Recombinant Cyanothece sp. Translation initiation factor IF-2 (infB), partial

What is the role of Translation Initiation Factor IF-2 in bacterial protein synthesis?

Translation Initiation Factor IF-2 is a critical protein that facilitates the initiation phase of protein synthesis in bacteria. It primarily functions to promote the binding of the initiator tRNA (fMet-tRNA) to the small ribosomal subunit and assists in the subsequent joining of the large ribosomal subunit to form the complete translation complex. Research has demonstrated that IF-2 binds GTP and interacts with both the initiator tRNA and the ribosome to enable proper positioning of the start codon in the ribosomal P-site. The factor also participates in maintaining the fidelity of translation by ensuring that protein synthesis begins at the correct position on the mRNA template.

To study IF-2 function experimentally, researchers often employ in vitro dipeptide synthesis assays using purified components, as has been documented with the infB gene in E. coli. These assays utilize templates containing the infB gene, along with fMet-tRNA and various labeled aminoacyl-tRNAs to monitor the formation of dipeptides that indicate successful translation initiation .

What experimental evidence supports the existence of multiple translational start sites in infB genes?

The existence of multiple translational start sites in infB genes has been primarily established through a combination of biochemical and genetic approaches. In E. coli, researchers purified both IF-2α and IF-2β and performed Edman degradation to determine their N-terminal sequences. The analysis revealed that these sequences matched perfectly with the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream, respectively .

Further evidence came from fusion experiments where researchers constructed a fusion between the proximal half of the infB gene and the lacZ gene. This fused gene expressed two products of 170,000 and 150,000 daltons, corresponding to the fused proteins IF-2α-β-galactosidase and IF-2β-β-galactosidase. This confirmed in vivo that the IF-2 forms differ at their N-terminus .

Additionally, a deletion of the 5'-non-translated region of the fused gene, including the Shine-Dalgarno ribosomal binding site, resulted in the expression of IF-2β-β-galactosidase but not IF-2α-β-galactosidase. This strongly suggested that IF-2β results from independent translation rather than from proteolytic cleavage of IF-2α .

What genetic tools are available for manipulating the infB gene in Cyanothece species?

Recent advances have established a genetic toolbox for Cyanothece PCC 7425 that can be applied to studying the infB gene. RSF1010-derived plasmid vectors have been demonstrated to work effectively in this organism for various genetic manipulations. These tools allow for:

• Promoter analysis using promoter probe vectors

• Constitutive or temperature-controlled overproduction of proteins

• Analysis of protein subcellular localization

• Introduction and expression of heterologous genes

This genetic system represents a significant advancement as no gene manipulation system had previously been developed for Cyanothece PCC 7425. The toolbox includes conjugation protocols for introducing foreign DNA, which is particularly valuable for manipulating genes like infB .

For studying infB specifically, these tools would enable:

• Creation of infB fusion constructs for localization studies

• Controlled expression of modified infB variants

• Analysis of infB promoter activity under different growth conditions

• Introduction of heterologous infB genes from other organisms for comparative studies

How can researchers isolate and characterize the infB gene from Cyanothece species?

Isolation and characterization of the infB gene from Cyanothece species would follow similar approaches to those used for other bacteria. A comprehensive methodology would include:

• Genomic DNA isolation: Using standard protocols optimized for cyanobacteria, which often require modifications to handle their robust cell walls.

• PCR amplification: Designing primers based on conserved regions of infB genes from related cyanobacteria. The G domain and C-terminus regions show high conservation across bacterial species and serve as good targets for primer design .

• Cloning and sequencing: The amplified infB gene can be cloned into suitable vectors and sequenced to determine its complete nucleotide sequence.

• Bioinformatic analysis: Comparing the sequence with known infB genes from other organisms to identify conserved domains, potential translational start sites, and regulatory elements.

• Expression analysis: Using Northern blotting or RT-PCR to study the expression patterns of infB under different growth conditions.

• Protein purification: Expressing the recombinant IF-2 protein with affinity tags for purification and subsequent biochemical characterization.

• Functional complementation: Testing the ability of the Cyanothece infB gene to complement infB mutations in E. coli, similar to studies performed with S. aurantiaca infB .

What approaches can be used to study IF-2 protein-protein interactions in Cyanothece?

