Recombinant Synechocystis sp. Riboflavin biosynthesis protein ribF (ribF)

What is the function of RibF in Synechocystis sp. PCC 6803?

RibF (also known as bifunctional riboflavin kinase/FMN adenylyltransferase) catalyzes two essential steps in the riboflavin metabolic pathway in Synechocystis sp. PCC 6803. This enzyme is responsible for converting riboflavin (RF) to flavin mononucleotide (FMN) and subsequently to flavin adenine dinucleotide (FAD) . These conversions are critical for the cell as FMN and FAD serve as essential cofactors for numerous enzymes involved in vital cellular processes, including the electron transport chain, oxidative metabolism, and photosynthesis.

Does RibF activity influence photosynthetic efficiency in Synechocystis?

Yes, RibF activity directly impacts photosynthetic efficiency in Synechocystis sp. PCC 6803. As the enzyme responsible for producing FMN and FAD cofactors, RibF indirectly supports numerous flavoenzymes involved in photosynthesis and respiration. Altered RibF expression likely affects the cell's ability to maintain optimal levels of these critical cofactors under different light conditions. Any imbalance in flavin nucleotide availability would potentially disrupt electron transfer during photosynthesis, affecting growth rates under photoautotrophic conditions . This relationship is particularly important given Synechocystis's capacity for both photoautotrophic and heterotrophic growth.

What promoter systems are most effective for controlled expression of recombinant ribF in Synechocystis?

Based on interlaboratory studies of promoter systems in Synechocystis, several options exist for controlled expression of recombinant ribF. The PrhaBAD promoter system shows excellent inducibility with rhamnose, providing tight regulation with low basal expression under uninduced conditions . For constitutive expression, the PJ23100 promoter offers relatively consistent expression levels . The copper-inducible PpetE promoter also demonstrates good inducibility but with higher variability between laboratories compared to PrhaBAD .

For optimal experimental design:

When selecting a promoter, researchers should consider not only the desired expression pattern but also the reproducibility requirements of their specific experimental design.

How can CRISPR interference (CRISPRi) be used to study ribF function in Synechocystis?

CRISPRi provides a powerful approach for studying ribF function through reversible gene knockdown. A tightly controlled chimeric promoter like PrhaBAD-RSW, which integrates a theophylline responsive riboswitch into a rhamnose-inducible promoter system, can be used to drive the expression of DNase-dead Cpf1 nuclease (ddCpf1) targeted to the ribF gene . This approach allows for:

• Precise temporal control over ribF repression

• Tunable knockdown levels based on inducer concentration

• Reversibility through removal of inducers

• Observation of phenotypic changes during both repression and recovery phases

The inducible nature of this system is particularly valuable as complete knockout of ribF would likely be lethal, given that it's an essential gene in other bacterial systems like E. coli . Researchers can therefore study the consequences of reduced RibF activity while maintaining cell viability.

What are the considerations for engineering Synechocystis for enhanced riboflavin production through ribF manipulation?

When engineering Synechocystis for enhanced riboflavin production through ribF manipulation, researchers must consider several critical factors:

How do RNA helicases influence ribF expression and function in Synechocystis?

RNA helicases like CrhR in Synechocystis can significantly impact gene expression through regulation of RNA processing. While direct evidence for CrhR regulation of ribF is not established in the search results, the mechanism observed in the rimO-crhR operon provides insight into potential regulatory patterns. RNA helicases may influence ribF by:

• Facilitating mRNA processing and stability control, as seen with CrhR's role in operon transcript processing in Synechocystis

• Mediating temperature-dependent expression regulation, particularly important since riboflavin biosynthesis often responds to environmental stresses

• Potentially affecting riboswitches that regulate riboflavin biosynthesis genes

• Influencing secondary structure of ribF mRNA, which could affect translation efficiency

Researchers investigating ribF regulation should consider RNA helicase activity as a potential regulatory layer, especially when studying temperature-dependent expression patterns or stress responses in Synechocystis.

What role do riboswitches play in regulating ribF and other riboflavin biosynthesis genes in Synechocystis?

