Recombinant Sensor protein CutS (cutS)

What is CutS and what is its role in bacterial physiology?

CutS is a sensor kinase that forms part of the CutRS two-component system in Streptomyces bacteria. This system mediates the secretion stress response, which is activated when bacteria detect protein misfolding in the extracellular environment. The CutRS system works alongside the CssRS two-component system to control secretion stress response by regulating the expression of HtrA-family chaperones. Together, these systems ensure proper protein folding in the fluctuating soil environment where Streptomyces bacteria typically reside .

How does the CutS protein structure relate to its function?

The CutS protein contains an extracellular sensor domain positioned between two transmembrane helices. This sensor domain features two highly conserved cysteine residues (C85 and C103 in S. venezuelae) that are invariant across all Streptomyces CutS homologues. These cysteines are positioned approximately 5Å apart, the optimal distance for forming disulfide bonds. This structural arrangement allows CutS to monitor the redox state of the extracellular environment, which serves as a proxy for correct disulfide bond formation in Sec-translocated proteins .

What are reliable methods for producing recombinant CutS protein?

Methodological Approach:

• Gene Synthesis and Optimization:

  • Synthesize the cutS gene with codon optimization for your expression system (E. coli, yeast, etc.)

  • Consider fusion tags (His6, GST, MBP) to facilitate purification and enhance solubility

• Expression Vector Selection:

  • For soluble expression: pET vectors with T7 promoter system for E. coli

  • For membrane-bound studies: Consider specialized vectors for membrane proteins

• Expression Conditions for Optimal Yield:

  Parameter	Recommended Conditions	Notes
  Expression host	E. coli BL21(DE3) or Rosetta(DE3)	Rosetta strain provides rare codons
  Temperature	16-18°C	Lower temperature reduces inclusion body formation
  Induction	0.1-0.5 mM IPTG	Start with lower concentration for membrane proteins
  Duration	16-20 hours	Overnight expression at lower temperature
  Media	LB or TB with appropriate antibiotics	TB provides higher biomass

• Purification Strategy:

  • For full-length CutS (membrane protein): Detergent solubilization (DDM, LMNG)

  • For sensor domain only: Standard affinity chromatography

  • Consider size exclusion chromatography as a final polishing step

How can the activity of recombinant CutS be measured in vitro?

Methodological Approach:

• Autophosphorylation Assay:

  • Incubate purified CutS with [γ-32P]ATP

  • Monitor phosphorylation by SDS-PAGE and autoradiography

  • Compare activity under different redox conditions (DTT, H2O2, oxidized/reduced glutathione)

• Phosphotransfer Assay:

  • Co-incubate phosphorylated CutS with purified CutR

  • Measure phosphotransfer rate under various conditions

  • Analyze by Phos-tag SDS-PAGE to separate phosphorylated and non-phosphorylated forms

• Thermal Shift Assays:

  • Use differential scanning fluorimetry to detect conformational changes

  • Compare thermal stability under reducing vs. oxidizing conditions

• Disulfide Bond Formation Analysis:

  • Use mass spectrometry to identify disulfide bond formation between C85 and C103

  • Employ non-reducing SDS-PAGE to assess mobility differences

How can site-directed mutagenesis of CutS conserved cysteines inform its mechanism?

Mutagenesis studies replacing the conserved cysteines in CutS provide critical insights into the activation mechanism. When both conserved cysteines (C85 and C103 in S. venezuelae) are replaced with serine residues (CutS(C85S,C103S)), the resulting variant exhibits constitutive activity. This is evidenced by higher expression of htrA3 and stronger repression of htrB, consistent with activation of the CutR regulatory pathway.

