Recombinant Gluconobacter oxydans Serine--tRNA ligase (serS)

What is Serine--tRNA ligase (serS) in Gluconobacter oxydans and what is its primary function?

Serine--tRNA ligase (serS), also known as Seryl-tRNA synthetase, is an essential enzyme in Gluconobacter oxydans responsible for catalyzing the attachment of serine to its cognate tRNA (tRNASer) during protein translation. This aminoacylation reaction is critical for accurate protein synthesis according to the genetic code. The serS gene is part of the G. oxydans genome, which contains 2,702,173 base pairs and 2,432 open reading frames . While its canonical function involves protein translation, emerging research suggests aminoacyl-tRNA synthetases may perform additional non-canonical functions in various organisms, as demonstrated by the seryl-tRNA synthetase in Trametes hirsuta, which regulates laccase gene transcription .

How does G. oxydans serS compare structurally to seryl-tRNA synthetases in other organisms?

While detailed structural information specific to G. oxydans serS remains limited in published research, comparative analysis suggests it likely shares the conserved domain architecture typical of class II aminoacyl-tRNA synthetases. A notable comparison point comes from Trametes hirsuta seryl-tRNA synthetase (ThserRS), which lacks the UNE-S domain at its carboxyl terminus . This structural difference is significant because domain organization often correlates with functional diversity, particularly regarding potential non-canonical functions like transcriptional regulation observed in ThserRS. Researchers investigating G. oxydans serS should conduct computational structural modeling and experimental structure determination to identify unique features that might confer organism-specific functions beyond its canonical aminoacylation activity.

What expression systems are most effective for recombinant production of G. oxydans serS?

Several expression systems can be optimized for recombinant G. oxydans serS production, with recent advances in G. oxydans-specific systems providing particularly valuable options:

• The tetracycline-inducible system featuring TetR repressor has demonstrated high tunability in G. oxydans with promoters of varying strengths (P0169, P264, and P452) .

• A bicistronic TetR expression system offers exceptional control with induction ratios exceeding 3,000-fold and minimal background expression when uninduced, making it ideal for proteins where tight regulation is critical .

• The L-arabinose-inducible AraC-PBAD system has shown low background leakiness and approximately 480-fold induction in G. oxydans according to recent literature .

For heterologous expression, standard E. coli systems can be employed, but researchers should consider codon optimization given G. oxydans' high GC content. The optimal system choice depends on experimental requirements, with the bicistronic TetR system providing the tightest regulation for studying potentially toxic proteins or those requiring precise expression control .

Does G. oxydans serS exhibit noncanonical functions similar to seryl-tRNA synthetases in other organisms?

While direct evidence for noncanonical functions of G. oxydans serS is not explicitly documented in the provided literature, several lines of investigation are warranted based on findings in related systems. In Trametes hirsuta, ThserRS functions as a transcriptional regulator, negatively regulating laccase (lacA) gene expression by directly binding to its promoter region through xenobiotic response elements .

To investigate potential noncanonical functions in G. oxydans serS, researchers should employ:

• DNA-binding assays to determine if G. oxydans serS interacts with specific genomic regions

• Transcriptome analysis comparing serS overexpression or knockdown strains

• Subcellular localization studies to determine if serS can enter the nucleus in G. oxydans

• Genetic reporter assays using potential target promoters

The discovery of such noncanonical functions would expand understanding of regulatory networks in G. oxydans and potentially explain how this industrially important organism adapts to environmental stresses.

How does copper ion exposure affect serS expression and function in G. oxydans?

While specific effects of copper ions on G. oxydans serS are not directly documented in the provided research, compelling parallels can be drawn from studies of ThserRS in Trametes hirsuta. In T. hirsuta, ThserRS expression displays a biphasic response to copper, decreasing during the first 36 hours of copper exposure followed by upregulation . This expression pattern inversely correlates with laccase gene expression, suggesting a regulatory relationship tied to copper metabolism .

To investigate copper effects on G. oxydans serS, researchers should:

• Monitor serS transcript and protein levels in G. oxydans cultures exposed to various copper concentrations over time using RT-qPCR and western blotting

• Analyze the serS promoter region for metal-responsive elements

• Measure aminoacylation activity of purified serS in the presence of copper

• Investigate potential DNA-binding activities under copper stress conditions

• Conduct comparative transcriptomics under copper stress to identify copper-responsive genes potentially regulated by serS

Understanding this relationship could reveal novel regulatory mechanisms and potential biotechnological applications, particularly given G. oxydans' industrial importance .

How can a bicistronic TetR autoregulation system be designed for controlled expression of G. oxydans serS?

Designing a bicistronic TetR autoregulation system for tightly controlled G. oxydans serS expression requires several strategic steps based on recently developed expression tools for acetic acid bacteria :

• Vector backbone selection: The pBBR1MCS-2 plasmid has demonstrated effectiveness in G. oxydans and provides a suitable foundation .

