Recombinant Shewanella violacea Guanylate kinase (gmk)

What is Shewanella violacea and why is its guanylate kinase of interest to researchers?

Shewanella violacea is a gram-negative, rod-shaped bacterium isolated from marine sediment in the Ryukyu Trench at a depth of 5,110m. It appears violet when grown on Marine Agar 2216 Plates and is a motile organism with flagella. As a facultative anaerobic extremophile, S. violacea has optimal growing conditions at 8°C and 30 MPa, making it valuable for studying adaptations to extreme environments .

The guanylate kinase from S. violacea is of particular interest because it functions under high pressure and low temperature conditions. This enzyme catalyzes the phosphorylation of GMP to GDP, playing an essential role in recycling GMP and indirectly affecting cGMP pathways . Studying this enzyme can provide insights into:

• Molecular adaptations to extreme environments

• Structural modifications that enable function under high pressure

• Evolutionary adaptations in nucleotide metabolism

• Comparative enzymatic mechanisms between extremophiles and mesophiles

How does S. violacea gmk differ from guanylate kinases in mesophilic bacteria?

Guanylate kinases from extremophiles like S. violacea differ from those in mesophilic bacteria in several ways that reflect adaptations to extreme environments:

• Oligomeric state and ionic sensitivity: While specific data for S. violacea gmk is not provided in the search results, related bacterial guanylate kinases like that of E. coli differ significantly from eukaryotic counterparts. E. coli guanylate kinase is multimeric, with its protomeric state dictated by ionic conditions - appearing as a tetramer under low ionic conditions and a dimer under high ionic conditions .

• Substrate binding characteristics: E. coli guanylate kinase binds GMP cooperatively, and this cooperativity changes with ionic strength, unlike eukaryotic guanylate kinases . S. violacea gmk likely has similar adaptations to function optimally in high-pressure, low-temperature environments.

• Structural adaptations: Deep-sea bacteria like S. violacea typically demonstrate structural modifications in their proteins, including increased flexibility in cold environments and pressure-resistant conformations.

• Co-factor requirements: While specific co-factor data for S. violacea gmk is not available in the search results, related Shewanella UshA enzymes involved in nucleotide metabolism require Mg²⁺ and Mn²⁺ as cofactors, suggesting similar metal ion dependencies for gmk .

What are the optimal conditions for recombinant expression of S. violacea gmk?

Based on related research with Shewanella proteins, the following methodological approach is recommended for recombinant expression of S. violacea gmk:

Expression System Selection:

• Heterologous expression in E. coli BL21(DE3) is generally effective for Shewanella proteins

• For proteins requiring cold adaptation, consider using cold-adapted expression hosts or cold-induction systems (16°C induction)

Vector Design Considerations:

• Include a 6×His tag for purification (N-terminal tags are typically less disruptive for kinases)

• Consider using pET-series vectors with T7 promoter systems for controlled expression

• Codon optimization may be necessary as S. violacea is a GC-rich organism

Expression Conditions:

• Initial induction at low temperature (16-20°C) to ensure proper folding

• Use low IPTG concentrations (0.1-0.5 mM) to prevent inclusion body formation

• Extend expression time (16-24 hours) at lower temperatures

Specialized Considerations for Extremophile Proteins:

• Addition of osmolytes (such as glycerol 5-10%) to stabilize protein structure

• Inclusion of appropriate divalent cations (Mg²⁺, Mn²⁺) in the growth medium

What purification strategy is most effective for obtaining high-quality recombinant S. violacea gmk?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant S. violacea gmk:

Initial Capture:

• Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol

  • Wash buffer: Same as lysis with 20-30 mM imidazole

  • Elution buffer: Same as lysis with 250-300 mM imidazole

Intermediate Purification:
2. Ion Exchange Chromatography

• Based on the theoretical pI of the protein (approximately 5.5-6.5 for most gmk proteins)

• Use Q-Sepharose (anion exchange) if the protein is negatively charged at pH 8.0

Polishing Step:
3. Size Exclusion Chromatography

• Buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 10% glycerol

• Critical for separating different oligomeric states and removing aggregates

Storage Recommendations:

• Add 20-50% glycerol for long-term storage at -80°C (aliquot to avoid freeze-thaw cycles)

• Typical shelf life is 6 months at -20°C/-80°C in liquid form and 12 months in lyophilized form

How can I verify the activity and integrity of purified recombinant S. violacea gmk?