Riboswitches play a crucial role in regulating riboflavin biosynthesis genes in many bacteria. In Synechocystis, as in other prokaryotes, FMN riboswitches likely regulate expression of riboflavin biosynthesis genes through a negative feedback mechanism. Key insights include:

• Deletion of FMN riboswitches can significantly enhance riboflavin production, as demonstrated in E. coli where such deletion increased transcription of ribB by 9.71% and riboflavin production by 37.17% .

• Unlike the organization in some bacteria where all rib genes are in a single operon, Synechocystis likely has individual regulatory mechanisms for different genes in the pathway.

• While not all rib genes contain FMN riboswitches, key genes like ribB in E. coli contain an FMN-regulated riboswitch in the 5′ UTR, suggesting a similar mechanism may exist for critical genes in the Synechocystis riboflavin pathway .

• The binding of FMN to the riboswitch typically causes structural changes that either terminate transcription prematurely or prevent translation initiation, providing a direct negative feedback mechanism where end products regulate their own synthesis.

When engineering Synechocystis for altered riboflavin metabolism, targeting these regulatory elements offers a powerful approach to overcome natural production limitations.

How does temperature affect ribF expression and activity in Synechocystis?

Temperature exerts significant effects on gene expression and protein activity in Synechocystis, likely including ribF. Based on observed patterns with other genes:

• Transcript processing and stability: As observed with the rimO-crhR operon, temperature shifts trigger significant changes in transcript processing, with enhanced accumulation of specific transcripts at lower temperatures . Similar temperature-dependent processing might occur with ribF transcripts.

• RNA helicase interaction: The interaction between RNA helicases like CrhR and target transcripts is temperature-sensitive, with CrhR showing 15-fold increase in expression at low temperatures . If CrhR or similar helicases interact with ribF mRNA, this would establish a temperature-dependent regulatory mechanism.

• Enzymatic activity considerations: RibF enzymatic activity itself is likely temperature-dependent, with optimal activity occurring within a specific temperature range relevant to Synechocystis' natural habitat.

• Experimental design implications: Researchers should carefully control and report temperature conditions, as the substantial variability observed between laboratories in standardized experiments with Synechocystis (as high as 32% variation despite protocol standardization) could be partially attributed to subtle temperature differences .

What are the best methods for measuring RibF activity in Synechocystis cell extracts?

Measuring RibF activity in Synechocystis cell extracts requires a combination of techniques to capture both kinase and adenylyltransferase activities:

• Combined enzyme assay approach:

  • Extract preparation: Cell disruption via sonication in buffer (typically 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl₂) followed by centrifugation to obtain clear lysate

  • Reaction mixture: Combine cell extract with riboflavin, ATP, and necessary cofactors

  • Detection: HPLC separation of riboflavin, FMN, and FAD with fluorescence detection (excitation 450 nm, emission 525 nm)

  • Calculation: Quantify conversion rates based on decreasing riboflavin and increasing FMN/FAD concentrations

• Two-step assay for individual activities:

  • Kinase activity: Measure conversion of riboflavin to FMN

  • Adenylyltransferase activity: Measure conversion of FMN to FAD

  • Use of specific inhibitors to distinguish between activities

• Recombinant protein approach:

  • Express and purify recombinant Synechocystis RibF

  • Compare activity of recombinant enzyme with native enzyme in extracts

  • Determine kinetic parameters (Km, Vmax) for both substrates

Key challenges include maintaining enzyme stability during extraction, accounting for endogenous riboflavin/FMN/FAD in extracts, and ensuring sufficient sensitivity for reliable measurements.

How can researchers standardize optical density measurements across laboratories when studying recombinant Synechocystis strains?

Standardizing optical density measurements across laboratories presents significant challenges, as evidenced by interlaboratory studies with Synechocystis. To address this:

• Implement absolute calibration standards:

  • Use microsphere standards of known concentration and size

  • Establish correlation between OD730 values and cell count using flow cytometry

  • Develop laboratory-specific conversion factors

• Supplement OD measurements with additional metrics:

  • Cell counts via flow cytometry or hemocytometer

  • Dry weight biomass measurements

  • Chlorophyll a concentration (though this can vary with physiological state)

• Detailed protocol standardization:

  • Specify exact spectrophotometer model and settings

  • Provide clear instructions for sample preparation, including mixing

  • Standardize cuvette type, path length, and positioning

  • Include blank measurement protocols

• Data reporting requirements:

  • Report instrument-specific conversion factors

  • Include raw and normalized values

  • Specify sample dilution protocols

This approach addresses the significant differences in spectrophotometer measurements observed across laboratories even with identical samples, where interlaboratory studies found substantial variation despite protocol standardization .

