC offers a more specific target for inhibitor development. Additionally, antisense RNA strategies or CRISPR-Cas9 based approaches delivered via phage vectors could specifically downregulate creC expression. The potential clinical advantage of targeting CreC lies in its role in tolerance rather than resistance—addressing a phenomenon that conventional susceptibility testing might miss but that nevertheless contributes to treatment failure. Developing these CreC-targeting strategies requires further characterization of CreC structure, binding partners, and activation mechanisms, representing an important frontier in antimicrobial research.

What are the optimal parameters for expressing recombinant CreC protein in different systems?

For all expression systems, the addition of 10% glycerol and 1 mM DTT to purification buffers significantly improves CreC stability. The incorporation of a C-terminal His-tag rather than N-terminal tagging preserves sensor function, yielding biologically active protein. Cell-free expression systems have demonstrated particular promise for producing functional CreC protein with proper membrane integration capabilities, making this method especially valuable for structural and functional studies despite its higher cost.

What are the confirmed interaction partners of CreC and their functional significance?

These interaction data highlight the central role of CreC in integrating multiple cellular processes, particularly through its primary phosphotransfer partner CreB and the subsequent regulation of CreD expression. The cross-talk with the PhoB response regulator demonstrates the interconnected nature of bacterial signaling networks. The essential role of CreD in mediating enhanced growth in antibiotic-containing environments reveals a critical downstream effector of the CreC-CreB system with significant implications for bacterial adaptation to antimicrobial stress.

How do different mutations in the creC gene affect its function and associated phenotypes?

These mutational data demonstrate that CreC function can be modulated in multiple ways, with distinct consequences for bacterial physiology and antibiotic responses. Activatory mutations, particularly those affecting the periplasmic sensing domain or signal transduction regions, confer enhanced growth in the presence of β-lactam antibiotics including cefoxitin, cefotaxime, and meropenem. This phenotype depends on downstream overproduction of CreD, an inner-membrane protein whose function remains incompletely characterized. The phenotypic consequences of creC mutations highlight the importance of this two-component system in bacterial adaptation to various environmental conditions, including antibiotic exposure.

What are the most significant unanswered questions about CreC function in E. coli?

Despite substantial progress in understanding CreC sensor kinase function, several crucial questions remain unanswered. The precise environmental signals directly detected by the CreC periplasmic sensing domain have not been definitively identified, though carbon source availability and cell envelope perturbations have been implicated. The structural mechanisms by which these signals trigger conformational changes leading to kinase activation remain poorly characterized, requiring further structural and biophysical studies. The complete composition of the cre regulon under various environmental conditions has not been comprehensively mapped, with most studies focusing on a limited subset of regulated genes. The molecular function of CreD, the inner-membrane protein essential for CreC-mediated enhanced growth in antibiotic environments, remains enigmatic—understanding its biochemical activities could reveal critical insights into bacterial antibiotic tolerance mechanisms. The physiological significance of the potential cross-talk between CreC and the PhoR-PhoB phosphate regulatory system requires further investigation, particularly regarding conditions under which CreC phosphorylates PhoB. The evolutionary history of the CreC-CreB system and its distribution across bacterial species might reveal important aspects of its core functions and species-specific adaptations. Finally, the clinical significance of CreC-mediated antibiotic tolerance requires evaluation in patient isolates to determine whether this phenomenon contributes to treatment failures despite apparent antibiotic susceptibility in standard testing. Addressing these questions will provide a more complete understanding of CreC function and its potential as a target for novel antimicrobial strategies.