Recombinant Synechococcus sp. Undecaprenyl pyrophosphate synthase (uppS)

What is the biochemical function of Undecaprenyl pyrophosphate synthase (uppS) in Synechococcus sp.?

Undecaprenyl pyrophosphate synthase (uppS) in Synechococcus sp., like in other bacteria, catalyzes the condensation between isopentenyl pyrophosphate (IPP) and allylic pyrophosphate to generate undecaprenyl pyrophosphate (UPP, C55-PP). This enzyme belongs to the prenyltransferase family and is crucial for bacterial cell wall synthesis. UPP contains a trans,cis-mixed isoprenoid chain and serves as a lipid carrier for glycosyl transfer in the biosynthesis of various cell wall polysaccharide components . In cyanobacteria like Synechococcus sp., uppS plays a similar essential role in cell envelope formation, although some species-specific variations may exist in substrate specificity and regulatory mechanisms.

How is the uppS gene organized and expressed in Synechococcus sp.?

The uppS gene in Synechococcus sp. shares homology with uppS genes identified in other bacterial species. Based on comparative genomic analysis, uppS is likely organized as a single-copy essential gene, similar to its homologs in other bacteria where it has been demonstrated to be indispensable for growth . Expression studies typically involve cloning the uppS gene into expression vectors with regulatable promoters. When heterologously expressed, the gene can be modified to include amino-terminal His-tags to facilitate purification through nickel affinity chromatography, as has been successfully demonstrated with uppS from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae .

What purification methods are most effective for recombinant Synechococcus sp. uppS protein?

The most effective purification strategy for recombinant Synechococcus sp. uppS typically involves a two-step chromatographic approach. First, express the protein with an amino-terminal His-tag in E. coli, followed by cell lysis and filtration through a 0.22-μm-pore-size membrane. The filtered lysate can then be applied to a Ni²⁺ affinity column, with bound proteins eluted using an imidazole gradient . For higher purity, a second chromatographic step using ion exchange chromatography (such as Phospho-Ultrogel A6R) can be employed, with proteins eluted using a linear NaCl gradient. This approach has proven successful with homologous uppS proteins and should be adaptable to Synechococcus sp. uppS with minimal modifications to account for potential differences in isoelectric point or hydrophobicity .

How does the kinetic mechanism of Synechococcus sp. uppS compare to homologous enzymes from other bacterial species?

The kinetic mechanism of Synechococcus sp. uppS likely follows similar patterns observed in homologous enzymes from other bacteria, though with potential adaptations reflecting the unique cellular environment of cyanobacteria. Key kinetic parameters to investigate include Km values for IPP and allylic substrate, catalytic efficiency (kcat/Km), and product chain length determination. Research indicates that bacterial uppS enzymes are absolutely dependent on detergents like Triton X-100 and divalent cations such as Mg²⁺ for activity . When designing kinetic experiments for Synechococcus sp. uppS, researchers should establish optimal detergent concentrations and metal cofactor requirements, as these may differ from those of other bacterial homologs. Radiometric assays tracking ¹⁴C-IPP incorporation or coupled enzyme assays monitoring pyrophosphate release represent established methodological approaches for kinetic characterization.

What structural features of Synechococcus sp. uppS determine substrate specificity and product chain length?

Substrate specificity and product chain length determination in uppS enzymes involve complex structural elements that may be investigated through site-directed mutagenesis and structure-function studies. Research on homologous uppS enzymes suggests the presence of conserved domains for substrate binding and catalysis. Critical structural features likely include:

To investigate these features in Synechococcus sp. uppS, researchers should employ a combination of X-ray crystallography, molecular dynamics simulations, and biochemical assays with mutant variants. Systematic comparison with structurally characterized uppS homologs will provide insights into unique features that may reflect adaptation to the cyanobacterial cellular environment .

How do post-translational modifications affect the activity and regulation of Synechococcus sp. uppS?