Studying IF-2 protein-protein interactions in Cyanothece can employ several methodological approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against IF-2 to precipitate the protein along with its interaction partners from Cyanothece cell lysates. This approach requires developing specific antibodies against Cyanothece IF-2 or using epitope-tagged recombinant IF-2.

• Bacterial two-hybrid assays: Adapting bacterial two-hybrid systems for use in Cyanothece or using E. coli as a surrogate host to screen for potential interaction partners of Cyanothece IF-2.

• Pull-down assays: Using recombinant IF-2 with affinity tags to pull down interacting proteins from cell lysates, followed by mass spectrometry identification.

• Fluorescence microscopy: Utilizing the genetic tools available for Cyanothece to create fluorescent protein fusions with IF-2 to study its subcellular localization and co-localization with potential interaction partners .

• Surface plasmon resonance (SPR): For quantitative analysis of IF-2 interactions with purified components of the translation machinery, such as ribosomes, GTP, and initiator tRNA.

These approaches can help elucidate the protein-protein interaction network of IF-2 in Cyanothece and provide insights into any unique aspects of translation initiation in this organism compared to model bacteria like E. coli.

How can researchers optimize the expression of recombinant Cyanothece IF-2 in heterologous systems?

Optimizing the expression of recombinant Cyanothece IF-2 in heterologous systems requires addressing several key factors:

• Codon optimization: Adjusting the codon usage of the Cyanothece infB gene to match the preferred codons of the expression host (e.g., E. coli, yeast) can significantly improve protein yield. This is particularly important for cyanobacterial genes, which often have different GC content compared to common expression hosts.

• Expression vector selection: Choosing appropriate vectors with promoters of suitable strength. For bacterial expression, T7 promoter-based systems often work well for large proteins like IF-2, while for expression in cyanobacteria, native or modified cyanobacterial promoters may be more effective.

• Induction conditions: Optimizing temperature, inducer concentration, and induction timing. Lower temperatures (16-25°C) often improve the solubility of large proteins like IF-2.

• N-terminal fusion tags: Adding solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO can improve the solubility and yield of recombinant IF-2. For purification purposes, His-tags or other affinity tags can be incorporated.

• Host strain selection: Using specialized E. coli strains that provide rare tRNAs or assist in proper protein folding can improve expression of challenging cyanobacterial proteins.

• Temperature-controlled expression: Utilizing temperature-sensitive promoters, as demonstrated in the genetic toolkit for Cyanothece PCC 7425, can provide fine control over protein expression levels .

For expression in Cyanothece itself, the recently developed RSF1010-derived plasmid vectors offer promising tools for controlled expression of native or modified IF-2 proteins .

What are the challenges in expressing functional recombinant IF-2 from Cyanothece species?

Expressing functional recombinant IF-2 from Cyanothece species presents several challenges that researchers must address:

• Protein size: IF-2 is a large protein (typically >70 kDa), which can lead to incomplete translation, premature termination, or inclusion body formation in heterologous expression systems.

• Protein solubility: Large, multi-domain proteins like IF-2 often have solubility issues when overexpressed, particularly in E. coli. This may require optimization of growth conditions, solubility tags, or refolding protocols.

• Post-translational modifications: If Cyanothece IF-2 undergoes specific post-translational modifications, these may not be properly executed in heterologous systems, potentially affecting protein function.

• Protein folding: The correct folding of IF-2 may depend on specific chaperones or folding conditions present in Cyanothece but absent in heterologous hosts.

• Functional assessment: Verifying that the recombinant IF-2 is functionally active requires appropriate assays, such as in vitro translation systems or complementation of IF-2 deficient strains, which need to be adapted specifically for the Cyanothece protein.

• Domain architecture preservation: Ensuring that all functional domains of the IF-2 protein are correctly expressed and folded, particularly the GTP-binding domain (G domain) which is critical for function .

Studies with IF-2 from other bacteria have shown that despite significant sequence differences, particularly in the N-terminal regions, functional complementation across species can be achieved. This suggests that focusing on preserving the conserved functional domains of IF-2 may be more important than achieving exact replication of the native protein .

How does nitrogen fixation capacity in Cyanothece affect translation initiation and IF-2 function?

The nitrogen fixation capacity of Cyanothece creates a unique physiological context that likely influences translation initiation and IF-2 function in several ways:

• Temporal segregation of processes: Cyanothece temporally separates nitrogen fixation (predominantly at night) from carbon fixation (during the day) to protect the oxygen-sensitive nitrogenase enzyme. This temporal segregation creates distinct metabolic states that may require differential regulation of translation initiation .