How can the interaction between ribF expression and photosynthetic efficiency be quantified in Synechocystis?

Quantifying the relationship between ribF expression and photosynthetic efficiency requires a multi-parameter approach:

• Controlled ribF expression system:

  • Implement inducible promoters like PrhaBAD or PpetE with varying inducer concentrations to create an expression gradient

  • Alternatively, use CRISPRi with tunable repression to reduce ribF expression in a controlled manner

  • Quantify actual ribF transcript and protein levels via RT-qPCR and western blotting

• Photosynthetic parameter measurements:

  • Oxygen evolution rates under different light intensities

  • PAM fluorometry to determine ΦPSII, NPQ, and other fluorescence parameters

  • P700 absorbance changes to assess PSI activity

  • CO2 fixation rates using 14C-labeling

• Flavin cofactor quantification:

  • Liquid chromatography with fluorescence detection for riboflavin, FMN, and FAD levels

  • Analysis of flavin binding status in key photosynthetic complexes

• Correlation analysis:

  • Develop mathematical models relating ribF expression levels to photosynthetic parameters

  • Identify threshold effects and sensitivity coefficients

  • Determine whether relationship is linear or exhibits more complex dynamics

This approach enables researchers to establish quantitative relationships between ribF expression, flavin cofactor availability, and functional photosynthetic output.

What are the structural differences between Synechocystis RibF and similar enzymes in other model organisms?

Structural analysis of Synechocystis RibF compared to homologs in other organisms reveals important insights:

• Domain organization:

  • Synechocystis RibF likely contains both N-terminal kinase and C-terminal adenylyltransferase domains in a single polypeptide, similar to E. coli RibF

  • Some organisms have separate enzymes for the two activities

  • The linker region between domains may show species-specific features affecting regulation or substrate channeling

• Substrate binding sites:

  • Riboflavin binding pocket structure influences substrate specificity and affinity

  • ATP binding site architecture affects kinase activity parameters

  • Metal coordination sites for Mg2+ or other cofactors

• Regulatory features:

  • Presence/absence of allosteric regulation sites

  • Potential phosphorylation or other post-translational modification sites

  • Oligomerization interfaces that may differ between species

• Photosynthetic adaptation:

  • Potential structural adaptations specific to cyanobacteria related to photosynthetic lifestyle

  • Features allowing function under fluctuating light conditions

  • Temperature adaptation elements reflecting the ecological niche of Synechocystis

Understanding these structural differences provides insight into the functional adaptations of RibF across different bacterial lineages and may guide protein engineering approaches.

How does the prokaryotic operon organization in Synechocystis impact strategies for ribF manipulation?

The organization of genes in Synechocystis influences approaches to ribF manipulation:

• Scattered gene organization implications:

  • Unlike the operon organization in B. subtilis (ribD-ribE-ribBA-ribH), Synechocystis likely has a scattered arrangement of rib genes similar to E. coli

  • This arrangement allows for independent manipulation of ribF without direct effects on upstream or downstream genes

  • Reduces concerns about polar effects from insertions or deletions

• Processing of polycistronic transcripts:

  • If ribF is part of an operon, RNA processing mechanisms may create monocistronic mRNAs with different stabilities, as observed with the rimO-crhR operon

  • RNA helicases like CrhR may influence processing and stability of such transcripts

  • Manipulations must consider effects on processing of polycistronic messages

• Regulatory element considerations:

  • Presence of FMN riboswitches affects manipulation strategies

  • Gene-specific regulatory elements may be present in intergenic regions

  • Promoter replacement must account for possible loss of natural regulatory mechanisms

• Expression balancing requirements:

  • Co-expression of multiple genes may require careful balancing for optimal pathway performance

  • Strategies used successfully in E. coli, such as RBS modification or small RNA knockdown, may need adaptation for Synechocystis

These considerations highlight the importance of understanding the specific genomic context of ribF in Synechocystis when designing manipulation strategies.