Post-translational modifications (PTMs) may significantly impact uppS activity and regulation in Synechococcus sp., particularly considering the unique photosynthetic metabolism of cyanobacteria. While specific PTMs for Synechococcus sp. uppS have not been extensively characterized, potential modifications to investigate include:

• Phosphorylation: May regulate enzyme activity in response to cellular energy status

• Redox modifications: Could link enzyme activity to photosynthetic electron transport

• Methylation or acetylation: Might influence protein-protein interactions or subcellular localization

Research methodologies should combine mass spectrometry-based proteomics to identify PTMs with site-directed mutagenesis to assess their functional significance. Temporal analysis of PTM patterns under varying light conditions or nutrient availability would provide insights into regulatory mechanisms specific to cyanobacterial metabolism. Comparative analysis with PTM patterns in homologous enzymes from non-photosynthetic bacteria would highlight adaptations specific to cyanobacterial physiology .

What are the optimal expression systems for producing active recombinant Synechococcus sp. uppS?

The selection of an appropriate expression system is critical for obtaining sufficient quantities of active recombinant Synechococcus sp. uppS. Based on experiences with homologous enzymes, E. coli remains the most versatile host for initial expression studies. When designing an expression strategy, consider the following methodological approaches:

For optimal results, clone the Synechococcus sp. uppS gene into vectors with tunable promoters (e.g., T7lac) and explore various fusion tags beyond the standard His-tag, such as MBP or SUMO, which may enhance solubility. Critical experimental parameters include induction temperature (typically lowered to 18-25°C), inducer concentration, and expression duration. Validation of proper folding and activity is essential following purification, regardless of the expression system selected .

How can researchers design activity assays to accurately measure Synechococcus sp. uppS function?

Accurate measurement of Synechococcus sp. uppS enzymatic activity requires careful assay design that accounts for the membrane-associated nature of the enzyme and its absolute dependence on detergents and divalent cations. Recommended methodological approaches include:

• Radiometric assays: Track incorporation of [¹⁴C]-IPP into UPP, with subsequent lipid extraction and quantification by scintillation counting or TLC analysis.

• Coupled enzyme assays: Monitor release of pyrophosphate using auxiliary enzymes and spectrophotometric detection.

• HPLC-based methods: Separate and quantify reaction products using reverse-phase chromatography with UV or mass spectrometric detection.

Critical assay parameters to optimize include:

• Detergent concentration (typically 0.1-0.5% Triton X-100)

• Mg²⁺ concentration (usually 5-10 mM)

• pH optimum (typically 7.5-8.5)

• Substrate concentrations (IPP and allylic pyrophosphate)

• Reaction time and temperature

Researchers should validate assay linearity with respect to enzyme concentration and time, and include appropriate controls to account for non-enzymatic reactions or interference from buffer components .

What approaches can be used to study the interaction of Synechococcus sp. uppS with other cellular components?

Understanding the interactions between Synechococcus sp. uppS and other cellular components requires integrating multiple experimental approaches:

• Affinity purification coupled with mass spectrometry (AP-MS): Identify protein-protein interactions by expressing tagged uppS in Synechococcus sp., followed by gentle lysis and affinity purification under near-native conditions.

• Bacterial two-hybrid assays: Screen for potential interacting partners by expressing uppS fused to one domain of a split reporter protein and a library of proteins fused to the complementary domain.

• Fluorescence microscopy: Visualize subcellular localization using fluorescent protein fusions or immunofluorescence with specific antibodies.

• Crosslinking studies: Capture transient interactions using chemical crosslinkers followed by mass spectrometric identification of crosslinked peptides.

• Lipidomics: Analyze changes in lipid profiles when uppS expression is modified using regulatable promoters or conditional mutants.

These approaches should be complemented with computational predictions of interaction networks based on homology to better-characterized bacterial systems. Given the essential nature of uppS, construction of conditional mutants using regulatable promoters would provide valuable tools for studying the consequences of uppS depletion on cellular physiology .

