Recombinant Staphylococcus epidermidis Uncharacterized sensor-like histidine kinase SE_0166 (SE_0166)

What is SE_0166 and what is its significance in bacterial signaling?

SE_0166 is an uncharacterized sensor-like histidine kinase found in Staphylococcus epidermidis (strain ATCC 12228) with a UniProt accession number Q8CU03 . This protein belongs to the two-component signal transduction system (TCS) family, which is critical for bacterial adaptation to environmental changes. Histidine kinases function as sensory proteins that, upon detecting specific stimuli, undergo autophosphorylation at a conserved histidine residue and subsequently transfer this phosphate group to a response regulator, which then mediates cellular responses .

The significance of SE_0166 lies in its potential role in S. epidermidis pathogenicity and adaptation. As S. epidermidis is both a common skin commensal and a nosocomial pathogen responsible for biofilm formation on medical devices and subsequent infections , understanding the function of its signaling proteins may reveal mechanisms underlying its transition from commensal to pathogen.

How does SE_0166 compare structurally to characterized histidine kinases?

While SE_0166 remains uncharacterized, its predicted structure can be compared to well-studied histidine kinases such as those from Thermotoga maritima and the EvgS histidine kinase from Escherichia coli :

• Membrane topology: Like most sensor histidine kinases, SE_0166 likely has a transmembrane region that anchors it to the bacterial membrane, with the sensing domain oriented toward the extracellular/periplasmic space and catalytic domains in the cytoplasm .

• Signal transduction mechanism: Similar to other histidine kinases, SE_0166 likely functions through conformational changes triggered by environmental stimuli. These changes are probably transmitted through the HAMP domain, which has been shown in other histidine kinases to undergo rotational movements or piston-like motions .

• Catalytic mechanism: The catalytic mechanism likely involves the formation of homodimers, with the histidine residue in the DHp domain of one monomer being phosphorylated by the CA domain of either the same monomer (cis-autophosphorylation) or the partner monomer (trans-autophosphorylation) .

What experimental designs are most suitable for studying the function of SE_0166?

To investigate the function of SE_0166, researchers should consider a multi-faceted experimental approach:

• True experimental design with control groups: This approach allows for rigorous testing of hypotheses about SE_0166 function by comparing wild-type S. epidermidis with SE_0166 knockout or modified strains .

• Solomon four-group design: For comprehensive analysis, researchers might employ this design which includes four randomly allocated groups: wild-type with and without treatment, and SE_0166 mutant with and without treatment, allowing multiple comparisons and control for testing effects .

• Sequential experimental phases:

  a) Bioinformatic characterization: Predict ligands and functions based on sequence homology with characterized histidine kinases .

  b) In vitro biochemical characterization: Express and purify recombinant SE_0166 to test for autokinase activity, phosphotransfer specificity, and potential cognate response regulators .

  c) Genetic manipulation: Create knockout and point mutants to study the role of SE_0166 in S. epidermidis physiology and pathogenicity .

  d) Environmental response profiling: Expose S. epidermidis to various stimuli to identify conditions that activate SE_0166 signaling, similar to methods used for the EvgS histidine kinase .

What methods can be used for expression and purification of recombinant SE_0166?

Based on available information and standard practices for histidine kinase proteins:

• Expression systems:

  • Prokaryotic expression in E. coli using vectors like pET or pBAD series

  • Cell-free expression systems for potentially toxic membrane proteins

  • Consideration of expressing only the cytoplasmic portion (lacking transmembrane domains) for improved solubility

• Purification strategy:

  • Affinity chromatography using added tags (His-tag, GST-tag)

  • Size exclusion chromatography for higher purity

  • For the complete protein containing transmembrane domains, detergent solubilization or nanodiscs may be required

• Storage considerations:

  • Store in Tris-based buffer with 50% glycerol at -20°C

  • Avoid repeated freeze-thaw cycles

  • For extended storage, maintain at -80°C

  • Working aliquots can be stored at 4°C for up to one week

How can researchers detect and measure SE_0166 activity in vitro?

