Recombinant Escherichia coli Sensor histidine kinase GlrK (glrK)

What is the fundamental role of histidine kinase GlrK in E. coli signaling?

GlrK functions as a membrane-bound sensor histidine kinase that participates in two-component signal transduction, similar to well-characterized systems like YehU/YehT and YpdA/YpdB. Upon activation by specific environmental stimuli, GlrK autophosphorylates at a conserved histidine residue and subsequently transfers this phosphoryl group to its cognate response regulator, typically GlrR (QseF). This phosphotransfer initiates downstream transcriptional changes, allowing bacteria to adapt to changing environmental conditions . As with other histidine kinases, GlrK likely contains transmembrane domains, a conserved histidine-containing phosphotransfer domain, and possibly additional sensory domains like GAF domains observed in related kinases such as YehU and YpdA .

How does GlrK differ structurally from other E. coli histidine kinases?

GlrK belongs to the larger family of sensor histidine kinases in E. coli but contains specific structural features that distinguish it from the 30 other histidine kinases. Like YehU and YpdA, GlrK likely contains multiple transmembrane helices in its input domain and potentially regulatory domains such as GAF, which can bind various ligands including cyclic nucleotides or specific ions . The precise arrangement of these domains and their structural differences from other histidine kinases determine GlrK's specificity for both its sensory inputs and its phosphotransfer partner. Based on studies of other E. coli histidine kinases, these structural differences contribute to the remarkable kinetic preference (estimated at 103-fold in terms of relative kcat/Km ratios) for phosphotransfer to its cognate response regulator over non-cognate targets .

What environmental signals activate the GlrK histidine kinase?

GlrK responds to specific environmental signals, likely similar to how other two-component systems in E. coli function. For instance, the YehU/YehT system responds to peptides/amino acids, while the YpdA/YpdB system is activated by extracellular pyruvate . GlrK may be particularly responsive to signals that occur during the transition to stationary phase, as observed with other nutrient-sensing histidine kinases. This activation typically occurs when certain carbon sources become limiting for growth, allowing bacteria to prioritize utilization of remaining nutrients. The activation of GlrK likely follows a pattern similar to other histidine kinases, with a single pulse of signaling sufficient to induce expression of its target genes .

How does phosphotransfer specificity between GlrK and its response regulator compare to other two-component systems?

The phosphotransfer specificity between GlrK and its cognate response regulator follows principles observed in other E. coli two-component systems. Studies of histidine kinases such as EnvZ, CheA, and CpxA demonstrate a strong kinetic preference for their cognate response regulators in vitro. For example, EnvZ shows phosphotransfer to 11 different response regulators after a 1-hour reaction time, but with a shorter 10-second reaction, phosphotransfer occurs only to its cognate partner OmpR . This kinetic preference is estimated to be at least 103-fold in terms of relative kcat/Km ratios.

For GlrK, we would expect a similar high specificity for its cognate response regulator, with promiscuous phosphotransfer only occurring after extended incubation times in vitro. This specificity is likely achieved through molecular recognition between complementary surfaces on the histidine kinase and response regulator, evolved to minimize cross-talk between different signaling pathways . The strong kinetic preference ensures accurate signal transduction in the cellular environment where multiple two-component systems operate simultaneously.

What are the interactions between GlrK and other components of bacterial signaling networks?

GlrK likely engages in complex interactions with other components of bacterial signaling networks, similar to the interconnected nature of YehU/YehT and YpdA/YpdB systems. In these systems, heteromeric interactions between histidine kinases and transporters have been detected using bacterial adenylate cyclase two-hybrid systems . For example, the histidine kinase YehU interacts strongly with both transporters YjiY and YhjX, while YpdA similarly interacts with both transporters .

Based on these findings, GlrK may also form protein-protein interactions with membrane transporters or other histidine kinases, potentially creating larger signaling complexes that integrate multiple inputs. These interactions might allow for cross-talk between different signaling pathways, enabling more sophisticated responses to environmental changes. Additionally, GlrK signaling is likely influenced by global regulators such as the carbon storage regulator A (CsrA), which has been shown to be involved in posttranscriptional regulation of target genes in other two-component systems .