Methodological Approach for Mutagenesis Studies:

• Design and Create Mutants:

  • Single mutants: C85S, C103S

  • Double mutant: C85S,C103S

  • Control mutants: mutations in non-conserved residues

• Expression Analysis:

  • Use qRT-PCR to measure expression of CutR target genes (htrA3, htrB)

  • Compare wild-type, ΔcutRS, and cysteine mutant strains

  • Analyze protein levels using Western blotting

• Functional Characterization:

  • Assess growth phenotypes of different mutants

  • Evaluate response to protein secretion stress inducers

  CutS Variant	htrA3 Expression	htrB Expression	Growth Phenotype
  Wild-type	Baseline	Baseline	Normal
  ΔcutRS	Decreased	Increased	Defective
  CutS(C85S,C103S)	Higher than WT	Lower than WT	Normal (rescued)

• Structural Analysis:

  • Use AlphaFold or other prediction tools to model the impact of mutations

  • Consider advanced biophysical techniques (e.g., X-ray crystallography, cryo-EM) to determine structure

What approaches can be used to study the interplay between CutRS and CssRS systems?

The research indicates that CutRS and CssRS systems work together in the secretion stress response, with potential opposing roles. This complex interplay requires sophisticated experimental approaches to untangle.

Methodological Approaches:

• Genetic Manipulation Strategies:

  • Create single and double deletion mutants (ΔcutRS, ΔcssRS, ΔcutRS/ΔcssRS)

  • Develop inducible expression systems for each component

  • Use CRISPR-Cas9 for precise genomic editing

• Transcriptomic Analysis:

  • RNA-seq under various stress conditions comparing the mutants

  • ChIP-seq for both CutR and CssR to identify genome-wide binding sites

  • Time-course experiments to capture dynamic responses

• Proteomics Approach:

  • TMT (Tandem Mass Tag) proteomics to quantify protein level changes

  • Phosphoproteomics to identify signaling cascades

  • Protein-protein interaction studies using crosslinking mass spectrometry

• In vivo Sensor Domain Studies:

  • FRET-based sensors to monitor disulfide bond formation in real-time

  • Native disulfide bond mapping using proteomic approaches

  • Redox state tracking during various stress conditions

How can CutS function be assessed in heterologous systems?

Methodological Approach:

• Selection of Appropriate Heterologous Hosts:

  • E. coli: Widely used but lacks endogenous Streptomyces protein secretion machinery

  • B. subtilis: Gram-positive with well-characterized secretion stress systems

  • Other Streptomyces species: For comparative studies across related organisms

• System Construction:

  • Clone the cutRS operon with native or controlled promoters

  • Include reporter systems fused to CutR-dependent promoters

  • Consider chimeric systems with components from different species

• Functional Validation:

  • Challenge with secretion stress inducers (e.g., DTT, misfolded protein overexpression)

  • Monitor activation using fluorescent or luminescent reporters

  • Compare response in wild-type vs. heterologous systems

• Cross-species Complementation:

  • Test if S. venezuelae CutRS can complement other bacterial TCS mutants

  • Examine if CutS sensors from diverse species respond to the same signals

  • Create domain-swapped chimeras to identify species-specific sensing mechanisms

What are common challenges in working with recombinant CutS and how can they be addressed?

Common Challenges and Solutions:

• Membrane Protein Expression Issues:

  Challenge	Solution
  Toxicity to expression host	Use tightly controlled inducible systems; lower induction levels
  Inclusion body formation	Lower expression temperature; use solubility-enhancing tags (MBP, SUMO)
  Low yield	Optimize codon usage; try different expression hosts; use specialized media

• Maintaining Proper Disulfide Bonds:

  • Express in the presence of appropriate redox buffers

  • Consider specialized E. coli strains designed for disulfide bond formation (e.g., SHuffle)

  • Optimize oxidative refolding protocols if purifying from inclusion bodies

• Activity Preservation:

  • Determine optimal buffer conditions (pH, salt, reducing agents) through stability screens

  • Include stabilizing agents (glycerol, specific lipids) when working with full-length CutS

  • Consider nanodiscs or liposomes for functional reconstitution

How should experiments be designed to distinguish between direct and indirect effects of CutS activity?

Methodological Approach:

• Direct Target Identification:

  • ChIP-seq for CutR binding sites (the direct output of CutS activation)

  • In vitro DNA binding assays with purified CutR and candidate promoters

  • Bacterial one-hybrid or EMSA to confirm direct interactions

• Distinguishing from Secondary Effects:

  • Time-course experiments to establish order of events

  • Inducible systems to achieve temporal control of CutS/CutR activation

  • Use of transcription/translation inhibitors to block secondary responses

• Epistasis Analysis:

  • Create strains with combinations of mutations in CutRS and downstream factors

  • Test synthetic phenotypes to establish pathway hierarchies

  • Use constitutively active variants to bypass upstream regulation

• Integration with Other Signaling Pathways:

  • Map the crosstalk between CutRS and other stress response systems

  • Investigate the relationship between CutRS and CssRS with phosphotransfer studies

  • Determine if other cellular processes affect CutS redox sensing

How might CutS function in detecting extracellular redox states compare across bacterial species?

