Recombinant Synechocystis sp. Uncharacterized protein ssl3177 (ssl3177)

How does ssl3177 relate to rare lipoprotein A proteins?

Ssl3177, when properly annotated as RepA, belongs to the rare lipoprotein A family, which plays a critical role in cell wall remodeling during bacterial cell division. Synechocystis 6803 has two rare lipoprotein A genes: ssl3177/repA and rlpA (slr0423) . The protein contains characteristic domains of rare lipoproteins A, though it was initially misannotated as having only a partial domain. Ssl3177/RepA likely functions in peptidoglycan modification during cell division, consistent with its genomic location downstream of ftsZ, a key cell division protein .

What is the evolutionary conservation pattern of ssl3177 across cyanobacterial species?

The ssl3177 gene shows interesting conservation patterns at both protein and RNA levels. The protein sequence reflects conservation typical of rare lipoprotein A homologs, while the 3' RNA region that interacts with YlxR/Ssr1238 shows strong sequence and structure conservation across different Synechocystis strains . This RNA conservation is particularly notable given that these strains differ by hundreds of genes (for example, Synechocystis 6803 and Synechocystis 6714 share 2838 protein-coding genes, while 845 genes are unique to Synechocystis 6803 and 895 genes to Synechocystis 6714) . This conservation pattern suggests functional constraints on both the protein and its associated RNA structure.

What is the functional significance of the alternative TTG start codon in ssl3177?

The alternative TTG start codon in ssl3177 results in a 110 amino acid protein rather than the previously annotated 90 amino acid truncated version . This longer protein contains a complete rare lipoprotein A domain, which is critical for its function in cell wall remodeling. The use of the alternative TTG codon rather than the canonical ATG may affect translation efficiency and regulation, potentially providing an additional layer of control over Ssl3177 expression in response to changing environmental conditions. The correct annotation using the TTG start codon is supported by comparative analysis with homologous proteins and by the functional coherence with its genomic position near cell division genes .

What approaches can be used to verify the correct annotation of ssl3177?

Verifying the correct annotation of ssl3177 requires multiple complementary approaches:

• Transcription analysis: RNA-seq and differential RNA-seq (dRNA-seq) to map transcription start sites precisely.

• Translation initiation confirmation: Ribosome profiling (Ribo-seq) to identify ribosome binding sites and protected fragments.

• Protein characterization: Mass spectrometry analysis of the native protein to determine its exact size and N-terminal sequence.

• Reporter constructs: Creating fusions with the predicted TTG start codon versus the previously annotated start, fused to a fluorescent protein, to test translation efficiency.

• Site-directed mutagenesis: Modifying the TTG codon to assess effects on translation.

• Complementation studies: Introducing the correctly annotated gene to an ssl3177 deletion mutant to restore phenotypes.

• Comparative genomics: Examining sequence conservation patterns across multiple cyanobacterial genomes.

This multi-faceted approach would conclusively resolve the current annotation conflicts.

What expression systems are optimal for producing recombinant Ssl3177 for structural studies?

For recombinant expression of Ssl3177, several systems should be considered:

• E. coli-based expression: Using BL21(DE3) strain with a pET-based vector containing a C-terminal His-tag to avoid interfering with potential N-terminal processing. Codon optimization for E. coli may improve yields.

• Controlled expression systems: Tightly regulated promoters like pBAD or pET with T7 lysozyme co-expression to manage potential toxicity.

• Alternative hosts: If E. coli expression proves problematic, expression in Synechocystis itself using inducible promoter systems could maintain native folding environments.

• Purification strategy: Implementing immobilized metal affinity chromatography followed by size exclusion chromatography, with careful buffer optimization including appropriate detergents (DDM, LDAO, or Triton X-100) to maintain solubility of this lipid-associated protein.

• Fusion partners: Expression as a fusion with solubility-enhancing partners like MBP or SUMO might improve yield and folding.

• Quality control: Verification by mass spectrometry, circular dichroism, and thermal stability assays before structural biology studies.

These considerations address the particular challenges of expressing membrane-associated lipoproteins while maintaining their native structure.

What is the nature of the interaction between YlxR/Ssr1238 and the ssl3177 transcript?

YlxR/Ssr1238, an RNA-binding protein in Synechocystis, has been found to interact with a specific RNA fragment derived from the 3' end of the ssl3177 gene . This RNA fragment is approximately 45 nucleotides in length and contains a potentially strong helical structure of 37 nucleotides resembling a rho-independent terminator . The transcript segment bound by YlxR/Ssr1238 overlaps the last two codons and the ssl3177 stop codon, extends 27 nucleotides into the 3'UTR, and lacks an oligo U-stretch at its end, making it a relatively untypical terminator structure . The conservation of this RNA fragment across different Synechocystis strains suggests it has an important functional role, potentially in post-transcriptional regulation .

How can the interaction between YlxR/Ssr1238 and the ssl3177 transcript be studied in vivo?

Investigating this RNA-protein interaction requires several complementary approaches:

• UV crosslinking and immunoprecipitation (CLIP-seq): This technique has already proven effective in identifying this interaction and can be further refined for detailed mapping.

• RNA Electrophoretic Mobility Shift Assays (EMSA): Using purified YlxR/Ssr1238 protein and labeled ssl3177 RNA fragments to determine binding affinity and specificity.