How does the expression of GlrK and its target genes change under different growth conditions?

The expression of GlrK and its target genes likely follows patterns similar to other two-component systems in E. coli, with growth phase-dependent regulation being particularly important. For the YehU/YehT and YpdA/YpdB systems, expression of target genes occurs at the onset of stationary phase when grown in LB medium . This timing suggests that GlrK signaling may be particularly important during transitions in nutrient availability.

The regulation of GlrK target genes is likely multilayered, involving both transcriptional and posttranscriptional mechanisms. Based on studies of similar systems, we can infer that CsrA might play a role in posttranscriptional regulation of GlrK target genes. CsrA itself is regulated via sequestration by Hfq-dependent small RNAs such as CsrB and CsrC . Additionally, degradation of target gene transcripts may be controlled by the interplay between ribosomal protein L4 and RNase E, as seen with the YjiY and YhjX transcripts . These multiple regulatory checkpoints ensure precise control over the production of proteins encoded by GlrK target genes.

What are the optimal protocols for cloning and expressing recombinant GlrK in E. coli?

For cloning and expressing recombinant GlrK in E. coli, researchers should consider the following optimized protocol:

• Gene Amplification: Amplify the glrK gene from E. coli genomic DNA using high-fidelity polymerase with primers containing appropriate restriction sites.

• Vector Selection: For efficient expression, use a vector with an inducible promoter system such as pET or pBAD series, which allow tight control of expression.

• Protein Solubility Considerations: Since GlrK is a membrane-associated protein, consider expressing only its soluble cytoplasmic domain (containing the catalytic core) as a fusion protein with solubility enhancers like thioredoxin-His6 or His6-MBP tags .

• Expression Optimization: Transform into an appropriate E. coli strain (BL21(DE3) or derivatives) and test multiple induction conditions varying temperature (16-37°C), inducer concentration, and duration to optimize soluble protein yield.

• Membrane Protein Considerations: If expressing full-length GlrK including transmembrane domains, use specialized E. coli strains designed for membrane protein expression (C41(DE3) or C43(DE3)) and include mild detergents during purification.

For purification, use metal affinity chromatography followed by size exclusion chromatography to obtain pure protein. When necessary, make successive N-terminal truncations while preserving the H-box and ATP binding domain to identify constructs that produce active kinase in vitro .

How can recombination systems be utilized to engineer GlrK mutations in the E. coli chromosome?

Engineering GlrK mutations directly in the E. coli chromosome can be efficiently accomplished using λ Red recombination system, which eliminates the need for standard cloning:

• Strain Selection: Use an E. coli strain containing a λ prophage harboring the recombination genes exo, bet, and gam under control of a temperature-sensitive λ cI-repressor .

• Designing Linear DNA for Recombination:

  • For point mutations: Synthesize complementary oligonucleotides (≥70 bp) homologous to glrK but containing your desired mutation .

  • For gene replacements: Create a PCR product containing a selectable marker (e.g., cat for chloramphenicol resistance) flanked by 50-bp homology arms targeting glrK .

• Recombination Protocol:

  • Induce λ recombination functions by shifting cultures to 42°C for 15 minutes.

  • Prepare electrocompetent cells from the induced culture.

  • Electroporate linear DNA (100 ng) into the cells.

  • Recover at 32°C and plate on selective media .

• Verification: Confirm successful recombination by PCR and sequencing. For gene replacements, verify phenotypic changes associated with the glrK modification.

This method has several advantages for GlrK engineering: it works in both recA+ and recA- backgrounds, requires short homology regions (50-70 bp), and allows precise genetic modifications without leaving scars in the chromosome .

What assays can be used to measure GlrK autophosphorylation and phosphotransfer activity?

Several robust assays can be employed to measure GlrK autophosphorylation and phosphotransfer activity:

• Radioactive Phosphotransfer Assay:

  • Incubate purified GlrK with [γ-32P]ATP to allow autophosphorylation.

