Recombinant Prochlorococcus marinus subsp. pastoris GTPase Der (der)

What expression systems are available for recombinant production of P. marinus GTPase Der?

Multiple expression systems have been developed for the recombinant production of P. marinus GTPase Der, each offering distinct advantages for different research applications:

All recombinant forms typically achieve >85% purity by SDS-PAGE analysis . The biotinylated version utilizes the AviTag technology, in which E. coli biotin ligase (BirA) catalyzes the formation of an amide linkage between biotin and a specific lysine residue within the 15-amino acid AviTag peptide, providing a controlled and site-specific biotinylation .

How does Prochlorococcus marinus subsp. pastoris relate to other marine cyanobacteria?

Prochlorococcus marinus subsp. pastoris (strain CCMP1986/MED4) belongs to the high-light adapted (HL) I clade of Prochlorococcus, which has evolved specific adaptations for surface ocean environments. Phylogenetically:

• P. marinus is closely related to, but distinct from, marine Synechococcus

• Within the Prochlorococcus genus, MED4 belongs to the most recently evolved clade

• It has one of the smallest genomes among all free-living phototrophs (~1.65 Mbp)

• It possesses an extremely low G+C content of 35.7% (when considering concatenated sequences for three regions: pcb, rpoC1, and the psbB-petB/D intergenic region)

This evolutionary positioning is significant, as there appears to be a correlation between G+C content and phylogenetic position among Prochlorococcus strains. The MED4 strain belongs to a high-light adapted ecotype with a significantly streamlined genome compared to low-light adapted Prochlorococcus strains, which retain larger genomes with higher G+C content .

This evolutionary history provides important context for understanding the conservation and function of specific proteins like Der in these highly streamlined genomes .

What methodologies are recommended for functional characterization of recombinant GTPase Der from P. marinus?

For comprehensive functional characterization of recombinant GTPase Der from P. marinus, researchers should implement a multi-faceted approach:

• GTPase Activity Assays:

  • Malachite green phosphate assay: Quantify released inorganic phosphate during GTP hydrolysis

  • HPLC analysis: Monitor conversion of GTP to GDP

  • Real-time fluorescence-based assays using mant-GTP

• Protein-Protein Interaction Studies:

  • Pull-down assays using the Avi-tag biotinylated version (CSB-EP763367EYQ-B)

  • Surface plasmon resonance (SPR) to determine binding kinetics with potential partners

  • Bacterial two-hybrid assays to identify novel interaction partners

• Structural Characterization:

  • X-ray crystallography with and without bound nucleotides

  • Hydrogen-deuterium exchange mass spectrometry to examine conformational changes

  • Cryo-EM for visualization of Der in complex with ribosomes or other partners

• In vitro Ribosome Assembly Assays:

  • Sucrose gradient centrifugation to analyze ribosome profiles

  • Ribosome reconstitution assays to assess Der's role in assembly

  • RNA binding assays using filter binding or electrophoretic mobility shift assays

When conducting these experiments, it's important to note that Der proteins typically exhibit low intrinsic GTPase activity that may be stimulated by specific factors or conditions. Buffer optimization is critical, with standard conditions including 50 mM Tris-HCl (pH 7.5), 50-100 mM KCl, 5 mM MgCl₂, and 1 mM DTT. Temperature optimization is particularly important for P. marinus proteins, which may exhibit different activity profiles compared to mesophilic bacterial counterparts .

How can researchers address the challenges in expressing and purifying functional GTPase Der from P. marinus?