The conservation of sensor kinases with extracellular cysteine residues appears to be widespread across bacterial species. Analysis of approximately 12,800 bacterial genomes revealed that 98.9% of bacterial strains across all classes have at least one sensor kinase with two or more extracellular cysteine residues, suggesting extracellular redox sensing is a conserved mechanism in bacteria.

Research Approaches:

• Comparative Genomics and Evolution:

  • Phylogenetic analysis of CutS homologs across bacterial phyla

  • Correlation with ecological niches and lifestyle (soil, host-associated, etc.)

  • Identification of co-evolving partners and conserved genomic contexts

• Structural Biology Approach:

  • Compare predicted structures of CutS-like sensor domains across species

  • Identify conserved structural features beyond primary sequence

  • Map the evolutionary conservation onto structural models

• Experimental Cross-Species Validation:

  Species	Sensor Kinase	Conserved Cysteines	Redox Sensitivity
  S. venezuelae	CutS	C85, C103	Confirmed
  S. coelicolor	CutS	Yes (position varies)	Predicted
  Other Streptomyces	CutS homologs	Yes (>100 species)	To be determined
  Other bacterial classes	Various SKs	98.9% of species have ≥1 SK with ≥2 cysteines	To be investigated

• Functional Divergence Studies:

  • Test response to different redox-altering conditions across species

  • Identify species-specific stress responses mediated by CutS-like proteins

  • Investigate adaptations to different environmental redox challenges

What are the implications of CutS redox sensing for antimicrobial development?

Research Directions:

• Target Validation:

  • Assess essentiality of CutS in different bacterial pathogens

  • Evaluate virulence attenuation in CutS mutants

  • Determine if CutS inhibition sensitizes bacteria to existing antibiotics

• Inhibitor Design Strategies:

  • Target the conserved cysteine-containing sensor domain

  • Design redox-active compounds that interfere with disulfide bond formation

  • Develop peptidomimetics that disrupt CutS-CutR interactions

• Screening Approaches:

  • High-throughput screens using reporter strains

  • Structure-based virtual screening against the sensor domain

  • Fragment-based approaches targeting the ATP-binding domain

• Potential Applications:

  • Antivirulence strategies that don't kill bacteria but reduce pathogenicity

  • Combination therapies with existing antibiotics

  • Species-specific targeting based on variations in sensor domains

How could artificial manipulation of CutS activity be used for biotechnological applications?

Research Applications:

• Protein Production Enhancement:

  • Engineering CutS to optimize secretion stress response

  • Creating strains with tunable secretion capacity for industrial protein production

  • Developing feedback systems to maintain optimal secretion efficiency

• Biosensor Development:

  • Creating CutS-based whole-cell biosensors for extracellular redox state

  • Developing in vitro biosensors for detecting improperly folded proteins

  • Engineering CutS variants with altered specificity for different redox states

• Synthetic Biology Applications:

  • Incorporating CutS into synthetic circuits responding to redox signals

  • Creating orthogonal two-component systems for programmable cell behavior

  • Engineering artificial stress response systems with predictable outcomes

• Implementation Strategies:

  Application	Engineering Approach	Expected Benefit
  Recombinant protein production	Tunable CutS-CutR system	Increased yield of correctly folded secreted proteins
  Bioremediation	CutS sensors tuned to specific pollutants	Bacteria that respond to and degrade environmental contaminants
  Diagnostic tools	CutS-based reporters	Detection of redox-altering conditions in clinical samples
  Cell-based therapy	Engineered probiotics with modified CutS	Responsive therapeutic delivery based on host redox state

How does the CutS redox sensing mechanism integrate with other bacterial stress response systems?