  • To measure phosphotransfer, add purified cognate response regulator.

  • Terminate reactions at different time points by adding SDS-PAGE sample buffer.

  • Separate proteins by SDS-PAGE and visualize phosphorylated proteins by autoradiography .

  • Compare phosphotransfer kinetics using different time points (10 seconds vs. 1 hour) to assess specificity .

• Phos-tag SDS-PAGE Assay:

  • Phos-tag acrylamide specifically retards the migration of phosphorylated proteins.

  • Perform autophosphorylation and phosphotransfer reactions with non-radioactive ATP.

  • Separate proteins on Phos-tag SDS-PAGE gels and visualize by Coomassie staining.

  • Identify phosphorylated species by their reduced mobility.

• FRET-Based Assays:

  • Engineer fluorescently labeled GlrK and response regulator pairs.

  • Monitor phosphotransfer in real-time through changes in FRET signal.

  • This approach allows continuous measurement of reaction kinetics.

For comparative analysis, test phosphotransfer to all E. coli response regulators to determine the specificity profile of GlrK, similar to how EnvZ, CheA, and CpxA have been characterized .

How should researchers interpret discrepancies in GlrK phosphotransfer data between in vitro and in vivo experiments?

When interpreting discrepancies between in vitro and in vivo phosphotransfer data for GlrK, researchers should consider several factors:

• Kinetic vs. Thermodynamic Control:

  • In vitro experiments often use extended incubation times that reveal thermodynamic compatibility rather than kinetic preference.

  • Short-time point assays (10 seconds) better reflect the kinetic preference observed in vivo .

  • Compare phosphotransfer at multiple time points (10 seconds, 1 minute, 10 minutes, 1 hour) to distinguish between kinetic and thermodynamic control.

• Cellular Context Factors:

  • In vivo, spatial organization and protein concentration ratios differ from in vitro conditions.

  • Additional proteins may form complexes with GlrK, as seen with YehU and YpdA interacting with transporters .

  • The presence of phosphatases can rapidly reverse phosphotransfer events in vivo.

• Analysis Framework:

  Parameter	In vitro	In vivo	Interpretation
  Phosphotransfer specificity	May show promiscuity with long incubation	Highly specific	Focus on short time point data
  Cross-talk	Often detected	Minimal under normal conditions	Physiological relevance questionable
  Effect of mutations	Direct effects on kinetics	May show compensatory mechanisms	Consider system-wide responses

• Reconciliation Strategies:

  • Validate in vitro findings with in vivo approaches such as phospho-specific antibodies or Phos-tag Western blotting.

  • Use genetic approaches to verify signaling relationships, including epistasis analysis with deletion strains.

  • Consider mathematical modeling to integrate both datasets and predict system behavior.

The approximately 103-fold kinetic preference for cognate response regulators observed in other histidine kinases suggests that in vivo, GlrK likely communicates primarily with its cognate partner despite potential in vitro cross-reactivity .

What bioinformatic approaches are useful for analyzing the evolutionary relationships between GlrK and other histidine kinases?

Several bioinformatic approaches provide valuable insights into the evolutionary relationships between GlrK and other histidine kinases:

• Sequence-Based Phylogenetic Analysis:

  • Multiple sequence alignment of histidine kinase domains using MUSCLE or MAFFT.

  • Construction of phylogenetic trees using maximum likelihood (RAxML, IQ-TREE) or Bayesian (MrBayes) methods.

  • Analysis of domain architecture using SMART or Pfam to identify conserved and variable regions.

  • Separation of catalytic domains from sensory domains for independent evolutionary analysis.

• Genomic Context Analysis:

  • Examination of gene neighborhood conservation across bacterial species.

  • Identification of co-evolved gene pairs (histidine kinase/response regulator).

  • Analysis of horizontal gene transfer events using methods like compositional bias detection.

• Structural Bioinformatics:

  • Homology modeling of GlrK based on crystal structures of related histidine kinases.