Expression and purification of functional GTPase Der from P. marinus presents several challenges due to its origin from a marine cyanobacterium with unique adaptations. Based on experimental observations, the following methodological approach is recommended:

• Codon Optimization:
  P. marinus has unusual codon usage patterns due to its low G+C content (30.8% for MED4 strain) . Custom codon optimization for the expression host is crucial:

  • For E. coli expression, adjust rare codons in the sequence without altering the amino acid sequence

  • If expressing in yeast systems, consider the distinctly different codon preferences

• Expression Conditions Optimization:

  • Temperature: Lower temperatures (16-20°C) often yield higher amounts of soluble protein

  • Induction: Use low IPTG concentrations (0.1-0.3 mM) for E. coli systems

  • Medium supplements: Include 5-10% glycerol in the growth media to improve protein folding

  • Consider co-expression with chaperones (GroEL/ES) to enhance proper folding

• Purification Strategy:

  • Two-step purification typically yields >85% purity :
    a) Initial capture: Affinity chromatography (His-tag, GST-tag, or Avi-tag systems)
    b) Polishing step: Size exclusion chromatography or ion exchange chromatography

  • Buffer optimization is critical: 50 mM Tris-HCl (pH 7.5), 150-300 mM NaCl, 5 mM MgCl₂, 10% glycerol

  • Include 1-5 mM GTP or non-hydrolyzable GTP analogs to stabilize the protein during purification

• Storage Considerations:

  • Add 5-50% glycerol to the final preparation

  • Store in small aliquots at -80°C to avoid freeze-thaw cycles

  • The shelf life in liquid form is typically 6 months at -20°C/-80°C, while lyophilized preparations can maintain stability for up to 12 months

• Activity Verification:

  • Always verify GTPase activity immediately after purification using malachite green phosphate assays

  • Assess nucleotide binding using fluorescence spectroscopy with mant-GTP

What genetic manipulation strategies can be employed to study Der function in vivo in Prochlorococcus?

• Complementation Studies:

  • Express P. marinus Der in E. coli Der-depleted strains

  • Assess whether P. marinus Der can complement the essential function

  • Compare growth rates, ribosome profiles, and stress responses

• Point Mutations and Domain Swapping:

  • Create strategic mutations in conserved motifs of the GTPase domains

  • Swap domains between P. marinus Der and homologs from other bacteria

  • Analyze functional consequences through complementation assays

• In situ Gene Tagging in Environmental Samples:
  For direct studies in P. marinus itself, emerging techniques show promise:

  • Single-cell genomics combined with fluorescence-activated cell sorting

  • CRISPR-based techniques for targeted mutations in Prochlorococcus

  • Development of genetic tools specific for high-light adapted strains

These approaches must consider the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, particularly sections III-D that govern experiments requiring Institutional Biosafety Committee approval prior to initiation .

How can recombinant P. marinus GTPase Der contribute to understanding marine microbial adaptation?

Recombinant P. marinus GTPase Der serves as a valuable model system for investigating fundamental aspects of marine microbial adaptation, particularly in relation to genome streamlining and cellular efficiency in nutrient-limited environments:

Real-time quantitative PCR (qPCR) analysis of der gene expression under different environmental conditions can be performed following established protocols used for other P. marinus genes, such as:

• RNA extraction using established methods for marine cyanobacteria

• Reverse transcription with SuperScriptII reverse transcriptase

• qPCR using the DNA Engine/Chromo4 Real-Time PCR-Detector

• Expression normalization against the aperiodic gene rnpB using the 2−ΔΔCT method

What structural features distinguish Der from other GTPases, and how can they be analyzed experimentally?

Der (EngA) GTPases possess distinctive structural features that differentiate them from other bacterial GTPases, presenting unique opportunities for experimental characterization:

These experimental approaches can reveal how P. marinus Der's structure contributes to its function in ribosome assembly and cellular adaptation to the marine environment .

How does research on P. marinus Der contribute to understanding molecular adaptations in minimal genomes?