• Energy allocation: Nitrogen fixation is an energy-intensive process, consuming ATP and reducing power. During nitrogen fixation, energy resources must be carefully allocated between nitrogen fixation and other cellular processes, including protein synthesis. This may influence the activity or regulation of translation factors like IF-2.

• Carbon storage utilization: During nitrogen fixation at night, Cyanothece relies on fixed carbon stored as polysaccharides to provide energy through respiration. The cellular carbon content fluctuates significantly during the diurnal cycle in nitrogen-fixing conditions, with substantial decreases during the dark period. This dynamic carbon economy likely affects protein synthesis rates and may require specific adaptations in translation initiation .

• Protein synthesis priorities: Under nitrogen-fixing conditions, the cell must prioritize the synthesis of nitrogenase and associated proteins, potentially requiring specialized regulation of translation initiation for these specific mRNAs.

• Redox state influence: The cellular redox state differs significantly between nitrogen-fixing and non-nitrogen-fixing conditions, which may affect the activity of translation factors through post-translational modifications or protein-protein interactions.

Experimental data shows that under nitrogen-fixing conditions, Cyanothece cells accumulate higher levels of carbon storage compounds during the light period compared to nitrate-supplemented conditions, but this storage is extensively depleted during the dark nitrogen-fixing period . These carbon dynamics likely influence translation rates and could affect the expression or activity of IF-2.

What is the relationship between carbon metabolism and translation initiation in Cyanothece?

Carbon metabolism and translation initiation in Cyanothece are intimately linked through several mechanisms:

• Carbon availability effect on protein synthesis: Dissolved inorganic carbon (DIC) limitation directly affects carbon fixation rates in Cyanothece, which subsequently influences the cell's capacity for protein synthesis. Under carbon limitation, cells must allocate fixed carbon judiciously between storage, growth, and maintenance functions .

• Diurnal carbon storage dynamics: Cyanothece exhibits pronounced diurnal patterns in carbon storage, particularly under nitrogen-fixing conditions. During the light period, cells accumulate polysaccharides, which are subsequently consumed during the dark period to support nitrogen fixation and cellular maintenance. These cyclical changes in carbon availability likely influence translation rates and may affect the expression or activity of translation factors like IF-2 .

• Energy coupling: Translation initiation is an energy-dependent process, with IF-2 utilizing GTP for its function. The availability of GTP is directly linked to cellular energetics and carbon metabolism, creating a direct connection between carbon metabolism and translation initiation efficiency.

• Regulatory interconnections: Carbon and nitrogen metabolism are coordinately regulated in cyanobacteria. This coordination likely extends to translation regulation, with carbon status potentially influencing translation initiation through various regulatory mechanisms.

Experimental data from Cyanothece cultures show that carbon storage (measured as polysaccharides) and optical density (OD₇₂₀, a proxy for total cellular carbon) fluctuate significantly under different growth conditions. Under nitrogen-fixing conditions, OD₇₂₀ decreases drastically during the dark period, reflecting the drop in polysaccharide content as carbon is consumed for nitrogen fixation. In contrast, under nitrate-supplemented conditions, OD₇₂₀ remains relatively constant during the dark period, indicating different carbon utilization patterns .

How does the unique physiology of Cyanothece affect recombinant protein expression strategies?

The unique physiology of Cyanothece presents both challenges and opportunities for recombinant protein expression strategies:

• Diurnal cycle considerations: Cyanothece's strong diurnal rhythm affects metabolism and gene expression patterns throughout the day-night cycle. Optimizing recombinant protein expression may require synchronizing induction with specific phases of this cycle, particularly for proteins that may interfere with nitrogen fixation or photosynthesis .

• Nitrogen source flexibility: Cyanothece can utilize various nitrogen sources, including atmospheric N₂, nitrate, and urea. This flexibility allows researchers to select optimal nitrogen sources for recombinant protein production. Studies have demonstrated that engineered Cyanothece PCC 7425 can produce the terpene limonene while growing on urea as the sole nitrogen source, indicating that urea may be a viable and potentially advantageous nitrogen source for recombinant protein production .

• Carbon storage capacity: Cyanothece's ability to accumulate carbon storage compounds provides a metabolic buffer that may support recombinant protein synthesis even under fluctuating external conditions. This capacity could be particularly valuable for continuous protein production strategies .