Several complementary approaches can be used to detect and quantify the activity of recombinant SE_0166:

• Autophosphorylation assays:

  • Incubate purified SE_0166 with [γ-32P]ATP or [γ-33P]ATP

  • Analyze incorporation of radiolabeled phosphate by SDS-PAGE and autoradiography

  • Non-radioactive alternatives include Phos-tag™ SDS-PAGE to detect phosphorylated species based on mobility shift

• Phosphotransfer profiling:

  • Perform in vitro phosphotransfer experiments between autophosphorylated SE_0166 and candidate response regulators

  • Monitor phosphotransfer kinetics to identify cognate response regulators

• ATPase activity measurements:

  • Measure ATP hydrolysis using colorimetric assays (e.g., malachite green assay)

  • Calculate kinetic parameters (Km, Vmax) under different conditions

• Conformational change detection:

  • Use fluorescence resonance energy transfer (FRET) with labeled protein to detect ligand-induced conformational changes

  • Employ circular dichroism spectroscopy to monitor secondary structure changes upon activation

What environmental signals might activate SE_0166 in S. epidermidis?

While the specific signals that activate SE_0166 remain unknown, insights can be drawn from studies of other bacterial histidine kinases:

• Potential activating signals:

  • pH changes (similar to EvgS in E. coli, which responds to mildly acidic conditions)

  • Redox state changes (like the PAS domain-containing histidine kinases that respond to oxygen or redox conditions)

  • Membrane stress (common for membrane-embedded sensor kinases)

  • Antimicrobial compounds (particularly relevant for a nosocomial pathogen)

• Experimental approaches to identify signals:

  • Transcriptional reporter assays using SE_0166-dependent promoters under various conditions

  • Phosphorylation state analysis of SE_0166 in response to environmental stimuli

  • Comparative analysis of wild-type and SE_0166 mutant growth under different stress conditions

• Contextual considerations:

  • The ecological niche of S. epidermidis (human skin and hospital environments) suggests SE_0166 might respond to host-derived signals or hospital-specific conditions

  • The difference between commensal and nosocomial strains might influence SE_0166 function

What role might SE_0166 play in S. epidermidis virulence and biofilm formation?

The potential role of SE_0166 in pathogenicity can be analyzed through several perspectives:

• Comparative genomics evidence:

  • Analysis of SE_0166 presence/absence or sequence variation across commensal versus nosocomial S. epidermidis strains

  • Studies have shown that S. epidermidis strains separate into two phylogenetic groups, with one consisting only of commensals

  • Genes like formate dehydrogenase serve as discriminatory markers between commensal and pathogenic strains

• Functional hypotheses:

  • SE_0166 may regulate the expression of virulence factors such as biofilm components

  • It could sense host environmental cues during infection

  • It might coordinate adaptation to antimicrobial stress

• Experimental evidence required:

  • Phenotypic characterization of SE_0166 knockout strains in biofilm formation assays

  • Virulence assessment in appropriate infection models

  • Transcriptomic analysis comparing wild-type and SE_0166 mutant strains under infection-relevant conditions

How can researchers identify the cognate response regulator of SE_0166?

Identifying the cognate response regulator is crucial for understanding the complete signal transduction pathway:

• Bioinformatic approaches:

  • Genomic context analysis (response regulators are often encoded adjacent to their cognate histidine kinases)

  • Phylogenetic profiling (co-evolution of histidine kinase and response regulator pairs)

  • Structure-based prediction of interaction specificity

• Experimental strategies:

  • In vitro phosphotransfer profiling with purified SE_0166 and candidate response regulators

  • Bacterial two-hybrid or protein-protein interaction assays

  • In vivo cross-linking followed by co-immunoprecipitation

  • Phenotypic comparison of SE_0166 and candidate response regulator mutants

• Validation methods:

  • Epistasis analysis of SE_0166 and response regulator mutants

  • Reconstitution of the signaling pathway in a heterologous host

  • Demonstration of in vivo phosphotransfer using Phos-tag™ Western blotting

What are the key challenges in studying an uncharacterized histidine kinase like SE_0166?

Researchers face several technical and conceptual challenges:

• Membrane protein challenges:

  • Difficulties in expression and purification of full-length membrane proteins

  • Maintaining native conformation and activity during solubilization

  • Reconstituting appropriate membrane environment for functional studies

• Unknown activation signals:

  • Identifying the specific environmental cues that activate SE_0166

  • Developing appropriate assay conditions that reflect physiological signaling

• Functional redundancy:

  • S. epidermidis likely has multiple histidine kinases with potentially overlapping functions

  • Isolating the specific contribution of SE_0166 requires careful experimental design

• Technical considerations:

  • Establishing reliable activity assays for an uncharacterized protein

  • Developing specific antibodies or detection methods

  • Genetic manipulation challenges in clinical S. epidermidis isolates

How can researchers design mutations to probe SE_0166 function?