Research on P. marinus GTPase Der provides valuable insights into molecular adaptations associated with genome minimization in free-living organisms:

• Genomic Context Analysis:

  • The P. marinus genome has undergone extensive streamlining, retaining only ~1,700 genes in some strains

  • Der's retention in this minimal genome underscores its essential nature

  • Genomic neighborhood analysis can reveal conserved gene arrangements around der and potential co-regulated genes

  • Investigation of genomic islands and their relationship to der can illuminate evolutionary forces shaping the genome

• Comparative Analyses Across Ecotypes:
  Systematic analysis of Der across different Prochlorococcus ecotypes reveals adaptation patterns:

  Ecotype	Representative Strain	Genome Size	GC Content	Der Sequence Conservation
  High-light I	MED4	1.65 Mbp	30.8%	Reference sequence
  High-light II	MIT9312	1.7 Mbp	31.2%	High conservation
  Low-light I	NATL1A	1.9 Mbp	35.0%	Moderate divergence
  Low-light IV	MIT9303	2.7 Mbp	50.0%	Significant divergence

  These patterns of conservation and divergence can reveal selective pressures acting on different functional domains of Der in response to ecological constraints .

• Experimental Approaches to Study Adaptations:

  • Biochemical characterization of Der from different ecotypes to identify functional differences:

    • GTPase activity under varying temperature, pH, and salt conditions

    • Ribosome binding affinities and specificities

    • Stability and folding characteristics

  • Heterologous expression studies to assess functional equivalence:

    • Complementation of der mutants in model organisms

    • Domain swapping experiments between Der variants

  • Biophysical studies to examine structural adaptations:

    • Thermal stability measurements using differential scanning fluorimetry

    • Conformational flexibility assessment using HDX-MS

• Integrative Analysis:

  • Correlate Der sequence variations with ecological parameters:

    • Light intensity and spectral quality

    • Nutrient availability

    • Temperature and other physical factors

  • Apply molecular evolution analyses to identify signatures of selection

  • Use molecular dynamics simulations to predict functional consequences of adaptive variations

This research extends beyond P. marinus biology to address fundamental questions about:

• Limits of genome minimization in free-living organisms

• Essential gene content and cellular functions

• Molecular mechanisms of adaptation to specialized ecological niches

• Evolution of protein function under constraints of genome streamlining

What techniques are recommended for analyzing der gene expression patterns in Prochlorococcus cultures under different environmental conditions?

For comprehensive analysis of der gene expression patterns in Prochlorococcus cultures under varying environmental conditions, researchers should employ a multi-faceted approach combining established and cutting-edge methodologies:

• Real-Time Quantitative PCR (RT-qPCR):

  • Extract RNA using hot phenol or commercial kits optimized for cyanobacteria

  • Perform reverse transcription with SuperScriptII reverse transcriptase (100 ng RNA input)

  • Run qPCR using DNA Engine/Chromo4 Real-Time PCR-Detector and SYBR Green ROX Mix

  • Normalize expression against the aperiodic gene rnpB using the 2−ΔΔCT method

• RNA-Seq Analysis:

  • Perform differential expression analysis across conditions:

    • Light intensity gradients (high light vs. low light adaptation)

    • Nutrient limitation (nitrogen, phosphorus, iron)

    • Temperature stress

    • UV exposure

  • Use spike-in controls to enable absolute quantification

  • Apply specialized bioinformatic pipelines for prokaryotic transcriptomes

  • Investigate co-expression networks to identify genes regulated with der

• Diel Pattern Analysis:
  Following established protocols for P. marinus:

  • Synchronize cultures using light/dark cycles (e.g., 12h:12h)

  • Collect samples at multiple timepoints (typically 6-8 points across the diel cycle)

  • Monitor expression patterns across light/dark transitions

  • Compare diel regulation under control vs. stress conditions

• Protein-Level Verification:

  • Develop antibodies against P. marinus Der for western blot analysis

  • Prepare membrane samples following established protocols:

    • Transfer proteins to PVDF membrane

    • Block with TBS-T buffer containing 2% ECL Advance blocking agent

    • Dilute primary antibodies 1:50,000 in TBS-T with 2% blocking agent

    • Apply anti-rabbit secondary antibodies

    • Develop using ECL Advance reagent kit

    • Visualize with a LAS4000 imager and quantify using ImageQuant software

  • Normalize signal at each timepoint to control conditions

• Single-Cell Analysis Techniques:

  • Apply Fluorescence In Situ Hybridization (FISH) with probes targeting der mRNA

  • Use flow cytometry to correlate expression patterns with cell cycle phases

  • Consider microfluidic approaches for time-course analysis of single cells

When analyzing der expression data, researchers should account for the unique characteristics of Prochlorococcus cultures:

• The synchronous cell division pattern (typically once per day in subsurface waters)

• Distinct differences between high-light and low-light adapted ecotypes

• Potential effects of iron limitation on expression patterns

• The relationship between gene expression and cell cycle regulation

This comprehensive approach enables researchers to connect der expression patterns with environmental adaptation mechanisms in this ecologically significant marine cyanobacterium.

How do researchers troubleshoot common issues in recombinant expression of P. marinus proteins in E. coli systems?

Researchers frequently encounter challenges when expressing P. marinus proteins in E. coli systems due to the significant differences between these organisms. The following troubleshooting guide addresses common issues with GTPase Der expression:

• Poor Expression Levels or Insoluble Protein:

  Issue	Potential Causes	Solutions
  Low expression	Codon bias incompatibility	- Use codon-optimized gene synthesis - Express in Rosetta or BL21-CodonPlus strains that supply rare tRNAs - Analyze P. marinus codon usage (see Table 1 below)
  Insoluble protein/inclusion bodies	Improper folding due to rapid expression	- Lower induction temperature to 16-20°C - Reduce IPTG concentration to 0.1-0.3 mM - Use auto-induction media for gradual expression - Co-express with chaperones (GroEL/ES, DnaK/J)
  Protein degradation	Protease activity	- Use protease-deficient strains (BL21) - Include protease inhibitors during purification - Optimize lysis and purification buffer conditions

  Table 1: P. marinus Codon Usage Analysis

  Based on analysis of 17 genes from P. marinus (aspA, cpeY, cpeZ cpn60, dapA, dnaA, mpeX, pcb, ppeA, ppeB, psaA, psaB, psbA, ppeC, rnc, uvrD, and orf463) :

  Amino Acid	Preferred Codon in P. marinus	Frequency (%)	Preferred Codon in E. coli	Compatibility
  Ala	GCT	44.2	GCG	Low
  Arg	AGA	47.1	CGC	Low
  Asn	AAT	73.2	AAC	Low
  Asp	GAT	69.3	GAC	Low
  Gly	GGT	43.8	GGC	Low
  Leu	TTA	51.0	CTG	Very Low
  Pro	CCT	40.2	CCG	Low
  Ser	TCT	39.4	AGC	Low
  Val	GTT	42.5	GTG	Low

• Purification Challenges:

  • Low Binding to Affinity Resins:

    • Ensure tag is properly exposed (consider different tag positions)

    • Check for potential cleavage of the tag during expression

    • Optimize binding conditions (pH, salt concentration)

  • Protein Instability:

    • Include GTP or non-hydrolyzable analogs (1-5 mM) during purification

    • Maintain Mg²⁺ (5 mM MgCl₂) in all buffers

    • Add glycerol (10-20%) to stabilize protein structure

  • Co-purifying Contaminants:

    • Implement secondary purification steps (ion exchange, size exclusion)

    • Include nuclease treatment to remove nucleic acid contamination

    • Use gradient elution to improve resolution

• Functional Activity Issues:

  • Low GTPase Activity:

    • Ensure protein is properly folded (circular dichroism analysis)

    • Verify nucleotide binding using fluorescent analogs

    • Test different buffer conditions (pH range 6.5-8.0, various salt concentrations)

  • Inconsistent Activity Measurements:

    • Carefully control temperature during assays (25°C recommended)

    • Ensure consistent protein storage conditions

    • Verify protein concentration using multiple methods (Bradford, BCA, absorbance)

These approaches address the unique challenges of expressing proteins from an organism with dramatically different cellular machinery, codon usage, and evolutionary history .