Multiple complementary approaches should be used to verify both the structural integrity and enzymatic activity of purified recombinant S. violacea gmk:

Structural Integrity Assessment:

• SDS-PAGE analysis to confirm molecular weight (expected ~23 kDa)

• Western blot using anti-His antibodies for tagged protein

• Circular dichroism spectroscopy to evaluate secondary structure

• Dynamic light scattering to assess homogeneity and oligomeric state

Enzymatic Activity Assays:

• Coupled spectrophotometric assay:

  • Measure the production of GDP from GMP and ATP

  • Couple the reaction to pyruvate kinase and lactate dehydrogenase

  • Monitor NADH oxidation at 340 nm

• Direct HPLC assay:

  • Separate and quantify GMP, GDP, and GTP

  • Use a C18 reverse-phase column with appropriate mobile phase

• Radioactive assay:

  • Use [γ-³²P]ATP as substrate

  • Measure incorporation of ³²P into GDP

Functional Parameters to Measure:

• Km for GMP and ATP substrates

• kcat and catalytic efficiency (kcat/Km)

• Effects of temperature and pressure on activity

• Metal ion dependencies (likely Mg²⁺ or Mn²⁺)

How can I design experiments to study pressure adaptation of S. violacea gmk?

Investigating the pressure adaptation of S. violacea gmk requires specialized approaches and equipment:

Experimental Design for Pressure Studies:

Data Interpretation Framework:

• Evaluate volume changes during catalysis (ΔV‡)

• Analyze compressibility factors

• Assess changes in oligomeric state with pressure

Researchers should note that S. violacea has demonstrated adaptive responses to pressure in its respiratory system, with an inhibitor- and pressure-resistant terminal oxidase expressed in cells grown under high pressure (50 MPa) . Similar adaptations may be present in gmk.

What mutagenesis approaches can be used to study structure-function relationships in S. violacea gmk?

Several mutagenesis strategies can be employed to investigate structure-function relationships in S. violacea gmk:

Site-Directed Mutagenesis Approaches:

• Alanine scanning mutagenesis:

  • Systematically replace conserved residues with alanine

  • Target the P-loop motif (GXXGXGKS/T) essential for ATP binding

  • Analyze effects on catalytic activity and substrate binding

• Domain swapping:

  • Create chimeric proteins with domains from mesophilic Shewanella gmk

  • Identify regions responsible for pressure and cold adaptation

• Conservative vs. non-conservative substitutions:

  • Replace residues with similar amino acids (conservative)

  • Compare with effects of dramatically different residues (non-conservative)

Random Mutagenesis Methods:

• Error-prone PCR:

  • Generate libraries with random mutations throughout the gene

  • Screen for variants with altered pressure or temperature optima

• Directed evolution:

  • Apply selection pressure (temperature, pressure) to identify adaptive mutations

  • Use multiple rounds of mutagenesis and selection

Functional Characterization of Mutants:

How does the G547W mutation in sensor histidine kinase relate to adaptive mechanisms in Shewanella species?

While not directly related to gmk, understanding adaptive mutations in Shewanella provides valuable context for studying extremophile enzymes. The G547W mutation in sensor histidine kinase (pdsS) in Shewanella algae offers insights into bacterial adaptation mechanisms:

Key Findings on G547W Mutation:

The G547W mutation in the sensor histidine kinase (pdsS) of S. algae was associated with:

• Overexpression of an OmpA-like protein (pdsO) within a proteobacteria-specific sortase system

• β-lactam resistance through maintenance of membrane integrity

• Adaptive subpopulation dynamics in response to antibiotics

Mechanistic Insights:

• Transcriptome analysis revealed the G547W mutation led to overexpression of multiple genes involved in:

  • Membrane integrity maintenance

  • Biofilm formation

  • Immune evasion

  • β-lactamase activation

• The study observed a recurrent switch between wild-type and G547W alleles, revealing expansion and contraction of cell subpopulations in response to environmental pressures

Relevance to S. violacea gmk Research:

This adaptive mechanism demonstrates how Shewanella species can respond to environmental stressors through genetic adaptations. Similar mechanisms may be involved in pressure adaptation of S. violacea enzymes including gmk. Researchers studying gmk should consider:

• The possibility of naturally occurring variants with enhanced function under extreme conditions

• Potential regulatory mechanisms controlling gmk expression under different pressure conditions

• The role of gmk in broader adaptive responses to environmental stressors

What approaches can be used to study the role of S. violacea gmk in nucleotide metabolism under high pressure?