• Large cell size advantage: Cyanothece PCC 7425's larger cell size (3-4 μm) compared to model cyanobacteria provides more cellular space for accumulation of recombinant proteins and may facilitate certain types of protein localization studies .

• Temperature-controlled expression systems: The genetic toolkit developed for Cyanothece PCC 7425 includes temperature-controlled expression vectors, allowing for precise regulation of recombinant protein production. This feature is particularly valuable for expressing proteins that may be toxic or detrimental to cellular functions when continuously produced .

For optimal recombinant IF-2 expression in Cyanothece, researchers should consider:

• Selecting appropriate promoters (constitutive vs. inducible)

• Timing expression to align with favorable metabolic states

• Choosing optimal nitrogen sources based on expression goals

• Utilizing temperature control for fine-tuning expression levels

• Considering the impact of recombinant IF-2 on native translation processes

How can structural biology approaches be applied to study Cyanothece IF-2?

Structural biology approaches can provide valuable insights into the structure-function relationships of Cyanothece IF-2:

By comparing the structure of Cyanothece IF-2 with its counterparts from other bacteria, researchers could identify unique structural features that might relate to Cyanothece's distinctive physiology. For instance, any adaptations that might facilitate translation under the fluctuating metabolic conditions associated with diurnal nitrogen fixation cycles could be of particular interest.

What experimental approaches can resolve contradictions in IF-2 functional data across different bacterial species?

Resolving contradictions in IF-2 functional data across different bacterial species requires systematic comparative approaches:

For example, studies with S. aurantiaca IF-2 revealed a unique 160-residue sequence near the N-terminus with unusual composition (primarily alanine, proline, valine, and glutamic acid) containing nine repeats of the pattern PXXXAP. Surprisingly, complete deletion of this sequence did not affect the factor's function in translation initiation and even increased its complementation capacity in E. coli . Similar studies with Cyanothece IF-2 could reveal unexpected functional properties specific to this cyanobacterium.

How might IF-2 function be integrated with nitrogen fixation and photosynthesis regulatory networks in Cyanothece?

The integration of IF-2 function with nitrogen fixation and photosynthesis regulatory networks in Cyanothece likely involves complex interactions and coordinated regulation:

• Temporal coordination: Cyanothece separates nitrogen fixation and photosynthesis temporally to protect nitrogenase from oxygen. Translation requirements differ during these distinct metabolic phases, suggesting that IF-2 activity may be regulated to support changing protein synthesis needs throughout the diurnal cycle .

• Energy allocation mechanisms: Both nitrogen fixation and translation are energy-intensive processes. During nitrogen fixation, cells must balance energy allocation between these competing demands. Potential regulatory mechanisms include:

  • Phosphorylation of IF-2 in response to energy status

  • Adjustments in IF-2 expression levels

  • Regulation of GTP availability for IF-2 function

  • Modulation of IF-2's interaction with ribosomes

• Redox signaling pathways: Photosynthesis and nitrogen fixation create distinct redox environments in the cell. Redox-sensitive mechanisms could regulate IF-2 activity to coordinate translation with these metabolic states:

  • Direct oxidation/reduction of cysteine residues in IF-2

  • Indirect regulation through redox-sensitive transcription factors

  • Interaction with redox-regulated binding partners

• Carbon storage interface: Cyanothece accumulates carbon storage compounds during photosynthesis and depletes them during nitrogen fixation. The regulation of translation initiation through IF-2 likely responds to carbon availability, potentially through:

  • Signaling molecules that reflect carbon storage status

  • Regulatory proteins sensitive to carbon/nitrogen balance

  • Direct sensing of metabolite concentrations by the translation machinery

• Integrated regulatory networks: Systems biology approaches could reveal how IF-2 function is integrated within the broader regulatory networks controlling nitrogen fixation and photosynthesis:

  • Transcriptomics to identify co-regulated genes

  • Proteomics to map protein-protein interaction networks

  • Metabolomics to identify key metabolites that may serve as regulatory signals

Experimental approaches to investigate these integrations could include:

• Creating reporter constructs to monitor infB expression throughout diurnal cycles

• Performing co-immunoprecipitation studies to identify IF-2 interaction partners under different metabolic conditions

• Using phosphoproteomics to detect post-translational modifications of IF-2 in response to changing metabolic states