Recombinant Mesoplasma florum GMP synthase [glutamine-hydrolyzing] (guaA), partial

What is Mesoplasma florum and why is it relevant for synthetic biology research?

Mesoplasma florum is a non-pathogenic bacterium closely related to Mycoplasma species and part of the Spiroplasma group. It has gained significant attention in synthetic biology research due to several advantageous characteristics. Unlike its Mycoplasma relatives which are typically Biosafety Level 2 organisms, Mesoplasma florum can be handled in BSL1 laboratories, making it more accessible for research . It exhibits remarkably fast growth rates, with doubling times of approximately 31-33 minutes (comparable to E. coli under similar conditions), enabling rapid experimental cycles3. Additionally, it requires relatively less expensive growth media compared to Mycoplasma species .

The organism possesses a small genome, making it an excellent candidate for synthetic genome research. Researchers have already developed various genetic tools for Mesoplasma florum, including integrative plasmids, selectable markers, and characterized promoters that facilitate genetic engineering approaches3. The FreeGenes project has synthesized 573 of the 680 total protein-coding sequences from Mesoplasma florum L1, standardized for MoClo assembly and codon-optimized for expression in Escherichia coli .

What is the function of GMP synthase (guaA) in Mesoplasma florum metabolism?

GMP synthase, encoded by the guaA gene, catalyzes the conversion of xanthosine monophosphate (XMP) to guanosine monophosphate (GMP) in the de novo purine biosynthesis pathway. This reaction requires glutamine as a nitrogen donor and ATP as an energy source. As a key enzyme in guanine nucleotide biosynthesis, GMP synthase plays a critical role in providing precursors necessary for DNA and RNA synthesis, as well as for various signaling pathways in the cell.

In Mesoplasma florum, the expression of the guaA gene is regulated by a variant guanine riboswitch (specifically the type III-B RNA), which selectively binds guanine to control the transcription of the GMP synthase gene . This regulatory mechanism allows the bacterium to modulate GMP synthase expression in response to changing intracellular guanine concentrations, providing precise control over nucleotide metabolism.

How does Mesoplasma florum guaA compare to homologous genes in other bacteria?

The guaA gene in Mesoplasma florum shares functional homology with GMP synthase genes in other bacterial species, though with distinct regulatory mechanisms. Like many bacterial species, Mesoplasma florum uses riboswitch-mediated regulation to control guaA expression. What makes the Mesoplasma florum system particularly interesting is that it employs a variant guanine riboswitch that retains the ability to sense guanine despite having mutations in regions that are typically highly conserved in other bacterial riboswitches .

What unique riboswitch features regulate GMP synthase expression in Mesoplasma florum?

Mesoplasma florum employs a variant guanine riboswitch (classified as type III-B) to regulate GMP synthase expression. This riboswitch has several distinguishing features compared to consensus guanine riboswitches found in other bacteria:

• The Mesoplasma florum guanine riboswitch contains shortened hairpin-loop sequences (particularly in regions L2 and L3) that would normally be involved in forming tertiary contacts critical for aptamer folding .

• Despite these structural alterations, the riboswitch maintains its ability to selectively bind guanine with high affinity, demonstrating remarkable adaptability in ligand recognition mechanisms .

• The riboswitch is located in the 5' untranslated region of the guaA gene, positioned immediately upstream of a putative intrinsic terminator stem, indicating that it controls gene expression through transcription termination .

• In vitro transcription termination assays have confirmed that the III-B riboswitch responds to guanine by increasing the percentage of terminated transcripts, consistent with its predicted role in regulating GMP synthase expression .

The preservation of guanine specificity despite significant sequence variations demonstrates the molecular adaptability of riboswitch structures and highlights the evolutionary plasticity of these regulatory RNA elements in bacteria.

How do in vitro transcription assays demonstrate riboswitch control of guaA expression?

In vitro transcription termination assays have provided valuable insights into how the variant riboswitches in Mesoplasma florum control gene expression. Researchers used DNA templates containing the III-B riboswitch sequence (associated with the guaA gene) to perform single-round transcription termination assays. These experiments revealed that:

• The addition of guanine to the transcription reaction resulted in the highest levels of transcription termination for the III-B construct, consistent with its preference for guanine as determined by in-line probing assays .

• While the in vitro system does not reproduce the full spectrum between 100% termination and 100% full-length transcription observed in vivo, it clearly demonstrates changes in the percentages of terminated transcripts in response to the presence of guanine .

• The specificity exhibited by the III-B RNA in both in-line probing and transcription termination assays corresponds to its predicted role in controlling GMP synthase expression .