Investigating the role of gmk in nucleotide metabolism under high pressure requires integrating molecular, biochemical, and systems biology approaches:

Experimental Approaches:

• Metabolomics under pressure:

  • Cultivate S. violacea under different pressures (0.1, 30, 50 MPa)

  • Extract and quantify nucleotides (GMP, GDP, GTP) using LC-MS/MS

  • Compare nucleotide pools and ratios at different pressures

• Gene expression analysis:

  • qRT-PCR to measure gmk expression under different pressure conditions

  • RNA-seq to identify co-regulated genes in the nucleotide metabolism pathway

  • Promoter analysis to identify pressure-responsive elements

• Genetic approaches:

  • Create gmk knockout or knockdown strains if possible (consider using CRISPR-Cas9)

  • Develop conditional expression systems for gmk

  • Perform complementation studies with gmk variants

Integrative Analysis:

• Correlate gmk expression/activity with global nucleotide metabolism

• Investigate interaction partners of gmk using pull-down assays

• Develop mathematical models of nucleotide flux under different pressure conditions

Technical Considerations:

Working with S. violacea under high pressure requires specialized equipment:

• High-pressure cultivation vessels

• Pressure-resistant sampling systems

• Methods for rapid decompression without disrupting cellular metabolism

Studies on S. violacea respiratory systems have shown that cells grown under high pressure (50 MPa) express specialized proteins adapting to these conditions . Similar adaptations likely exist in nucleotide metabolism pathways.

How does S. violacea gmk compare structurally and functionally to gmk from other Shewanella species?

Comparative analysis of gmk across Shewanella species reveals insights into evolutionary adaptations to different environments:

Sequence and Structural Comparison:

While complete comparative data specific to gmk is not provided in the search results, we can infer information based on related Shewanella proteins:

• Sequence conservation:

  • Gmk is highly conserved across Shewanella species with approximately 80-95% sequence identity expected

  • The core catalytic domain and P-loop motif are likely invariant

  • Most variation would occur in surface-exposed regions

• Species-specific adaptations:

  • S. violacea (deep-sea, high pressure): Likely contains adaptations for pressure stability

  • S. denitrificans (strain OS217): Sequence is provided in search result

  • Shewanella sp. (strain MR-4): Sequence is provided in search result

Functional Comparison Based on Environment:

Different Shewanella species inhabit diverse environments, likely leading to functional adaptations:

Evolutionary Implications:

The observation that S. violacea belongs to Group 1 Shewanella (extremophiles) while many other species belong to Group 2 (mesophiles) suggests distinct evolutionary trajectories. Gmk likely shows adaptations reflecting these environmental specializations.

What can comparative genomics tell us about the evolution of guanylate kinase in extremophiles?

Comparative genomics provides valuable insights into how guanylate kinase has evolved in extremophiles like S. violacea:

Genomic Context and Organization:

• Gene neighborhood conservation:

  • In E. coli, gmk is found in an operon with rpoZ, spoT, and recG

  • Analysis of synteny across extremophiles can reveal conserved gene clusters

  • Changes in gene organization may indicate adaptive pressures

• Regulatory elements:

  • Promoter regions may contain extremophile-specific regulatory elements

  • Pressure and temperature-responsive elements could be identified through comparative analysis

Evolutionary Patterns in Extremophiles:

• Molecular signatures of adaptation:

  • Higher frequency of specific amino acids (e.g., glycine for flexibility in cold environments)

  • Modified surface charge distribution for function under extreme conditions

  • Altered oligomerization interfaces for stability

• Horizontal gene transfer vs. vertical evolution:

  • Examine whether gmk shows evidence of horizontal acquisition

  • Compare evolutionary rates in extremophile vs. mesophile lineages

Specific Adaptations in S. violacea:

The complete genome sequence analysis of S. violacea revealed adaptations for life in deep-sea environments . Though specific details about gmk are not provided in the search results, the bacterium's general adaptations include:

• Modified respiratory systems with inhibitor- and pressure-resistant terminal oxidases

• Higher percentage of polyunsaturated fatty acids in membranes compared to mesophilic Shewanella

• Likely modifications to enzyme systems for function under high pressure

How can S. violacea gmk be utilized in coupled enzyme assays for studying pressure effects on other enzymes?

S. violacea gmk can serve as a valuable auxiliary enzyme in coupled assay systems to study pressure effects on other enzymes:

Methodological Approach:

• Development of pressure-resistant coupled assay systems:

  • Use S. violacea gmk as an auxiliary enzyme in assays requiring GMP phosphorylation

  • Create a fully pressure-adapted enzyme cascade with other extremophile enzymes

  • Design spectrophotometric assays functional at high pressures (30-50 MPa)

• Application to study non-adapted enzymes:

  • Compare activity of mesophilic enzymes using either pressure-adapted or standard auxiliary enzymes

  • Isolate pressure effects on the target enzyme from effects on the detection system

Experimental Design Considerations:

• Ensure S. violacea gmk is not rate-limiting in the coupled assay

• Validate that gmk activity is stable at the experimental pressure range

• Include appropriate controls to account for pressure effects on substrates and products

Potential Applications:

• Nucleotide metabolism enzymes:

  • Nucleoside kinases

  • Nucleotide phosphatases

  • DNA/RNA polymerases

• Signal transduction components:

  • cGMP-dependent pathways

  • Protein kinases

  • Phosphodiesterases

Can S. violacea gmk be used to create auxotrophic strains for genetic manipulation studies?

Creating auxotrophic strains using gmk as a selective marker is a valuable approach for genetic manipulation of Shewanella species:

Strategy for Creating gmk Auxotrophs:

• Gene deletion approaches:

  • CRISPR-Cas9-based deletion of the native gmk gene

  • Traditional homologous recombination-based gene replacement

  • Transposon mutagenesis screening for gmk disruption

• Complementation system:

  • Plasmid-based expression of functional gmk

  • Integration of gmk under control of inducible promoters

  • Use of gmk orthologs from related species for complementation studies

Considerations for S. violacea:

The creation of auxotrophic mutants in extremophiles presents unique challenges:

• Growth media must be supplemented with appropriate metabolites

• Growth conditions must account for pressure and temperature requirements

• Transformation efficiencies may be lower than in model organisms

Examples from Related Research:

While specific gmk auxotrophy data for Shewanella is not provided in the search results, related examples include:

• Creation of a quadruple auxotrophic mutant (ura3, trp1, leu2, and his3) in Saccharomyces cerevisiae using CRISPR-Cas9, which achieved knockouts in up to 60% of colonies despite the need to disrupt multiple alleles simultaneously

• Development of NAD⁺-auxotrophic E. coli strains by disrupting essential genes in the NAD⁺ biosynthesis pathway, allowing precise control of intracellular NAD⁺ levels

• Generation of a uracil auxotroph of Saccharomyces boulardii using classical UV mutagenesis and selection with 5-FOA (5-Fluoroorotic acid)

These approaches could be adapted for creating gmk auxotrophs in S. violacea or related Shewanella species.

What are the key challenges in expressing active S. violacea gmk in heterologous systems?

Expressing active S. violacea gmk in heterologous systems presents several challenges due to its extremophilic origin:

Major Challenges and Solutions:

• Protein folding at non-native temperatures and pressures:

  • Challenge: S. violacea proteins evolved to fold at low temperatures (8°C) and high pressures (30 MPa)

  • Solutions:

    • Express at reduced temperatures (16-20°C)

    • Use chaperone co-expression systems (GroEL/GroES, DnaK/DnaJ/GrpE)

    • Add osmolytes to mimic pressure effects (glycerol, TMAO)

• Codon usage bias:

  • Challenge: Differences in codon preferences between S. violacea and expression hosts

