Recombinant Synechocystis sp. Ycf53-like protein (sll0558)

What is the functional role of Slr0058 in Synechocystis sp. PCC 6803?

Slr0058 is a protein involved in polyhydroxybutyrate (PHB) metabolism in Synechocystis sp. PCC 6803. Research indicates that it shows significant similarities with the regulatory phasin PhaF and plays a crucial role in PHB granule formation. The protein is encoded within an operon (slr0058-slr0061) containing several genes putatively related to PHB metabolism. Functionally, Slr0058 appears to regulate PHB granule surface-to-volume ratio during nitrogen starvation conditions, suggesting it serves as a key regulatory component in the PHB production pathway .

What cellular localization pattern does Slr0058 exhibit?

Fluorescence microscopy studies using GFP-tagged Slr0058 have demonstrated distinct localization patterns that vary with growth conditions. During vegetative growth, Slr0058 aggregates in specific foci within the cell. Interestingly, during nitrogen starvation conditions, when PHB granules are being formed, Slr0058 does not co-localize with these granules. This spatial separation suggests that Slr0058 may influence PHB granule formation through indirect regulatory mechanisms rather than by direct association with the granules themselves .

What molecular mechanisms underlie Slr0058's role in PHB granule formation?

While current research has established Slr0058's influence on PHB granule formation, the precise molecular mechanisms remain incompletely understood. Based on its similarity to phasin proteins like PhaF, Slr0058 likely acts as a regulatory scaffold that influences the nucleation and growth of PHB granules. Potential mechanisms include:

• Modulation of the initial nucleation events of PHB polymerization

• Regulation of the polymerization rate by interacting with PHB synthase

• Control of granule coalescence through surface interactions

• Influence on the physical properties of the granule surface

Research approaches to elucidate these mechanisms should combine structural biology, protein-protein interaction studies, and high-resolution microscopy to visualize the dynamics of granule formation in real-time .

How can transcriptomic and proteomic approaches be integrated to understand Slr0058 regulatory networks?

To comprehensively understand Slr0058's role in cellular regulation, researchers should implement a multi-omics strategy:

• RNA-Seq analysis comparing wild-type and Δslr0058 mutant strains under various conditions (nitrogen-replete, nitrogen-starved) to identify differentially expressed genes

• Quantitative proteomics to detect changes in protein abundance and post-translational modifications

• Metabolomics focusing on PHB precursors and related metabolic intermediates

• ChIP-seq if Slr0058 potentially interacts with DNA or chromatin components

• Integration of these datasets using computational network analysis to identify key nodes and pathways affected by Slr0058

This integrated approach would reveal both direct and indirect regulatory effects of Slr0058, potentially identifying previously unknown connections to other cellular processes beyond PHB metabolism .

What analytical techniques provide the most accurate quantification of PHB in Synechocystis?

For robust PHB quantification in Synechocystis research, a multi-method approach is recommended:

When studying Slr0058's effects on PHB metabolism, combining chemical quantification with microscopy techniques provides the most comprehensive assessment by capturing both total PHB content and granule distribution patterns at the single-cell level .

What is the optimal protocol for expressing and purifying recombinant Slr0058?

Based on research practices with similar cyanobacterial proteins, the following protocol is recommended:

• Cloning strategy:

  • Clone the slr0058 gene into a pET-based expression vector with an N-terminal His-tag

  • Include a TEV protease cleavage site for tag removal if needed for functional studies

• Expression conditions:

  • Transform into E. coli BL21(DE3) or Rosetta(DE3) for rare codon optimization

  • Culture in LB or 2×YT medium at 37°C until OD600 reaches 0.6-0.8

  • Induce with 0.5 mM IPTG and shift to 18°C for 16-20 hours (lower temperature improves protein folding)

• Purification steps:

  • Harvest cells and lyse in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Apply to Ni-NTA affinity column, wash with increasing imidazole concentrations

  • Further purify using size exclusion chromatography

  • Verify purity by SDS-PAGE and identity by Western blot or mass spectrometry

• Storage conditions:

  • Store at -80°C in buffer containing 20% glycerol to maintain stability

  • Avoid repeated freeze-thaw cycles

This protocol can be adapted based on specific experimental requirements and protein behavior during purification .

