Recombinant Synechocystis sp. 30S ribosomal protein S9 (rpsI)

What is the genetic context of rpsI in Synechocystis sp. PCC 6803?

The rpsI gene in Synechocystis sp. PCC 6803 encodes the 30S ribosomal protein S9, a critical component of the small ribosomal subunit. This gene is classified as one of the essential housekeeping genes used in phylogenetic analyses alongside other ribosomal and metabolic genes (dnaG, frr, infC, nusA, pgk, pyrG, and others) . When constructing phylogenetic trees for cyanobacterial species, rpsI is particularly valuable due to its high conservation and relatively slow evolutionary rate. The gene is typically extracted from genomic DNA using specific primers designed to amplify the complete rpsI coding sequence, followed by PCR amplification and sequencing verification.

How do I properly annotate the rpsI gene when working with newly sequenced Synechocystis strains?

When annotating rpsI in newly sequenced Synechocystis strains, researchers should:

• Extract the genome sequence using standard DNA isolation protocols similar to those used for PCR verification, where cell pellets are resuspended in distilled water, treated with RNase, and disrupted using glass beads .

• Use comparative genomic approaches with Average Nucleotide Identity (ANI) analysis. For Synechocystis strains, an ANI value threshold of 95% or higher indicates the same species .

• Locate the rpsI gene using homology-based searches against reference genomes.

• Verify gene boundaries through multiple sequence alignment with other cyanobacterial rpsI sequences.

• Confirm annotation through experimental validation, such as RT-PCR or proteomics.

What are the optimal expression vectors for recombinant rpsI in Synechocystis?

For optimal expression of recombinant rpsI in Synechocystis, the SEVA (Standard European Vector Architecture) plasmids have proven particularly effective. These self-replicative vectors offer several advantages:

• Size efficiency: SEVA vectors (e.g., pSEVA251: 5275 bp, pSEVA351: 5120 bp, pSEVA451: 5334 bp) are relatively small compared to other Synechocystis vectors (typically >8 kb), making them easier to handle during cloning procedures .

• Modular design: These vectors contain three variable modules (cargo, replication origin, and antibiotic marker) separated by permanent regions (T0 and T1 terminators and oriT conjugation origin) .

• Multiple antibiotic options: Researchers can select from vectors conferring resistance to kanamycin (pSEVA251), chloramphenicol (pSEVA351), or spectinomycin/streptomycin (pSEVA451) .

• Broad-host-range replication: The RSF1010 replicon enables stable maintenance in Synechocystis without integration into the genome .

These vectors can be successfully transformed into Synechocystis using natural transformation, electroporation, or conjugation methods, with electroporation offering the fastest results (colonies appear in approximately 1 week) .

How do different transformation methods compare when introducing recombinant rpsI constructs into Synechocystis?

For natural transformation (most commonly used):

• Grow Synechocystis cells to OD730 ≈ 0.5

• Harvest cells by centrifugation (10 min at 3850 g)

• Resuspend in BG11 to OD730 ≈ 2.5

• Incubate with plasmid DNA (20 μg/ml final concentration) for 5 hours under light at 25°C

• Spread onto membranes resting on solid BG11 plates

• Transfer to selective media after 24 hours

Electroporation offers faster results and requires less DNA, making it preferable when working with precious recombinant constructs .

What is the most effective purification strategy for obtaining high-purity recombinant rpsI from Synechocystis?

The most effective purification strategy for recombinant rpsI involves:

• Expression optimization: Using a strong, controllable promoter. From the characterized promoters in Synechocystis, select one with appropriate strength from the available range (e.g., three promoters that can be efficiently repressed as mentioned in the literature) .

• Initial extraction:

  • Harvest cells during exponential growth phase (OD730 ≈ 1.0-1.5)

  • Resuspend in lysis buffer containing protease inhibitors

  • Disrupt cells using glass beads or sonication as described for DNA extraction protocols

  • Centrifuge to remove cell debris

• Affinity chromatography: Using His-tag or other affinity tags for initial capture

• Ion exchange chromatography: For further purification based on rpsI's theoretical isoelectric point

• Size exclusion chromatography: As a final polishing step to achieve high purity

• Purity assessment: SDS-PAGE analysis with Coomassie staining and Western blotting

How can I verify that my recombinant rpsI maintains its native structure and function?

To verify that recombinant rpsI maintains its native structure and function:

• Structural analysis:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to probe folding integrity

• Functional assays:

  • In vitro translation assays using Synechocystis extracts

  • 30S ribosomal subunit reconstitution assays

  • RNA binding assays to confirm interaction with 16S rRNA

• Complementation studies:

  • Introduce the recombinant rpsI into an rpsI-deficient strain

  • Monitor growth recovery under standard conditions (30°C, 100 rpm, under 12h light/12h dark cycles)

  • Assess ribosome profiles by sucrose gradient centrifugation

What are the best approaches for creating rpsI knockout or modification mutants in Synechocystis?