Strategic mutation design can provide insights into SE_0166 function:

• Catalytic residue mutations:

  • Histidine to alanine mutation in the DHp domain to abolish autophosphorylation

  • Mutations in the CA domain ATP-binding site (e.g., conserved N-box or G-box) to prevent ATP binding

  • These mutations create catalytically inactive versions for dominant-negative or phosphatase-only variants

• Signal perception mutations:

  • Cysteine mutations in potential redox-sensing regions, similar to C671A and C683A mutations that affected EvgS activation under anaerobic conditions

  • Chimeric proteins with sensing domains from characterized histidine kinases to test signal specificity

• Structure-guided mutations:

  • Based on crystal structures of homologous histidine kinases like those from T. maritima

  • Mutations at dimerization interfaces to test oligomerization requirements

  • Alterations in the HAMP domain to affect signal transduction, as these domains have been shown to be critical in signal transmission

What experimental controls are essential when working with recombinant SE_0166?

Proper controls ensure reliable and interpretable results:

• Protein quality controls:

  • Verification of proper folding using circular dichroism or fluorescence spectroscopy

  • Size exclusion chromatography to confirm expected oligomeric state

  • Mass spectrometry to verify protein identity and detect potential modifications

• Activity assay controls:

  • Catalytically inactive mutant (H→A in DHp domain) as negative control

  • Well-characterized histidine kinase as positive control for assay validation

  • Time-course experiments to establish reaction kinetics

  • Metal ion dependency tests (Mg²⁺, Mn²⁺) to optimize reaction conditions

• Specificity controls:

  • Testing multiple potential response regulators to confirm specificity

  • Competition assays with unlabeled ATP to verify binding site specificity

  • Phosphatase-dead versions to distinguish kinase and phosphatase activities

How should researchers interpret contradictory results in SE_0166 activation studies?

When faced with contradictory results, consider these analytical approaches:

• Systematic analysis of variables:

  • Environmental conditions (pH, redox state, temperature) may significantly affect SE_0166 activity, as seen with the EvgS histidine kinase that is activated only under aerobic and mildly acidic conditions

  • Strain-specific differences may exist, particularly between commensal and nosocomial isolates of S. epidermidis

  • Experimental setup differences (in vitro vs. in vivo, buffer components, protein tags)

• Regulatory complexity considerations:

  • Histidine kinases can have both kinase and phosphatase activities

  • The activation state may depend on multiple inputs integrated at different domains

  • Cross-talk with other two-component systems may occur

• Reconciliation strategies:

  • Design experiments that directly test competing hypotheses

  • Employ multiple detection methods to confirm results

  • Consider time-dependent effects and the dynamic nature of signaling systems

What troubleshooting approaches can researchers use when SE_0166 shows no detectable activity?

Several strategies can address lack of detectable activity:

• Protein quality assessment:

  • Verify protein integrity by SDS-PAGE, Western blotting, and mass spectrometry

  • Check for proper folding using spectroscopic methods

  • Ensure appropriate storage conditions (avoid repeated freeze-thaw cycles)

• Assay optimization:

  • Test different buffer conditions (pH, salt concentration, reducing agents)

  • Vary metal ion cofactors (Mg²⁺, Mn²⁺) and their concentrations

  • Adjust protein and substrate concentrations

  • Extend incubation times for slow reactions

• Signal identification:

  • Systematically test potential activating signals (pH changes, redox conditions)

  • Consider that EvgS histidine kinase is activated only during aerobic growth conditions

  • Test the effect of ubiquinone or other electron carriers, which have been shown to be required for some histidine kinase activation

• Domain analysis:

  • Express and test individual domains separately

  • Consider expressing only the cytoplasmic portion if the full-length protein is problematic

  • Create chimeric proteins with domains from well-characterized histidine kinases

How can transcriptomic data be used to infer SE_0166 function?

Transcriptomic approaches offer powerful insights into SE_0166 function:

• Experimental design for transcriptomics:

  • Compare wild-type and SE_0166 knockout strains under various conditions

  • Analyze conditional SE_0166 overexpression or constitutively active mutants

  • Time-course experiments following exposure to potential activating signals

• Data analysis strategies:

  • Identify differentially expressed genes between wild-type and mutant strains

  • Perform gene ontology and pathway enrichment analysis

  • Look for conserved promoter motifs in co-regulated genes to identify potential binding sites for the cognate response regulator

• Functional validation:

  • Confirm direct regulation of key targets using reporter assays

  • Perform chromatin immunoprecipitation of the response regulator to identify binding sites

  • Test phenotypic effects of manipulating downstream genes

How does SE_0166 compare to histidine kinases in other Staphylococcal species?