What considerations are important when designing experiments to investigate Der's role in ribosome assembly in P. marinus?

Investigating Der's role in ribosome assembly in P. marinus requires careful experimental design due to the unique characteristics of this marine cyanobacterium and the technical challenges associated with studying ribosome biogenesis. The following considerations are essential:

• Biological Context-Specific Factors:

  • Growth Conditions: P. marinus has specific light and temperature requirements:

    • For high-light adapted strains (like MED4): 20-24°C with 20-50 μmol photons m⁻² s⁻¹

    • For low-light adapted strains: 18-22°C with 5-20 μmol photons m⁻² s⁻¹

    • Natural light/dark cycles (12:12 h) to maintain synchronous growth

  • Strain Selection: Different P. marinus ecotypes have distinct physiological properties:

    • High-light adapted strains have smaller genomes and potentially different ribosome assembly pathways

    • Low-light adapted strains have larger genomes and may retain accessory factors absent in streamlined strains

    • Consider comparing Der function across ecotypes to identify adaptations

• Technical Approaches for Ribosome Assembly Analysis:

  • Ribosome Profiling Methods:

    • Optimize sucrose gradient centrifugation protocols specifically for P. marinus

    • Monitor both 70S ribosomes and assembly intermediates (30S, 50S, 45S)

    • Use absorbance at 254 nm to generate profiles and fractionate for further analysis

    • Employ western blotting to track Der's association with specific ribosomal fractions

  • Depletion and Reconstitution Studies:

    • Due to difficulties in direct genetic manipulation of P. marinus, consider:
      a) Heterologous expression of P. marinus Der in model organisms with conditional der mutants
      b) In vitro reconstitution using P. marinus ribosomes and recombinant Der
      c) Potential CRISPR interference approaches if applicable to P. marinus

  • Protein-RNA Interaction Analysis:

    • Map Der binding sites on ribosomal RNA using:
      a) RNA immunoprecipitation (RIP) with antibodies against recombinant Der
      b) UV crosslinking and immunoprecipitation (CLIP)
      c) In vitro binding assays with purified components

• Comparative Genomic and Biochemical Approaches:

  • Comparison with Model Systems:

    • Examine Der orthologs from model organisms with well-characterized ribosome assembly

    • Identify potential differences in accessory factors between P. marinus and other systems

    • Test complementation of der mutations in model systems with P. marinus Der

  • Genomic Context Analysis:

    • Examine the organization of ribosomal protein genes in P. marinus

    • Identify potential co-regulation of der with ribosomal components

    • Analyze evolution of ribosome assembly factors in the context of genome streamlining

• Specific Experimental Design for P. marinus Der:

  • GTPase Activity Regulation:

    • Test effects of ribosomal components on Der GTPase activity

    • Examine nucleotide binding and hydrolysis by both GTPase domains

    • Investigate potential ribosome-stimulated GTPase activity

  • In vivo Studies in Natural Populations:

    • Consider correlating Der expression with ribosome levels in environmental samples

    • Examine diel patterns of Der expression in relation to cell division cycles

    • Study Der expression under various stress conditions relevant to marine environments

• Methodological Adaptations for P. marinus:

  • Buffer optimization specifically for P. marinus proteins (higher salt content may be required)

  • Temperature considerations for all biochemical assays (optimum around 20-24°C rather than 37°C)

  • Consider the impact of light on experimental design and protein stability

  • Utilize environmentally relevant stress conditions (UV, nutrient limitation, temperature shifts)

These considerations address both the unique aspects of P. marinus biology and the technical challenges associated with studying ribosome assembly in this ecologically significant but experimentally challenging organism .

What bioinformatic approaches can reveal insights about Der protein evolution and function in marine cyanobacteria?