• TetR cassette construction:

  • Amplify the tetR gene from a source plasmid like pASK-IBA3

  • Place tetR under control of a constitutive promoter (P0169, P264, or P452)

  • P0169 and P264 promoters provide stronger expression and better repression

• Bicistronic design implementation:

  • Optimize the arrangement for low background activity when uninduced

  • Include appropriate ribosomal binding sites (e.g., iGEM BBa_B0034)

  • Incorporate a transcriptional terminator (e.g., iGEM BBa_B0010)

• serS expression cassette:

  • Place serS under control of the Ptet promoter (repressed by TetR)

  • Include appropriate tags for detection and purification

• Transformation: Introduce the construct into G. oxydans via conjugation with E. coli S17-1 or electroporation

The resulting system provides anhydrotetracycline-inducible expression with induction ratios potentially exceeding 3,000-fold based on similar constructs, offering precise control over serS expression for functional studies .

What potential biotechnological applications exist for recombinant G. oxydans serS?

The biotechnological potential of recombinant G. oxydans serS extends beyond its canonical role in translation, particularly if it exhibits noncanonical functions similar to those observed in other seryl-tRNA synthetases:

• Enhanced industrial strains: If G. oxydans serS influences oxidative stress resistance similar to ThserRS in T. hirsuta , engineered serS expression could create more robust strains for industrial bioprocesses, including vitamin C production for which G. oxydans is already utilized .

• Synthetic biology tools: If G. oxydans serS possesses DNA-binding capabilities, it could be engineered as a novel transcriptional regulator for synthetic biology applications in acetic acid bacteria, expanding the toolkit for genetic circuit design.

• Biosensor development: The potential responsiveness to copper or other metals could be harnessed to develop whole-cell biosensors for environmental monitoring or bioprocess control.

• Protein engineering platform: The unique properties of G. oxydans serS could serve as a foundation for engineering aminoacyl-tRNA synthetases with novel specificities for applications in expanding the genetic code.

• Metabolic engineering: Understanding serS regulation networks could provide new targets for optimizing metabolic pathways in G. oxydans for enhanced production of industrially relevant compounds.

Each application would require thorough characterization of both canonical and potential noncanonical functions of G. oxydans serS using the advanced expression systems now available for this organism .

What are the optimal conditions for purifying recombinant G. oxydans serS?

Purification of recombinant G. oxydans serS requires careful optimization of expression and chromatographic conditions:

• Expression system optimization:

  • Utilize the bicistronic TetR autoregulation system for tight expression control

  • Add affinity tags (His6 or Strep-tag II) for single-step purification

  • Consider fusion with solubility-enhancing tags if expression yields are low

• Cell lysis optimization:

  • Buffer composition: 50 mM Tris-HCl pH 7.5-8.0, 150-300 mM NaCl, 5-10% glycerol

  • Include protease inhibitors and reducing agents (5 mM β-mercaptoethanol or 1 mM DTT)

  • Optimize lysis method (sonication, high-pressure homogenization, or chemical lysis)

• Purification strategy:

  • Affinity chromatography as the initial capture step (Ni-NTA for His-tagged or Strep-Tactin for Strep-tagged protein)

  • Ion exchange chromatography as an intermediate purification step

  • Size exclusion chromatography for final polishing and buffer exchange

  • Consider including low concentrations (1-5 mM) of ATP and MgCl₂ in purification buffers to stabilize the enzyme

• Quality assessment:

  • SDS-PAGE analysis for purity evaluation

  • Western blot for identity confirmation

  • Dynamic light scattering for homogeneity assessment

  • Aminoacylation activity assays for functional verification

Researchers should systematically optimize each parameter while monitoring protein yield, purity, stability, and enzymatic activity to establish a robust purification protocol.

How can the aminoacylation activity of recombinant G. oxydans serS be quantitatively assessed?

Comprehensive assessment of G. oxydans serS aminoacylation activity requires multiple complementary approaches:

• Radioactive aminoacylation assay:

  • Incubate purified serS with [³H] or [¹⁴C]-labeled serine, ATP, and tRNASer

  • Sample at various time points and precipitate with trichloroacetic acid

  • Filter through nitrocellulose and measure radioactivity by scintillation counting

  • Calculate initial velocities and determine kinetic parameters (KM, kcat, kcat/KM)

• Pyrophosphate release assay:

  • Couple pyrophosphate release to enzymatic reactions producing measurable signals

  • Use commercial kits (e.g., EnzChek Pyrophosphate Assay Kit) or develop coupled enzyme systems

  • Monitor reaction progress in real-time using spectrophotometric or fluorescent detection

  • Advantageous for high-throughput screening applications

• ATP consumption assay:

  • Measure ATP consumption using luciferase-based detection systems

  • Provides an alternative approach that doesn't require radioactive materials

  • Can be adapted for microplate format for multiple condition testing

• Acid gel electrophoresis:

  • Separate charged aminoacylated tRNA from uncharged tRNA

  • Visualize using staining or radioactive detection

  • Particularly useful for qualitative assessment of specificity

• Mass spectrometry:

  • Detect mass shift upon aminoacylation

  • Provides direct evidence of product formation

  • Useful for identifying misacylation events

These methods enable thorough characterization of both the amino acid activation and tRNA aminoacylation steps, providing insights into substrate specificity, catalytic efficiency, and potential regulatory mechanisms.