  • Solutions:

    • Codon optimization for the expression host

    • Use of specialized strains with rare tRNA genes

    • Expression in related Shewanella species rather than E. coli

• Post-translational modifications:

  • Challenge: Potential differences in post-translational processing

  • Solutions:

    • Characterize native modifications in S. violacea gmk

    • Select expression hosts with similar modification machinery

    • Engineer constructs to minimize modification requirements

• Protein solubility and stability:

  • Challenge: Aggregation or misfolding in non-native conditions

  • Solutions:

    • Fusion with solubility-enhancing tags (MBP, SUMO)

    • Addition of stabilizing agents in purification buffers

    • Directed evolution for improved expression

Documented Approaches from Related Research:

Researchers working with deep-sea bacteria have developed specialized approaches:

• S. violacea has been cultured under various pressures (0.1, 30, 50, 65 MPa) to study pressure adaptation of its respiratory system

• High-pressure cultivation equipment can be used to maintain native conditions during expression

How can I design assays to measure S. violacea gmk activity under high pressure conditions?

Designing assays for measuring enzyme activity under high pressure requires specialized equipment and methodological adaptations:

High-Pressure Enzyme Assay Methodologies:

• Spectrophotometric assays under pressure:

  • Equipment: High-pressure optical cells with sapphire windows

  • Method:

    • Coupled enzyme assays using pressure-resistant auxiliary enzymes

    • Direct monitoring of substrate disappearance or product formation

    • Stopped-flow systems modified for high-pressure work

• Discontinuous assays with sampling:

  • Equipment: High-pressure reactors with sampling capability

  • Method:

    • Withdraw samples at defined time points

    • Rapidly decompress and analyze by HPLC or other methods

    • Calculate activity based on reaction progression

• NMR spectroscopy under pressure:

  • Equipment: High-pressure NMR tubes and probes

  • Method:

    • Real-time monitoring of reaction progress

    • Identification of potential intermediates

    • Analysis of reaction kinetics

Data Analysis Considerations:

• Account for pressure effects on substrate and product concentrations

• Consider pressure-induced pH shifts in buffer systems

• Apply appropriate models for extracting kinetic parameters under pressure

Adaptation Strategy Based on S. violacea Research:

Research on S. violacea terminal oxidases demonstrated significant differences in activity profiles under various pressures . Similar approaches could be applied to gmk:

• Prepare membrane fractions or purified enzyme from cells grown under different pressures

• Measure activity across a pressure range (0.1-65 MPa)

• Compare relative activities to identify pressure optima and ranges

What are the best methods for studying substrate specificity of S. violacea gmk compared to mesophilic homologs?

A comprehensive approach to studying substrate specificity of S. violacea gmk should include:

Methodological Approaches:

• Substrate screening:

  • Traditional nucleotide substrates: GMP, dGMP, IMP, XMP

  • Nucleotide analogs: Modified bases, sugar modifications, fluorescent analogs

  • High-throughput screening: Microplate-based activity assays with substrate libraries

• Kinetic parameter determination:

  • Measure Km, Vmax, kcat for each viable substrate

  • Determine inhibition constants for competitive inhibitors

  • Calculate specificity constants (kcat/Km) to quantify preference

• Structural analysis of enzyme-substrate complexes:

  • X-ray crystallography with substrate or substrate analogs

  • Molecular docking studies

  • Molecular dynamics simulations under varying pressure conditions

Comparative Experimental Design:

Integration with Evolutionary Analysis:

• Compare substrate preferences across evolutionary diverse gmk enzymes

• Correlate specificity changes with habitat adaptation

• Identify key residues controlling specificity through sequence analysis

This approach would build on understanding of other Shewanella enzymes, such as UshA from S. violacea, which has been shown to utilize AMP, ATP, and GTP as substrates with Mg²⁺ and Mn²⁺ as cofactors .

What are promising research directions for studying the role of S. violacea gmk in high-pressure adaptation?