  • Structural alignment to identify conserved catalytic residues and variable sensory regions.

  • Prediction of protein-protein interaction interfaces between GlrK and its response regulator.

• Coevolution Analysis:

  • Direct coupling analysis (DCA) to identify co-evolving residue pairs between GlrK and its response regulator.

  • Statistical coupling analysis (SCA) to identify networks of co-evolving amino acids.

  • These methods help identify specificity-determining residues that maintain signaling fidelity.

These approaches reveal that GlrK likely belongs to the LytS/LytTR family like YehU and YpdA, with high conservation among gammaproteobacteria . The analysis of domain architecture would show whether GlrK contains GAF domains or other sensory modules similar to related histidine kinases .

How can researchers differentiate between direct and indirect effects of GlrK mutations on bacterial phenotypes?

Differentiating between direct and indirect effects of GlrK mutations requires a systematic experimental approach:

• Genetic Complementation Analysis:

  • Create a clean deletion of glrK using λ Red recombination system .

  • Complement with wild-type or mutant glrK expressed from a plasmid.

  • Direct effects should be rescued by wild-type complementation but not by inactive mutants.

  • Use inducible promoters with varying strengths to test dose-dependency.

• Phosphotransfer Profiling:

  • Perform in vitro phosphotransfer assays with purified wild-type and mutant GlrK proteins.

  • Compare phosphotransfer to cognate and non-cognate response regulators using short (10 seconds) and long (1 hour) time points .

  • Direct effects should show altered phosphotransfer kinetics to the cognate response regulator.

• Transcriptomic Analysis:

  • Compare RNA-seq profiles of wild-type, ΔglrK, and point mutant strains.

  • Primary GlrK targets should show similar expression changes in deletion and inactive point mutants.

  • Secondary effects may differ between deletion and point mutants.

  • Time-course analysis following GlrK activation can separate immediate from delayed responses.

• Protein-Protein Interaction Mapping:

  • Use bacterial two-hybrid assays to identify proteins interacting with GlrK, similar to those done for YehU and YpdA .

  • Compare interaction profiles of wild-type and mutant GlrK.

  • Direct effects involve proteins physically interacting with GlrK.

• Separation of Functions:

  GlrK Function	Experimental Approach	Expected Outcome for Direct Effects
  Autophosphorylation	In vitro kinase assay	Immediate impact on phosphorylation state
  Phosphotransfer	Phosphotransfer assay	Altered phosphorylation of cognate regulator
  Protein interactions	Bacterial two-hybrid	Changed interaction with known partners
  Gene regulation	Reporter fusions	Altered expression of direct target genes

By integrating these approaches, researchers can build a comprehensive understanding of the direct signaling pathway controlled by GlrK and distinguish it from downstream indirect effects.

What are the most common pitfalls in purifying active recombinant GlrK, and how can they be avoided?

Purifying active recombinant GlrK presents several challenges that researchers commonly encounter:

• Protein Solubility Issues:

  • Problem: GlrK, being a membrane-associated protein, often forms inclusion bodies.

  • Solution: Express only the cytoplasmic domain as done with other histidine kinases . Make successive N-terminal truncations while preserving the H-box and ATP binding domain. Use solubility-enhancing fusion tags like thioredoxin-His6 or His6-MBP .

• Loss of Activity During Purification:

  • Problem: Histidine kinases often lose activity during purification due to improper folding or loss of essential cofactors.

  • Solution: Include ATP or non-hydrolyzable ATP analogs in purification buffers to stabilize the active conformation. Purify at 4°C and include glycerol (10-20%) to maintain protein stability.

• Aggregation During Storage:

  • Problem: Purified GlrK may aggregate during storage, leading to activity loss.

  • Solution: Store in small aliquots at -80°C with cryoprotectants such as glycerol. Avoid repeated freeze-thaw cycles. Test different buffer conditions (pH, salt concentration) to optimize stability.

• Inconsistent Activity Measurements:

  • Problem: Variable activity in different preparations leads to irreproducible results.