This experimental approach confirms that the variant guanine riboswitch upstream of the guaA gene functions as a transcription terminator that responds specifically to guanine. The correlation between ligand binding preferences and transcriptional control provides strong evidence for the regulatory role of this riboswitch in modulating GMP synthase expression in Mesoplasma florum.

What is the evolutionary significance of using a guanine riboswitch to control GMP synthase?

The use of a guanine-sensing riboswitch to control GMP synthase expression in Mesoplasma florum raises interesting evolutionary questions. While it might seem more logical for the riboswitch to sense the ribonucleotide product of the enzyme (GMP), the data indicate that Mesoplasma florum, like at least 15 other bacterial species, uses a guanine-sensing riboswitch to control GMP synthase expression .

This conservation suggests several possibilities:

• Sensing guanine rather than GMP may provide more precise control over the purine biosynthesis pathway, as guanine represents a key branch point in nucleotide metabolism.

• The widespread occurrence of guanine riboswitches controlling GMP synthase genes across diverse bacterial species suggests that this regulatory strategy confers a significant evolutionary advantage.

• The variant riboswitch in Mesoplasma florum represents an example of convergent evolution, where similar regulatory logic has been maintained despite sequence divergence.

The discovery of variant riboswitches in Mesoplasma florum with altered ligand specificities suggests that riboswitch diversity in bacteria may be greater than previously appreciated, with some of these variants potentially having very narrow phylogenetic distributions . This finding indicates that the common fold and consensus sequence for guanine riboswitches can undergo mutations that allow it to bind different purine-related compounds while maintaining its secondary structure, highlighting the versatility of these regulatory RNA elements.

What expression systems are most suitable for recombinant Mesoplasma florum GMP synthase?

Based on current research approaches for Mesoplasma florum proteins, several expression systems can be considered for recombinant GMP synthase production:

• E. coli-based expression systems: The FreeGenes project has generated codon-optimized versions of 573 Mesoplasma florum protein-coding sequences specifically for expression in E. coli . These have been standardized for MoClo assembly, making them readily adaptable for various expression vectors. For GMP synthase specifically, common E. coli expression strains such as BL21(DE3) or Rosetta strains (for rare codon usage) would be appropriate starting points.

• Cell-free expression systems: Given the relatively small size of the GMP synthase enzyme and the potential challenges in folding, cell-free protein synthesis systems might offer advantages for producing active enzyme while avoiding inclusion body formation.

The optimal expression conditions would likely include using the codon-optimized sequence provided by the FreeGenes project, expression at lower temperatures (16-20°C) to promote proper folding, and the addition of appropriate cofactors during purification to maintain enzyme stability.

What are the key considerations for purifying active recombinant Mesoplasma florum GMP synthase?

Purifying active GMP synthase from Mesoplasma florum requires attention to several critical factors:

• Protein solubility and stability: GMP synthase typically contains multiple domains with distinct folding requirements. Purification buffers should include stabilizing components such as glycerol (10-20%), reducing agents (DTT or β-mercaptoethanol), and appropriate salt concentrations (typically 150-300 mM NaCl) to maintain proper folding.

• Cofactor requirements: GMP synthase activity depends on metal ion cofactors, typically magnesium or manganese. Including these ions at appropriate concentrations (1-5 mM) during purification and storage can help maintain the enzyme in its active conformation.

• Purification strategy: A multi-step purification approach is recommended:

  • Initial capture using affinity chromatography (His-tag or GST-tag)

  • Intermediate purification using ion exchange chromatography

  • Polishing step using size exclusion chromatography

• Activity preservation: GMP synthase activity is sensitive to oxidation and pH changes. Buffer systems that maintain pH stability (such as HEPES or Tris at 50 mM, pH 7.5-8.0) and include reducing agents are essential for preserving enzymatic activity throughout the purification process.

• Storage conditions: Purified enzyme should be stored in small aliquots at -80°C with cryoprotectants such as glycerol to prevent freeze-thaw damage. Activity should be verified after storage using standardized activity assays.

What assay methods are most appropriate for measuring Mesoplasma florum GMP synthase activity?

Several complementary approaches can be used to assess the activity of recombinant Mesoplasma florum GMP synthase:

• Spectrophotometric assays: GMP synthase activity can be monitored by coupling the release of PPi to enzymes that generate detectable products. The standard coupled enzyme assay uses pyrophosphatase to convert PPi to Pi, followed by colorimetric detection of Pi using malachite green or a similar reagent.

• HPLC-based assays: Direct monitoring of substrate consumption (XMP) and product formation (GMP) using reverse-phase HPLC provides a more direct measure of enzyme activity without potential interference from coupling enzymes.