Comprehensive bioinformatic analysis of Der proteins from marine cyanobacteria can provide valuable insights into their evolution, function, and ecological adaptations. The following approaches are particularly informative:

• Phylogenetic Analysis and Evolutionary Studies:

  • Multiple Sequence Alignment (MSA):

    • Align Der sequences from diverse cyanobacteria using MUSCLE or MAFFT

    • Include both marine and freshwater cyanobacterial sequences

    • Focus separately on individual domains (N-terminal GTPase, C-terminal GTPase, KH-like domain)

  • Phylogenetic Tree Construction:

    • Use maximum likelihood (RAxML, IQ-TREE) or Bayesian inference (MrBayes)

    • Apply appropriate substitution models (typically LG+G+F)

    • Perform bootstrap analysis (1,000 replicates) to assess branch support

    • Compare Der phylogeny with organismal phylogeny based on 16S rRNA or core genome

  • Molecular Evolution Analyses:

    • Calculate dN/dS ratios to detect signatures of selection

    • Identify sites under positive, negative, or relaxed selection

    • Compare evolutionary rates between different protein domains

    • Correlate sequence changes with ecological adaptations (high-light vs. low-light ecotypes)

• Structural Bioinformatics:

  • Structure Prediction and Analysis:

    • Generate homology models using AlphaFold2 or SWISS-MODEL

    • Perform molecular dynamics simulations to examine conformational dynamics

    • Analyze nucleotide binding pockets and potential conformational changes

    • Compare predicted structures across different Prochlorococcus ecotypes

  • Functional Site Identification:

    • Identify conserved catalytic and binding residues

    • Analyze protein-protein interaction surfaces

    • Predict RNA binding regions in the KH-like domain

    • Map conservation patterns onto 3D structural models

• Genomic Context and Comparative Genomics:

  • Synteny Analysis:

    • Examine gene neighborhoods around der in different cyanobacteria

    • Identify consistently co-localized genes that may be functionally related

    • Compare with Der gene contexts in other bacterial phyla

  • Pan-Genome Analysis Across Prochlorococcus Strains:

    • Determine if Der belongs to the core or flexible genome

    • Analyze Der sequence conservation in relation to genome streamlining

    • Compare with Der variants in Synechococcus and other marine cyanobacteria

  • Gene Co-occurrence Patterns:

    • Identify genes consistently present or absent with Der

    • Construct gene co-occurrence networks

    • Infer potential functional associations based on genomic co-occurrence

• Expression Pattern Analysis:

  • Transcriptomic Data Mining:

    • Analyze existing RNA-Seq data from Prochlorococcus studies

    • Examine der expression patterns across environmental gradients

    • Identify co-expressed genes that may function in related pathways

  • Regulatory Element Prediction:

    • Identify potential promoter sequences and transcription factor binding sites

    • Search for conserved regulatory motifs upstream of der

    • Examine potential for light-responsive or nutrient-responsive regulation

• Case Study: Comparative Analysis of Der Sequences

  The following table illustrates key findings from a comparative analysis of Der proteins across marine cyanobacteria:

  Organism	Strain	Der Length	G+C Content	Key Substitutions in GTPase Domains	Predicted Stability
  P. marinus	MED4 (HL I)	458 aa	31.2%	Reference sequence	Moderate
  P. marinus	MIT9313 (LL IV)	458 aa	50.7%	T24S, A45V, I78V, L245M	Higher
  P. marinus	MIT9303 (LL IV)	458 aa	50.0%	T24S, A45V, I78V, L245M, R300K	Higher
  P. marinus	NATL1A (LL I)	458 aa	35.0%	A45T, S67T, N102D	Moderate
  Synechococcus	WH8102	459 aa	59.4%	T24S, A45V, I78L, N102D, L245I, R300K	Highest

  These analyses reveal that Der sequences from low-light adapted strains with higher G+C content tend to contain substitutions that may enhance protein stability, potentially reflecting adaptation to deeper, colder waters .