Several promising research directions could advance our understanding of S. violacea gmk's role in high-pressure adaptation:

Fundamental Mechanistic Studies:

• Pressure-induced conformational changes:

  • High-pressure X-ray crystallography or NMR studies

  • Molecular dynamics simulations under varying pressure

  • FRET-based studies of protein dynamics under pressure

• Volume change analysis:

  • Determine activation volumes (ΔV‡) for the gmk reaction

  • Compare with mesophilic homologs

  • Identify structural elements contributing to pressure adaptation

• Hydration shell dynamics:

  • Examine water-protein interactions under pressure

  • Study the role of hydration in pressure adaptation

  • Use neutron scattering to probe hydration changes

Systems-Level Investigations:

• Metabolic network analysis under pressure:

  • Map nucleotide metabolism fluxes at varying pressures

  • Identify rate-limiting steps under pressure

  • Develop computational models of pressure effects on metabolism

• Transcriptional and translational regulation:

  • Investigate pressure-responsive elements controlling gmk expression

  • Study post-transcriptional regulation mechanisms

  • Examine protein-protein interactions affected by pressure

Applied Research Directions:

• Engineering pressure-adapted enzymes:

  • Use insights from S. violacea gmk to create pressure-resistant biocatalysts

  • Apply directed evolution to enhance pressure resistance

  • Develop chimeric proteins with pressure-adapted domains

• Biotechnological applications:

  • Develop high-pressure enzymatic processes using gmk and related enzymes

  • Create biosensors for pressure detection

  • Apply insights to enhance biocatalysis under non-conventional conditions

How can systems biology approaches enhance our understanding of S. violacea gmk in cellular context?

Systems biology approaches offer powerful tools for understanding S. violacea gmk in its broader cellular context:

Multi-Omics Integration Strategies:

Computational Modeling Approaches:

• Constraint-based metabolic modeling:

  • Develop genome-scale metabolic models for S. violacea

  • Perform flux balance analysis under different conditions

  • Predict metabolic adaptations to pressure changes

• Kinetic modeling of nucleotide metabolism:

  • Incorporate pressure effects on enzyme kinetics

  • Simulate nucleotide fluctuations under varying conditions

  • Identify control points in the network

Integration with Experimental Data:

This systems approach would build on existing knowledge about S. violacea adaptations, such as:

• Expression of inhibitor- and pressure-resistant terminal oxidases under high pressure

• Modifications to respiratory pathways with parallel electron transport chains

• Membrane adaptations with increased polyunsaturated fatty acids

Understanding gmk within this context would provide a more comprehensive view of nucleotide metabolism adaptation in deep-sea bacteria.

What evolutionary insights can be gained from studying S. violacea gmk mutations and their effects on enzyme function?

Evolutionary studies of S. violacea gmk can provide significant insights into adaptation mechanisms:

Evolutionary Analysis Approaches:

• Ancestral sequence reconstruction:

  • Infer ancestral gmk sequences across the Shewanella phylogeny

  • Resurrect ancestral enzymes through recombinant expression

  • Compare ancestral vs. modern enzyme properties

• Positive selection analysis:

  • Identify sites under positive selection using dN/dS ratios

  • Map selected sites onto protein structure

  • Correlate with functional/structural features

• Experimental evolution:

  • Culture S. violacea under altered pressure regimes

  • Monitor gmk sequence changes over generations

  • Characterize evolved variants

Specific Research Questions:

• Convergent evolution across extremophiles:

  • Compare gmk adaptations in distantly related piezophiles

  • Identify common strategies for pressure adaptation

  • Distinguish between convergent and divergent solutions

• Trade-offs in enzyme adaptation:

  • Characterize activity-stability trade-offs in gmk variants

  • Examine specialization vs. generalization in substrate use

  • Determine costs of pressure adaptation on other enzyme properties

• Epistatic interactions:

  • Study how gmk mutations interact with other genetic changes

  • Identify compensatory mutations in the nucleotide metabolism network

  • Map the adaptive landscape for gmk evolution

Parallel with Known Adaptive Mechanisms:

The study of adaptive mutations in Shewanella algae providing β-lactam resistance offers a model for understanding adaptive evolution:

• The G547W mutation in sensor histidine kinase (pdsS) led to overexpression of an OmpA-like protein and subsequent resistance

• The mutation showed dynamic behavior, with recurrent switching between wild-type and mutant alleles

• Subpopulation dynamics played a crucial role in adaptation