What approaches can reveal potential non-canonical functions of G. oxydans serS?

Investigating potential non-canonical functions of G. oxydans serS requires a multifaceted research strategy:

• Transcriptional regulation studies:

  • Perform chromatin immunoprecipitation (ChIP) followed by sequencing to identify potential DNA binding sites

  • Conduct electrophoretic mobility shift assays (EMSA) with various DNA fragments, similar to studies with ThserRS that demonstrated DNA binding with a dissociation constant of 919.9 nM

  • Utilize reporter gene assays to assess transcriptional regulation of candidate target genes

• Subcellular localization analysis:

  • Generate fluorescent protein fusions to track serS localization under various conditions

  • Perform subcellular fractionation followed by western blotting

  • Compare with ThserRS, which localizes to both cytoplasm and nucleus in T. hirsuta

• Protein interaction studies:

  • Conduct yeast two-hybrid or bacterial two-hybrid screening to identify interacting partners

  • Perform co-immunoprecipitation followed by mass spectrometry to identify protein complexes

  • Utilize proximity-labeling approaches (BioID or APEX) to identify transient interactions

• Physiological impact assessment:

  • Generate serS overexpression and knockdown strains using the bicistronic TetR system

  • Assess phenotypic changes in growth, stress resistance, and metabolism

  • Measure oxidative stress resistance, as ThserRS overexpression enhanced this trait in T. hirsuta

• Response to environmental stimuli:

  • Monitor serS expression and localization under various stress conditions

  • Test responses to copper exposure based on ThserRS findings

  • Analyze global transcriptional responses using RNA-seq in different serS expression backgrounds

Integrating these approaches can provide comprehensive insights into potential non-canonical functions of G. oxydans serS and their physiological significance.

How can site-directed mutagenesis be designed to probe G. oxydans serS structure-function relationships?

A systematic approach to site-directed mutagenesis of G. oxydans serS should target key functional domains and residues:

• Target residue selection:

  • Catalytic residues in the active site responsible for ATP binding and aminoacylation

  • tRNA recognition elements, particularly those interacting with the acceptor stem and anticodon

  • Putative DNA-binding residues if investigating transcriptional regulation functions

  • Residues unique to G. oxydans serS compared to well-characterized orthologs

• Mutation design strategy:

  • Alanine-scanning mutagenesis to neutralize side chain functions

  • Conservative substitutions to assess the importance of specific chemical properties

  • Non-conservative substitutions to test structural versus chemical requirements

  • Domain swapping with serS from organisms showing non-canonical functions

• Expression and purification:

  • Utilize the bicistronic TetR expression system for tight regulation

  • Include appropriate tags for efficient purification and detection

  • Verify proper folding using circular dichroism or thermal shift assays

• Functional analysis pipeline:

  • Aminoacylation assays to assess canonical function

  • DNA-binding assays to test potential transcriptional regulation activity

  • Structural analysis using thermal stability assays

  • In vivo complementation to assess biological functionality

• Data analysis framework:

  • Correlation of structural perturbations with functional changes

  • Computational modeling to interpret experimental findings

  • Comparative analysis with serS mutants from other organisms

This comprehensive approach enables detailed mapping of structure-function relationships in G. oxydans serS, potentially revealing unique features associated with both canonical and non-canonical functions.

How can evolutionary analysis inform G. oxydans serS research?

Evolutionary analysis of G. oxydans serS can provide valuable insights into its functional adaptation and specialization:

• Phylogenetic analysis:

  • Construct comprehensive phylogenetic trees of serS across diverse taxa

  • Identify clades with potential functional specialization

  • Position G. oxydans serS within the evolutionary context of acetic acid bacteria

  • Compare with serS from organisms with known non-canonical functions like T. hirsuta

• Sequence conservation mapping:

  • Identify highly conserved regions likely essential for canonical function

  • Detect variable regions potentially associated with species-specific functions

  • Map conservation onto structural models to identify surface-exposed variable regions

  • Focus on differences between G. oxydans serS and serS from organisms with known non-canonical functions

• Selection pressure analysis:

  • Calculate dN/dS ratios across serS sequences to identify regions under positive or purifying selection

  • Correlate selection patterns with functional domains

  • Identify potential adaptations specific to the G. oxydans lifestyle

• Domain architecture comparison:

  • Analyze presence/absence of domains across serS orthologs

  • Determine if G. oxydans serS lacks the UNE-S domain found in some eukaryotic serS enzymes

  • Compare with ThserRS which lacks this domain but still functions in transcriptional regulation

• Horizontal gene transfer assessment:

  • Investigate potential horizontal acquisition of serS or specific domains

  • Analyze GC content and codon usage patterns for evidence of recent transfer

This evolutionary context provides a framework for understanding the unique features of G. oxydans serS and guides experimental design for functional characterization.