Comparative analysis provides evolutionary and functional insights:

• Sequence conservation:

  • Analyze sequence homology with histidine kinases from S. aureus and other staphylococci

  • Identify conserved and variable regions that might reflect shared or species-specific functions

  • Assess whether SE_0166 belongs to the core or variable genome of S. epidermidis

• Genomic context analysis:

  • Compare the genomic neighborhood of SE_0166 across staphylococcal species

  • Identify conserved gene clusters that might indicate functional relationships

  • Consider horizontal gene transfer events that might have contributed to species-specific adaptations

• Functional comparison:

  • Evaluate whether SE_0166 homologs in other species have been characterized

  • Compare phenotypes of corresponding mutants across species

  • Consider the ecological niches of different staphylococci and how this might relate to SE_0166 function

What can be learned from the crystal structure of related histidine kinases?

Structural insights from related proteins inform SE_0166 research:

• Key structural features from homologs:

  • The crystal structure of the complete cytoplasmic region of a sensor histidine kinase from Thermotoga maritima at 1.9 Å resolution revealed the relative disposition of domains in a state poised for phosphotransfer

  • This structure inspired hypotheses for the mechanisms of autophosphorylation, phosphotransfer, and response-regulator dephosphorylation

• Functional implications:

  • Structural data suggests histidine kinases access multiple conformational states for different catalytic activities

  • The structure-based scheme for multiple activities indicates both symmetric ground states and asymmetric intermediate structures

  • Coiled-coil regions may play a critical role in signal transduction, with conformational changes transmitted through these elements

• Modeling approaches:

  • Homology modeling of SE_0166 based on crystal structures of related proteins

  • Molecular dynamics simulations to predict conformational changes upon activation

  • Docking studies to identify potential ligands or interaction partners

What emerging technologies could advance understanding of SE_0166 function?

Several cutting-edge approaches may accelerate SE_0166 research:

• Cryo-electron microscopy:

  • Determine the full-length structure of SE_0166 in different activation states

  • Visualize conformational changes associated with signaling

• Single-molecule techniques:

  • FRET-based approaches to monitor conformational dynamics in real-time

  • Magnetic tweezers to study force-dependent structural changes

• Genome editing technologies:

  • CRISPR-Cas9 for precise genetic manipulation of S. epidermidis

  • Multiplexed genome engineering to study combinatorial effects with other signaling systems

• Synthetic biology approaches:

  • Reconstitution of minimal signaling systems

  • Engineering sensor specificity for biotechnological applications

How might understanding SE_0166 function contribute to addressing S. epidermidis infections?

Potential translational implications include:

• Therapeutic targeting:

  • If SE_0166 regulates virulence or antibiotic resistance, it could be a target for anti-virulence therapies

  • Histidine kinase inhibitors could potentiate existing antibiotics or prevent biofilm formation

• Diagnostic applications:

  • SE_0166 activity or expression patterns might serve as biomarkers for virulent strains

  • Detection of SE_0166-regulated genes could indicate active infection

• Vaccine development:

  • If exposed regions of SE_0166 are immunogenic, they might be included in multi-component vaccines

  • Studies have identified prospective candidate antigens from S. epidermidis for prophylaxis or immunotherapy

What interdisciplinary approaches might yield new insights into SE_0166 function?

Cross-disciplinary collaboration offers novel perspectives:

• Systems biology:

  • Integrate transcriptomic, proteomic, and metabolomic data to place SE_0166 in a broader regulatory network

  • Mathematical modeling of two-component system dynamics

• Host-pathogen interaction studies:

  • Investigate how host factors influence SE_0166 activation

  • Study the impact of SE_0166 signaling on host immune responses

• Environmental microbiology:

  • Examine SE_0166 function in biofilms and mixed microbial communities

  • Investigate how SE_0166 contributes to competitive fitness in different environments

• Comparative genomics:

  • Analyze SE_0166 conservation and variation across the pan-genome of S. epidermidis

  • Studies have shown that commensal S. epidermidis strains have an open pan-genome with 80% core genes and 20% variable genes