• Mutational analysis: Systematic modification of the ~45 nt binding region to identify critical nucleotides for the interaction.

• Fluorescence in situ hybridization (FISH): Combined with immunofluorescence to visualize co-localization in vivo.

• RNA structure probing: Techniques like SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) to determine how binding affects RNA structure.

• Reporter gene assays: Using the ssl3177 3' region fused to a reporter to assess functional outcomes of the interaction.

These approaches would provide comprehensive insights into both the molecular nature and functional consequences of this RNA-protein interaction.

How does Ssl3177 contribute to cell wall remodeling during cyanobacterial cell division?

As a rare lipoprotein A homolog, Ssl3177/RepA likely plays a crucial role in cell wall dynamics during division . Its genomic location downstream of ftsZ (a key cell division protein) supports this function . To investigate this role, researchers should implement:

• Mutant phenotyping: Characterization of deletion and conditional expression mutants, examining growth rates, cell morphology using electron microscopy, and peptidoglycan composition using HPLC or mass spectrometry.

• Localization studies: Using Ssl3177-fluorescent protein fusions to determine subcellular distribution during the cell cycle, particularly relative to the division plane and FtsZ ring.

• Biochemical assays: Testing purified recombinant Ssl3177 for peptidoglycan binding or modifying activities.

• Interaction mapping: Identifying protein partners among cell division and cell wall synthesis proteins using bacterial two-hybrid or co-immunoprecipitation approaches.

• Cell wall labeling: Employing fluorescent D-amino acids (FDAAs) to visualize nascent peptidoglycan incorporation patterns in wildtype versus ssl3177 mutant strains.

These approaches would clarify Ssl3177's specific contribution to maintaining cell envelope integrity during the complex process of cyanobacterial division.

What is the relationship between ssl3177 expression and cellular responses to various stress conditions?

The ssl3177 gene exhibits dynamic regulation under multiple environmental stressors, including carbon metabolism manipulation, iron starvation, osmotic stress, and in response to depletion of the FtsH1/3 proteolytic complex . This multi-stress responsiveness suggests ssl3177 may function as part of a general stress response network. To investigate this relationship methodically, researchers should:

• Expression profiling: Perform time-course RT-qPCR analysis of ssl3177 expression under controlled stress conditions.

• Reporter systems: Create strains with the ssl3177 promoter driving fluorescent protein expression to monitor regulation in real-time.

• Transcription factor binding: Conduct ChIP-seq to identify regulatory proteins binding to the ssl3177 promoter region under different stress conditions.

• Gene manipulation: Implement CRISPR interference to downregulate ssl3177 expression and assess the impact on stress tolerance.

• Systems biology approaches: Use metabolomic and proteomic profiling of ssl3177 mutants under stress to identify affected pathways.

This systematic approach would clarify whether ssl3177 represents a convergence point for multiple stress response pathways or has stress-specific roles.

How can CRISPR-Cas9 be used to modify ssl3177 for functional studies in Synechocystis?

CRISPR-Cas9 technology enables precise genetic manipulation of ssl3177, with several strategies particularly valuable:

• Complete knockout: Using two guide RNAs flanking ssl3177 with a homology-directed repair template for scarless deletion, carefully designed to avoid polar effects on neighboring genes.

• Point mutations: Targeting specific features such as the alternative TTG start codon to validate the re-annotation hypothesis , conserved functional residues in the rare lipoprotein A domain, or modifications to the 3' region that interacts with YlxR/Ssr1238 .

• CRISPRi/CRISPRa: Using catalytically inactive Cas9 (dCas9) for tunable repression or activation of ssl3177 expression.

• Inducible systems: Implementing temporally controlled CRISPR systems regulated by theophylline riboswitches or similar elements for dynamic studies.

When applying these approaches in Synechocystis, researchers must optimize codon usage for Cas9, use cyanobacteria-compatible selection markers, carefully titrate Cas9 expression, and ensure complete segregation of all chromosome copies due to Synechocystis' polyploid nature.

What microscopy techniques are most suitable for studying Ssl3177 localization during cell division?

Investigating Ssl3177's subcellular localization requires specialized microscopy approaches:

• Fluorescent protein fusions: C-terminal tags using monomeric fluorescent proteins with spectra distinct from cyanobacterial pigments, such as mTurquoise2 or mScarlet.

• Super-resolution microscopy: SIM, STED, or PALM to precisely map Ssl3177 positioning relative to the division plane.

• Time-lapse imaging: Synchronized with cell cycle progression to reveal dynamic changes in localization.

• Co-localization studies: With fluorescently labeled FtsZ and other division proteins to establish temporal and spatial relationships within the divisome.

• Correlative Light and Electron Microscopy (CLEM): Connecting fluorescence localization data with ultrastructural features of the developing septum.

• Complementary approaches: Immunofluorescence with anti-Ssl3177 antibodies or Proximity Ligation Assay (PLA) to address potential artifacts from fluorescent tags.

• Advanced image analysis: Incorporating cell segmentation algorithms and fluorescence distribution quantification for objective characterization across multiple cells.

These approaches would provide comprehensive insights into Ssl3177's spatial and temporal dynamics during cyanobacterial cell division.