• Radiometric assays: Using radioisotope-labeled substrates (typically 14C-labeled glutamine or 32P-labeled ATP) allows for highly sensitive detection of product formation, particularly useful for kinetic studies.

• Mass spectrometry-based assays: LC-MS/MS approaches can provide detailed information about reaction products and potential intermediates, offering insights into the reaction mechanism.

The choice of assay should be guided by the specific research questions being addressed and the available equipment, with consideration given to sensitivity requirements and the need for direct versus indirect activity measurements.

How can researchers design experiments to study the interaction between GMP synthase and its riboswitch regulator?

Investigating the relationship between Mesoplasma florum GMP synthase and its riboswitch regulator requires experimental approaches spanning molecular biology, biochemistry, and structural biology:

• In-line probing assays: This approach has been successfully used to characterize the binding properties of Mesoplasma florum riboswitches . Researchers can design in-line probing experiments using synthetic RNA constructs corresponding to the III-B riboswitch to:

  • Determine binding affinities for guanine and related metabolites

  • Identify structural changes induced by ligand binding

  • Map the precise nucleotides involved in ligand recognition

• Transcription termination assays: Single-round transcription termination assays with DNA templates containing the riboswitch sequence can demonstrate how ligand binding affects transcriptional outcomes . These experiments can:

  • Quantify the percentage of terminated versus full-length transcripts

  • Determine the concentration-dependent effects of various ligands

  • Evaluate the impact of mutations in the riboswitch or coding sequence

• Reporter gene assays: Constructing fusion constructs that place reporter genes (such as fluorescent proteins or luciferase) under the control of the GMP synthase riboswitch allows for:

  • Real-time monitoring of gene expression in living cells

  • High-throughput screening of conditions affecting riboswitch function

  • Quantitative assessment of regulatory dynamics

• Structure-function studies: Combining site-directed mutagenesis with functional assays can reveal:

  • Critical nucleotides for ligand binding and structural switching

  • The molecular basis for ligand selectivity

  • Potential for engineering altered specificities

When designing these experiments, researchers should consider the fast growth rate of Mesoplasma florum (doubling every 31-33 minutes)3 and leverage the available genetic tools, including integrative plasmids and selection markers that have been developed for this organism3.

What approaches can be used to study the impact of mutations on GMP synthase function?

Several complementary approaches can be employed to investigate how mutations affect GMP synthase function and regulation:

• Site-directed mutagenesis: Targeted mutations can be introduced into:

  • The guaA coding sequence to alter enzyme activity or substrate specificity

  • The riboswitch sequence to modify ligand binding or structural switching

  • The promoter region to alter baseline expression levels

• Biochemical characterization: Comparing wild-type and mutant enzymes through:

  • Steady-state kinetic analysis (determining Km, kcat, and substrate specificity)

  • Stability assessments (thermal shift assays, circular dichroism)

  • Structural studies (X-ray crystallography, cryo-EM, or NMR)

• In vivo functional studies: Evaluating the physiological impact of mutations through:

  • Growth rate measurements under various nutrient conditions

  • Metabolomic profiling to assess changes in purine metabolism

  • Competition assays to determine fitness effects

• Transposon mutagenesis: Building on previous transposon mutagenesis work in Mesoplasma florum3, researchers can:

  • Identify genetic interactions with guaA

  • Discover compensatory mutations that rescue defects in GMP synthase function

  • Map the genetic network influenced by GMP synthase activity

These approaches should be implemented with consideration for the unique characteristics of Mesoplasma florum, including its fast growth rate and the availability of defined media that allows for precise control of nutrient conditions3.

How can recombinant Mesoplasma florum GMP synthase contribute to synthetic biology applications?

Recombinant Mesoplasma florum GMP synthase, along with its regulatory elements, offers several valuable applications in synthetic biology:

• Minimal genome projects: As part of efforts to construct minimal synthetic genomes, understanding the function and regulation of essential enzymes like GMP synthase is crucial. The knowledge gained from studying Mesoplasma florum GMP synthase can inform decisions about which genes to include in minimal genome designs and how to regulate them efficiently.

• Engineered biosensors: The guanine riboswitch that regulates GMP synthase could be repurposed as a biosensor for detecting guanine or related compounds in various applications. The variant riboswitches from Mesoplasma florum demonstrate that natural metabolite-sensing RNA motifs can accrue mutations that expand the diversity of ligand detection , suggesting potential for engineering riboswitches with novel specificities.

• Metabolic engineering: GMP synthase plays a key role in nucleotide metabolism, which is critical for cell growth and proliferation. Engineering GMP synthase activity or its regulation could allow for precise control of growth rates in synthetic biological systems or enable the production of valuable nucleotide-derived compounds.