  • Solution: Standardize protein concentration measurements and develop a specific activity assay. Include positive controls (other well-characterized histidine kinases) in parallel experiments .

• Contaminating Phosphatase Activity:

  • Problem: Bacterial phosphatases co-purifying with GlrK can interfere with phosphorylation assays.

  • Solution: Include phosphatase inhibitors in purification buffers. Perform additional purification steps like ion exchange chromatography to remove contaminants.

By addressing these common pitfalls, researchers can significantly improve the quality and consistency of their recombinant GlrK preparations, leading to more reliable experimental results.

How can researchers troubleshoot unsuccessful gene modifications of glrK in the E. coli chromosome?

When troubleshooting unsuccessful gene modifications of glrK in the E. coli chromosome using recombination-based methods, consider these systematic approaches:

• Recombination Efficiency Issues:

  • Verification: Confirm proper induction of recombination functions by testing the system with a control modification known to work efficiently .

  • Solution: Ensure proper temperature shift (42°C for 15 minutes) to induce the λ Red functions. Optimize electroporation conditions for maximum DNA uptake. Increase homology arm length beyond the minimal 50 bp .

• DNA Design Problems:

  • Verification: Sequence your PCR products to confirm correct homology arms and mutation.

  • Solution: Design homology arms to avoid repetitive or highly structured regions. For point mutations, place the mutation in the center of the oligonucleotide. Use high-quality oligonucleotides and high-fidelity polymerase for PCR .

• Selection Issues:

  • Verification: Test the selection marker on control strains to confirm it works properly.

  • Solution: For non-selectable modifications (like point mutations), include a linked selectable marker that can later be removed. Consider using CRISPR-Cas9 to counter-select against non-modified cells.

• Essential Gene Complications:

  • Verification: Determine if glrK is essential under your experimental conditions.

  • Solution: Use conditional approaches, such as introducing the modification alongside an inducible copy of the wild-type gene, then removing the wild-type copy after successful recombination.

• Troubleshooting Workflow:

  Problem	Diagnostic Test	Solution
  No recombinants	Test with control target	Optimize induction and electroporation
  False positives	PCR verification of locus	Redesign primers away from homology arms
  Growth defects	Compare growth curves	Consider polar effects on nearby genes
  Mixed populations	Single colony isolation	Increase selection stringency

By systematically addressing these potential issues, researchers can significantly improve their success rate in engineering precise modifications to the glrK gene in the E. coli chromosome .

What emerging technologies will advance our understanding of GlrK function in bacterial signaling networks?

Several emerging technologies are poised to revolutionize our understanding of GlrK function in bacterial signaling networks:

• CRISPR-Based Tools:

  • CRISPR interference (CRISPRi) for tunable repression of glrK expression.

  • CRISPR activation (CRISPRa) for controlled overexpression.

  • Base editors for precise genome editing without double-strand breaks.

  • These approaches allow dynamic control of GlrK levels and rapid generation of mutant libraries.

• Single-Cell Analysis Technologies:

  • Time-lapse fluorescence microscopy with GlrK-fluorescent protein fusions to track localization and dynamics.

  • Single-cell RNA-seq to reveal cell-to-cell variability in GlrK-dependent responses.

  • Mass cytometry to simultaneously measure multiple parameters in individual cells.

  • These methods will reveal heterogeneity in GlrK signaling across bacterial populations.

• Structural Biology Advances:

  • Cryo-electron microscopy for structure determination of full-length GlrK, potentially in complex with its response regulator.

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon activation.

  • Cross-linking mass spectrometry to identify interaction interfaces in signaling complexes.

  • These approaches will elucidate the molecular mechanisms of GlrK activation and signaling.

• Synthetic Biology Approaches:

  • Engineered histidine kinase scaffolds with modular sensing domains.

  • Optogenetic control of GlrK activity using light-sensitive domains.

  • Synthetic GlrK circuits to probe network architecture and dynamics.

  • These tools will allow precise manipulation of signaling pathways for both basic research and applications.