For creating rpsI knockout or modification mutants in Synechocystis:

• Knockout strategy:

  • Since rpsI is likely essential, consider using an inducible promoter system for conditional knockouts

  • Design constructs with 500-1000 bp homology arms flanking the rpsI gene

  • Insert an antibiotic resistance cassette (e.g., kanamycin) for selection

  • Transform using natural transformation as described earlier

  • Verify complete segregation through multiple rounds of selection with increasing antibiotic concentrations (up to 500 μg/ml for kanamycin)

  • Confirm segregation by Southern blot or PCR analysis

• CRISPR-Cas based modification:

  • Synechocystis contains native CRISPR-Cas systems that can be leveraged for genome editing

  • Design sgRNAs targeting the rpsI locus with minimal off-target effects

  • Provide a repair template containing desired modifications

  • Co-transform sgRNA and repair template using electroporation for faster results

• Site-directed mutagenesis:

  • For specific amino acid changes, design overlapping primers containing the desired mutation

  • Amplify the entire plasmid containing the rpsI gene

  • Transform into E. coli for plasmid propagation

  • Verify the mutation by sequencing before transforming into Synechocystis

How can I establish an inducible expression system for studying rpsI function in Synechocystis?

To establish an inducible expression system for studying rpsI function:

• Vector selection:

  • Choose an appropriate self-replicative vector like pSEVA251, pSEVA351, or pSEVA451

  • These vectors contain modular components that can be customized for inducible expression

• Promoter options:

  • Select from characterized promoters that show inducible properties

  • Consider using heterologous and redesigned promoters with a wide range of activities, particularly those that can be efficiently repressed

• Construction methodology:

  • Clone the rpsI gene into the multiple cloning site of the vector

  • Transform into Synechocystis using electroporation for rapid results

  • Select transformants on appropriate antibiotic-containing media

• Expression verification:

  • Monitor expression levels under induced vs. non-induced conditions using RT-qPCR

  • Assess protein levels using Western blotting with anti-rpsI antibodies

  • Evaluate impact on growth under standard conditions (30°C, 100 rpm, 12h light/12h dark regimen)

How does rpsI interact with photosynthetic machinery in Synechocystis?

While rpsI's primary function is in translation as part of the 30S ribosomal subunit, there may be interactions with photosynthetic machinery:

• Differential expression analysis:

  • Compare rpsI expression levels under different light conditions

  • Analyze potential co-regulation with photosynthetic genes

  • Examine expression changes in photosystem mutants (such as NDH-1 mutants where PSI function is affected)

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation experiments using tagged rpsI

  • Use bacterial two-hybrid systems to identify interaction partners

  • Conduct pull-down assays followed by mass spectrometry

• Phenotypic analysis of rpsI mutants:

  • Measure photosynthetic parameters (similar to those used to study NDH-1 mutants)

  • Monitor fraction of functional PSI using P700 change (Pm) measurements

  • Assess impact on electron transport using techniques described for NDH-1 studies

What is the impact of environmental stressors on rpsI expression and function?

To assess the impact of environmental stressors on rpsI:

• Expression analysis under stress conditions:

  • Monitor rpsI transcript levels under heat stress (similar to 45°C treatments used in NDH-1 studies)

  • Examine expression under salt stress (using 0%, 3%, 5%, and 7% NaCl as in halotolerance studies)

  • Assess changes during nutrient limitation

• Stress response experiments:

  • Conduct growth experiments under various stress conditions (temperature, salinity, light intensity)

  • Compare wild-type and rpsI mutant strains

  • Monitor growth by measuring OD730 over time (e.g., 16-day period)

• Proteomic analysis:

  • Quantify changes in rpsI protein levels under stress

  • Identify potential post-translational modifications

  • Examine changes in ribosome composition during stress response

How can rpsI be used as a phylogenetic marker for cyanobacterial studies?

The rpsI gene is valuable as a phylogenetic marker due to:

• Conservation characteristics:

  • rpsI is a housekeeping gene with relatively slow evolutionary rates

  • It is consistently used alongside other markers (dnaG, frr, infC, nusA, etc.) for phylogenetic analyses

• Methodological approach:

  • Extract genomic DNA from cyanobacterial samples

  • Amplify rpsI using specific primers

  • Sequence the amplicons and align with reference sequences

  • Construct phylogenetic trees using maximum likelihood methods (e.g., RAxML v7.04, bootstrap = 100)

  • Visualize using tools like iTOL (Interactive Tree Of Life)

• Species determination:

  • Use Average Nucleotide Identity (ANI) analysis with TETRA support

  • For Synechocystis strains, ANI values above 95.3% indicate the same species

  • Complement with CRISPR loci analysis for strain-level differentiation, as these show strain-specific patterns

What role does rpsI play in ribosome biogenesis and how can this be studied in Synechocystis?

To investigate rpsI's role in ribosome biogenesis:

• Assembly analysis:

  • Create conditional rpsI mutants using inducible systems

  • Isolate ribosomal particles by sucrose gradient centrifugation

  • Analyze accumulation of assembly intermediates using quantitative mass spectrometry

  • Perform in vitro reconstitution assays with and without rpsI

• Structural studies:

  • Use cryo-electron microscopy to visualize ribosomes from wild-type and rpsI mutant strains

  • Identify structural alterations in the small subunit

  • Map rpsI's position and interactions within the assembled ribosome

• Functional impact assessment:

  • Conduct in vitro translation assays using S30 extracts from wild-type and mutant strains

  • Measure translation efficiency and fidelity

  • Assess polysome profiles under various growth conditions

• Interaction network mapping:

  • Identify rpsI binding partners during different stages of ribosome assembly

  • Characterize the kinetics of rpsI incorporation into pre-ribosomal particles

  • Determine the hierarchy of assembly events involving rpsI