How does Synechococcus sp. uppS differ from homologous enzymes in other photosynthetic and non-photosynthetic bacteria?

Synechococcus sp. uppS likely exhibits both conserved features essential for function and unique adaptations reflecting its cyanobacterial lineage. Comparative analysis with homologs from other bacteria reveals:

Research indicates that despite sequence divergence, the fundamental biochemical characteristics of uppS—including detergent and MgCl₂ dependence—are conserved across bacterial species . For Synechococcus sp. uppS, investigation should focus on potential adaptations related to the photosynthetic lifestyle, including:

• Light-dependent regulation mechanisms

• Coordination with photosynthetic membrane biogenesis

• Adaptations to variable environmental conditions typical of cyanobacterial habitats

Methodologically, researchers should combine sequence analysis, homology modeling, and biochemical characterization to identify both conserved features and unique adaptations that distinguish Synechococcus sp. uppS from its homologs in other bacterial species .

What insights can structural biology provide about the evolution of uppS across bacterial lineages including Synechococcus sp.?

Structural biology approaches offer powerful insights into the evolutionary relationships between uppS enzymes across bacterial lineages. For Synechococcus sp. uppS, X-ray crystallography or cryo-EM studies would reveal:

• Conservation of the core catalytic domain across bacterial species

• Lineage-specific structural adaptations

• Substrate binding pocket architecture

• Potential oligomerization interfaces

Phylogenetic analysis combined with structural comparison would help reconstruct the evolutionary history of uppS enzymes and identify selective pressures operating in different bacterial lineages. Homologous uppS genes have been identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans, suggesting ancient evolutionary origins and fundamental biological importance .

Researchers investigating Synechococcus sp. uppS should consider:

• Generating high-resolution structures in multiple conformational states

• Performing molecular dynamics simulations to understand conformational flexibility

• Using ancestral sequence reconstruction to test evolutionary hypotheses

• Comparing structures with homologs from diverse bacterial phyla to identify cyanobacteria-specific features

These approaches would contribute to understanding how uppS has evolved while maintaining its essential function across diverse bacterial lineages including Synechococcus sp. .

How can CRISPR-Cas9 technologies be applied to study uppS function in Synechococcus sp.?

CRISPR-Cas9 technologies offer powerful tools for investigating uppS function in Synechococcus sp., particularly given the essential nature of this gene. Methodological approaches for applying CRISPR technologies include:

When designing CRISPR experiments for Synechococcus sp., researchers should optimize transformation protocols, guide RNA design for the AT-rich cyanobacterial genome, and establish appropriate selection markers. These approaches would provide unprecedented insights into uppS function in the context of cyanobacterial cell biology .

What are the implications of uppS research for understanding cyanobacterial cell envelope biogenesis?

Research on Synechococcus sp. uppS has significant implications for understanding cyanobacterial cell envelope biogenesis, which differs from that of model heterotrophic bacteria due to the presence of thylakoid membranes and unique peptidoglycan architecture. Key research directions include:

• Coordination between uppS activity and photosynthetic membrane formation: Investigate how undecaprenyl pyrophosphate synthesis is coordinated with thylakoid membrane development during cell division and under changing light conditions.

• Lipid carrier availability as a regulatory checkpoint: Examine whether UPP levels serve as a metabolic bottleneck that regulates the rate of cell wall synthesis in response to environmental signals.

• Interaction with cyanobacteria-specific cell division proteins: Identify potential interactions between uppS and components of the cell division machinery unique to cyanobacteria.

• Response to environmental stressors: Determine how uppS activity modulates cell envelope remodeling during exposure to changing environmental conditions typical of cyanobacterial habitats.

Methodologically, these questions require integrating genetics, biochemistry, advanced microscopy, and systems biology approaches. The research would bridge fundamental enzymology with cellular physiology to develop a comprehensive understanding of how uppS contributes to the distinctive features of cyanobacterial cell envelopes .