The CutS/CutR system appears to be integrated with multiple stress response pathways. Notably, there's significant crosstalk between the CutRS and CssRS systems, with evidence that cssRS is overexpressed in ΔcutRS mutants. Additionally, deletion of cssRS in the ΔcutRS background restored the mutant to wild-type growth, suggesting these systems play complementary but potentially opposing roles in monitoring and controlling extracellular protein folding.

Integration Mechanisms:

• Cross-Regulation:

  • CutRS may regulate expression of other stress response systems

  • Shared target genes (e.g., htrB) suggest coordinated regulation

  • Potential phosphorylation crosstalk between two-component systems

• Signal Integration:

  • CutS likely responds to redox changes caused by various stressors

  • Multiple stress inputs may converge on the CutRS system

  • The system may function as a node in a larger stress response network

• Experimental Approaches:

  • Global transcriptomic analysis under various stress conditions

  • Synthetic genetic array analysis to map genetic interactions

  • Phosphoproteomics to identify signaling cascades

  • Chromatin immunoprecipitation to identify regulatory networks

What is the role of CutS in coordinating the expression of HtrA-family proteins?

CutS plays a crucial role in coordinating the expression of HtrA-family chaperone/proteases, which are essential for proper protein folding in the extracellular environment. Specifically, CutRS directly regulates two of the four conserved htrA-like genes in Streptomyces: it activates htrA3 expression and represses htrB expression.

Regulatory Mechanisms:

• Direct Transcriptional Control:

  • CutR binds to a consensus sequence (TAWATAAA) in target promoters

  • The position of this binding site relative to the transcription start site determines whether CutR activates or represses transcription

  • ChIP-seq and proteomics data confirm this dual regulatory role

• Coordination with CssRS System:

  • CssR directly activates htrA1, htrA2, and htrB expression

  • CutR activates htrA3 and represses htrB

  • This creates a complex regulatory network with potential for fine-tuning the stress response

• Functional Specialization Model:

  HtrA Protein	Primary Regulator	Regulation by CutR	Regulation by CssR	Proposed Function
  HtrA1	CssR	Not directly	Activated	General chaperone/protease
  HtrA2	CssR	Not directly	Activated	General chaperone/protease
  HtrA3	CutR	Activated	Not directly	Specialized redox-related function
  HtrB	Both	Repressed	Activated	Shared function with complex regulation

What open questions remain about CutS structure-function relationships?

Despite significant advances in understanding CutS function, several fundamental questions remain unanswered regarding its structure-function relationship:

• Detailed Structural Analysis Needs:

  • High-resolution structure of the full-length CutS protein

  • Conformational changes upon disulfide bond formation/breakage

  • Structural basis for signal transduction across the membrane

• Molecular Mechanism Questions:

  • Precise chemical nature of the sensed redox signal

  • Kinetics of the disulfide bond formation/reduction in vivo

  • Mechanism of transmembrane signal transduction to the kinase domain

• Research Approaches:

  • Advanced structural biology techniques (cryo-EM, X-ray crystallography)

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • FRET-based sensors to monitor conformational changes in real-time

  • Molecular dynamics simulations to predict conformational changes

How can systems biology approaches enhance our understanding of CutS function in bacterial physiology?

Systems Biology Strategies:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Create comprehensive regulatory networks centered on CutS

  • Identify emergent properties not apparent from individual analyses

• Quantitative Modeling:

  • Develop mathematical models of the CutS signaling pathway

  • Simulate pathway dynamics under different environmental conditions

  • Predict system behavior in response to perturbations

• Single-Cell Analysis:

  • Investigate cell-to-cell variability in CutS activity

  • Study stochastic effects in stress response activation

  • Track dynamic responses using fluorescent reporters

• Advanced Data Integration:

  Data Type	Technique	Information Gained
  Genomics	Comparative genomics	Evolutionary context of CutS across species
  Transcriptomics	RNA-seq, NET-seq	Dynamic gene expression changes
  Proteomics	MS-based proteomics	Protein abundance and modifications
  Metabolomics	LC-MS, NMR	Metabolic consequences of CutS activation
  Phenomics	High-throughput phenotyping	Physiological effects of CutS perturbation
  Interactomics	AP-MS, BioID, PLA	Protein-protein interaction network