How can microscopy techniques be optimized for analyzing Slr0058 localization and PHB granule formation?

For comprehensive visualization of Slr0058 localization and PHB granules:

• Sample preparation:

  • For GFP-tagged Slr0058: fix cells with 4% paraformaldehyde to preserve fluorescence

  • For PHB granules: stain with Nile Red (1 μg/mL for 10 minutes) or BODIPY 493/503

  • Mount samples in anti-fade medium to prevent photobleaching

• Imaging techniques:

  • Confocal microscopy with Z-stack acquisition (0.2 μm steps) for 3D reconstruction

  • Super-resolution microscopy (STED or STORM) for detailed spatial relationships

  • Time-lapse imaging during nitrogen starvation to capture dynamic localization changes

• Quantification parameters:

  • Number and size of Slr0058 foci per cell

  • Number, size, and distribution of PHB granules

  • Colocalization coefficients if applicable

  • Changes in these parameters over time or under different conditions

• Controls:

  • Wild-type cells (negative control for GFP signal)

  • Cells expressing free GFP (control for protein localization)

  • Known membrane markers to provide cellular context

This methodological approach enables quantitative analysis of how Slr0058 influences PHB granule formation at the single-cell level .

What genetic engineering approaches can be used to study Slr0058 function and enhance PHB production?

Several genetic strategies can be employed to investigate Slr0058 function:

• Gene knockout and complementation:

  • Create complete gene deletion using homologous recombination

  • Complement with wild-type and mutated versions to identify critical domains

  • Use the zinc-inducible promoter system (ziaA) for controlled expression levels

• Domain mapping:

  • Generate truncated variants to identify functional regions

  • Create chimeric proteins with other phasins to determine domain-specific functions

  • Introduce point mutations at conserved residues to disrupt specific interactions

• Protein tagging strategies:

  • C-terminal vs. N-terminal tags to minimize functional disruption

  • Split-GFP system for protein-protein interaction studies

  • Proximity labeling (BioID or APEX) to identify interaction partners in vivo

• For enhanced PHB production:

  • Overexpress Slr0058 under nitrogen-starvation-specific promoters

  • Engineer Slr0058 variants with altered granule size control properties

  • Co-express Slr0058 with other PHB pathway components for coordinated enhancement

These approaches can be combined with analytical techniques to comprehensively assess how genetic modifications affect both PHB production and granule characteristics .

How should researchers design experiments to investigate the relationship between Slr0058 and other proteins in the PHB metabolic pathway?

To effectively investigate protein interactions within the PHB pathway:

• In vivo interaction studies:

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions

  • Förster Resonance Energy Transfer (FRET) for detecting proximity in living cells

  • Co-immunoprecipitation followed by mass spectrometry to identify binding partners

• Experimental design considerations:

  • Compare multiple growth conditions (normal, nitrogen starvation, phosphate limitation)

  • Include time-course analysis to capture dynamic interactions

  • Create double mutants (Slr0058 with other PHB-related genes) to assess genetic interactions

• Functional assays:

  • Enzyme activity measurements of PHB synthase in presence/absence of Slr0058

  • PHB granule isolation followed by proteomics to identify granule-associated proteins

  • In vitro reconstitution of granule formation with purified components

The experimental design should include appropriate controls and standardized conditions to ensure reproducibility, particularly when comparing wild-type and mutant phenotypes across different growth phases and stress conditions .

What bioinformatic approaches can predict functional domains in Slr0058?

For comprehensive structural and functional prediction of Slr0058:

• Sequence analysis tools:

  • Multiple sequence alignment with related phasins using MUSCLE or T-Coffee

  • Conservation analysis to identify functionally important residues

  • Hydrophobicity plots to identify potential membrane or granule-interacting regions

• Structure prediction methods:

  • AlphaFold2 or RoseTTAFold for 3D structure prediction

  • SWISS-MODEL for homology modeling if suitable templates exist

  • PrDOS or DISOPRED for predicting intrinsically disordered regions

• Functional prediction:

  • InterProScan for domain and motif identification

  • GPS for post-translational modification sites

  • CELLO for subcellular localization prediction similar to analysis done for Ycf1

• Protein-protein interaction prediction:

  • SPRINT or PIPE for predicting potential interaction partners

  • Molecular docking simulations with known PHB pathway components

These bioinformatic analyses should precede experimental work to guide hypothesis formation and experimental design. The predictions should be validated through targeted mutagenesis of key residues identified through computational analysis .