These technologies will help address fundamental questions about how GlrK integrates into larger signaling networks, similar to the nutrient-sensing regulatory network discovered for YehU/YehT and YpdA/YpdB systems .

How might understanding GlrK signaling contribute to developing novel antibacterial strategies?

Understanding GlrK signaling could contribute to novel antibacterial strategies through multiple pathways:

• Targeted Inhibition of Two-Component Systems:

  • Two-component systems are absent in mammals, making them attractive antibacterial targets.

  • Structure-based design of small molecules that specifically inhibit GlrK autophosphorylation or phosphotransfer.

  • Development of peptide inhibitors that disrupt GlrK interactions with its response regulator.

  • These approaches could disable bacterial adaptation responses while minimizing effects on host cells.

• Virulence Modulation:

  • If GlrK regulates virulence factors, as many LytS/LytTR family members do in bacterial pathogens , targeting it could attenuate pathogenicity without directly killing bacteria.

  • This anti-virulence approach may impose less selective pressure for resistance compared to conventional antibiotics.

  • Compounds that lock GlrK in an inactive conformation could prevent appropriate bacterial responses during infection.

• Sensitization to Existing Antibiotics:

  • Inhibition of stress-responsive signaling pathways may prevent adaptation to antibiotic stress.

  • Combination therapies targeting both GlrK signaling and conventional targets could enhance efficacy.

  • This approach could potentially restore sensitivity in resistant strains by blocking adaptive responses.

• Metabolic Vulnerability Exploitation:

  • Given that GlrK likely functions in nutrient sensing similar to other histidine kinases , its inhibition could create metabolic vulnerabilities.

  • Forcing bacteria into disadvantageous metabolic states through manipulation of GlrK signaling.

  • Targeting bacteria during specific growth phases when they rely most heavily on GlrK signaling.

• Research Applications:

  Application	Mechanism	Potential Impact
  Biofilm disruption	Interference with cell-cell communication	Increased antibiotic susceptibility
  Host colonization prevention	Blocking adaptation to host environment	Reduced infection establishment
  Metabolic reprogramming	Altering nutrient utilization priorities	Growth inhibition in specific niches

These strategies represent promising directions for turning fundamental research on GlrK signaling into practical antibacterial approaches, particularly against pathogens with antibiotic resistance.

What are the most significant recent advances in understanding histidine kinase signaling networks in E. coli?

Recent significant advances in understanding histidine kinase signaling networks in E. coli have transformed our view of bacterial signal transduction:

• Network-Level Integration: The discovery that two-component systems like YehU/YehT and YpdA/YpdB form interconnected networks rather than operating as isolated pathways represents a paradigm shift . This interconnectedness involves cross-talk at multiple levels, including transcriptional regulation, protein-protein interactions, and shared downstream targets .

• Kinetic Specificity Mechanisms: The elucidation of kinetic preference mechanisms (estimated at 103-fold in terms of relative kcat/Km ratios) that maintain signaling fidelity between histidine kinases and their cognate response regulators has resolved the paradox of how cells maintain pathway insulation despite the structural similarity of signaling components .

• Temporal Coordination: The observation that nutrient-sensing histidine kinase systems are activated at specific growth phases (e.g., transition to stationary phase) highlights the temporal coordination of signaling events . This timing ensures appropriate adaptation to changing environmental conditions.

• Post-Transcriptional Regulation: The identification of regulators like CsrA that control the expression of histidine kinase target genes at the post-transcriptional level adds another layer of complexity to signaling networks . This regulation allows for rapid and fine-tuned responses to environmental changes.

• Physical Interaction Networks: The discovery of heteromeric interactions between histidine kinases and transporters using bacterial adenylate cyclase two-hybrid systems reveals the physical basis for signal integration . These interactions suggest the formation of larger signaling complexes that coordinate multiple inputs and outputs.

These advances collectively point toward a more integrated view of bacterial signaling, with GlrK likely participating in similar network-level interactions that coordinate nutrient sensing and metabolic adaptation in E. coli.