• Synthetic regulatory circuits: The riboswitch-based regulation of GMP synthase provides a model for designing synthetic gene regulatory circuits that respond to small molecule signals. The understanding gained from studying natural variants in Mesoplasma florum can inform the design of synthetic riboswitches with tailored properties.

The standardized, codon-optimized version of the guaA gene available through the FreeGenes project provides a valuable resource for researchers interested in incorporating this enzyme into synthetic biology applications.

What can comparative studies between different Mesoplasma species reveal about GMP synthase evolution?

Comparative studies of GMP synthase across different Mesoplasma species and related organisms can provide significant insights into enzyme evolution and adaptation:

• Functional conservation versus divergence: Analysis of sequence conservation in the catalytic domains versus regulatory regions can reveal which aspects of GMP synthase function are most constrained by selection pressures.

• Riboswitch evolution: The discovery of variant riboswitches in Mesoplasma florum that have altered ligand specificities and affinities suggests that riboswitch diversity in bacteria may be greater than previously appreciated, with some variants potentially having very narrow phylogenetic distributions . Comparative studies can help trace the evolutionary history of these regulatory elements.

• Adaptation to different ecological niches: Differences in GMP synthase sequence, structure, or regulation across Mesoplasma species may reflect adaptations to different environmental conditions or host associations.

• Coevolution with other cellular components: Examining how changes in GMP synthase correlate with changes in other components of purine metabolism or broader cellular systems can reveal coevolutionary relationships.

What are common challenges in working with recombinant Mesoplasma florum proteins and how to address them?

Researchers working with recombinant Mesoplasma florum proteins, including GMP synthase, may encounter several challenges:

• Protein solubility issues: Mesoplasma proteins may fold improperly when expressed in heterologous systems, leading to insolubility or inclusion body formation.

  • Solution: Optimize expression conditions by lowering temperature (16-20°C), using solubility-enhancing fusion tags (SUMO, MBP), or adding folding chaperones as co-expression partners.

• Codon usage bias: Although the FreeGenes project provides E. coli codon-optimized sequences , expression in other systems may be hampered by codon usage differences.

  • Solution: Use host-specific codon optimization or expression strains that supply rare tRNAs.

• Activity preservation: Maintaining enzymatic activity throughout purification can be challenging.

  • Solution: Include appropriate cofactors and stabilizing agents in all buffers, minimize exposure to oxidizing conditions, and validate activity at each purification step.

• Riboswitch functionality in heterologous systems: The regulatory elements controlling GMP synthase expression may function differently in heterologous hosts.

  • Solution: Design careful control experiments to validate riboswitch function in each experimental system, potentially using reporter gene constructs to monitor activity.

These challenges can be addressed through careful experimental design, leveraging the tools and resources specifically developed for Mesoplasma florum research, including the standardized gene constructs from the FreeGenes project and the defined media formulations that have been optimized for Mesoplasma growth3.

How can researchers validate the specificity and functionality of recombinant Mesoplasma florum GMP synthase?

Validating the specificity and functionality of recombinant Mesoplasma florum GMP synthase requires a multi-faceted approach:

• Enzymatic activity assays: Multiple complementary assays should be employed to confirm activity:

  • Direct measurement of XMP to GMP conversion using HPLC or MS

  • Coupled enzyme assays to monitor ATP consumption or glutamine utilization

  • Comparison with commercially available GMP synthase from other sources as a benchmark

• Substrate specificity testing: Evaluate the enzyme's ability to utilize various substrates:

  • Test alternative purine precursors beyond XMP

  • Assess glutamine versus ammonia as nitrogen donors

  • Determine nucleotide specificity (ATP, GTP, etc.) for the energy source

• Inhibitor studies: Characterize the enzyme's response to known GMP synthase inhibitors:

  • Measure IC50 values for standard inhibitors

  • Compare inhibition profiles with GMP synthases from other organisms

  • Identify potentially unique inhibition patterns

• Functional complementation: Test whether the recombinant enzyme can restore function in:

  • E. coli guaA mutants lacking functional GMP synthase

  • Other bacterial species with guaA deficiencies

  • Cell-free systems requiring de novo GMP synthesis

• Structural validation: Confirm proper folding and assembly using:

  • Circular dichroism to assess secondary structure content

  • Size exclusion chromatography to verify oligomeric state

  • Limited proteolysis to probe domain organization

By implementing these validation approaches, researchers can ensure that their recombinant Mesoplasma florum GMP synthase retains the functional properties of the native enzyme and identify any differences that may result from heterologous expression or purification procedures.