How can researchers troubleshoot issues with Slr0058 expression or PHB quantification?

Common challenges in Slr0058 research and their solutions include:

When troubleshooting, systematic modification of one variable at a time while maintaining appropriate controls is essential for identifying the source of technical issues .

What are the best approaches for studying Slr0058 regulation under different environmental conditions?

To comprehensively investigate environmental regulation of Slr0058:

• Transcriptional regulation:

  • qRT-PCR to measure slr0058 expression under various conditions

  • Reporter gene assays (lacZ fusions) to monitor promoter activity

  • ChIP-seq to identify transcription factors binding to the slr0058 promoter

• Environmental variables to test:

  • Nutrient availability (nitrogen, phosphorus, carbon sources)

  • Light intensity and quality (including day/night cycles)

  • Temperature fluctuations

  • Osmotic and oxidative stress conditions

• Post-translational regulation:

  • Western blotting to detect protein levels and modifications

  • Pulse-chase experiments to determine protein stability

  • Phosphoproteomics to identify regulatory phosphorylation sites

• Experimental design matrix:

  Environmental Factor	Range to Test	Analysis Methods
  Nitrogen	0-17.6 mM NO₃⁻	PHB quantification, expression analysis
  Light intensity	10-200 μmol photons m⁻² s⁻¹	Localization studies, transcriptomics
  Carbon source	Air vs. 1-5% CO₂	Metabolomics, PHB production
  Temperature	25-37°C	Protein stability, activity assays

This comprehensive approach allows for identifying condition-specific regulatory mechanisms and potential environmental triggers for Slr0058 activity changes .

What emerging technologies could advance our understanding of Slr0058 function?

Several cutting-edge technologies show promise for elucidating Slr0058 function:

• Single-cell technologies:

  • Single-cell RNA-seq to capture cell-to-cell variability in slr0058 expression

  • Microfluidics combined with time-lapse microscopy for dynamic studies

  • Flow cytometry with fluorescent reporters for high-throughput phenotyping

• Genome editing advancements:

  • CRISPR interference (CRISPRi) for tunable gene repression

  • Base editors for precise amino acid substitutions without double-strand breaks

  • Multiplexed CRISPR systems for simultaneous manipulation of multiple PHB-related genes

• Structural biology methods:

  • Cryo-electron tomography for in situ visualization of PHB granules

  • Hydrogen-deuterium exchange mass spectrometry for protein dynamics

  • In-cell NMR for studying protein structure in the native environment

• Systems biology approaches:

  • Multi-omics data integration using machine learning algorithms

  • Genome-scale metabolic modeling to predict PHB flux changes

  • Synthetic biology platforms for rapid prototyping of engineered Slr0058 variants

These technologies can provide unprecedented insights into the molecular mechanisms of Slr0058 function and its integration into cellular metabolism .

How might understanding Slr0058 contribute to sustainable bioplastic production?

The fundamental knowledge gained about Slr0058 could translate to biotechnological applications:

• Engineering opportunities:

  • Rational design of Slr0058 variants that optimize PHB granule size and number

  • Development of inducible systems for controlled PHB production

  • Creation of synthetic regulatory circuits that link PHB production to photosynthetic activity

• Production enhancements:

  • Manipulation of Slr0058 expression levels to increase PHB yield

  • Engineering of PHB granule properties for easier extraction and processing

  • Development of continuous production systems based on understanding of regulatory mechanisms

• Sustainable advantages of cyanobacterial systems:

  • Carbon-neutral PHB production using photosynthesis

  • Potential for wastewater remediation coupled with bioplastic production

  • Reduced energy requirements compared to heterotrophic fermentation

Understanding the fundamental biology of Slr0058 provides the foundation for these biotechnological applications, highlighting the importance of basic research for addressing sustainability challenges .