Recombinant Gluconobacter oxydans 30S ribosomal protein S6 (rpsF)

Advanced Research Questions

• How can recombinant G. oxydans ribosomal protein S6 be expressed and purified for structural and functional studies?

  Recombinant expression and purification of G. oxydans ribosomal protein S6 can be accomplished using several strategies adapted from successful approaches with other ribosomal proteins:

  Methodology:

  1. Expression System: The SUMO fusion method has proven effective for ribosomal proteins . Clone the rpsF gene from G. oxydans into a vector containing an N-terminal His-tagged SUMO fusion partner.

  2. Expression Conditions: Express in E. coli BL21(DE3) at lower temperatures (16-20°C) to enhance proper folding.

  3. Purification Protocol:

     • Lyse cells in buffer containing appropriate protease inhibitors

     • Perform initial purification using Ni-NTA affinity chromatography

     • Cleave the His-SUMO tag using Ulp1 protease

     • Perform secondary purification via ion-exchange or size-exclusion chromatography

  4. Quality Assessment: Verify protein purity by SDS-PAGE and identity by mass spectrometry.

  This approach allows for production of native S6 protein without additional amino acids, as "the His-tagged SUMO components were removed by the active domain of Ulp1 protease (Ulp1p), which cleaves peptide bonds after the SUMO coding end" .

• What approaches can be used to study post-translational modifications of G. oxydans ribosomal protein S6?

  S6 is known to undergo phosphorylation on five evolutionarily conserved serine residues in eukaryotes . While bacterial S6 phosphorylation is less studied, similar modifications may occur in G. oxydans.

  Methodological approach:

  1. Phosphorylation Site Mapping:

     • Purify ribosomes from G. oxydans grown under various conditions

     • Isolate S6 protein via SDS-PAGE or immunoprecipitation

     • Perform tryptic digestion followed by titanium dioxide enrichment of phosphopeptides

     • Analyze by LC-MS/MS to identify phosphorylation sites

  2. Phosphorylation Dynamics Analysis:

     • Western blotting using phospho-specific antibodies

     • Phosphoproteomic analysis under different growth conditions

     • Radioactive 32P-labeling to track phosphorylation kinetics

  3. Kinase Identification:

     • In vitro kinase assays with recombinant S6 and candidate kinases

     • Chemical genetic approaches using kinase inhibitors

     • Pull-down assays to identify interacting kinases

  Research suggests multiple kinases potentially target S6, including S6K1, RSK, PKA, PKC, PKG, and DAPK , though their presence and activity in G. oxydans would require verification.

• How does rpsF mutation or deletion impact G. oxydans growth characteristics and industrial applications?

  Since rpsF appears to be non-essential in some bacteria , engineering this protein could potentially improve G. oxydans industrial properties.

  Research findings and methodological approach:

  G. oxydans is currently limited by slow growth and low biomass yields , with engineered strains showing improved characteristics:

  Strain Modification	Growth Rate Improvement	Biomass Yield Increase	Reference
  mgdH::kan (Glucose dehydrogenase mutant)	+39%	+110%
  ΔmgdH sgdH::kan (Double glucose dehydrogenase mutant)	+78%	+271%
  Potential rpsF modification	Unknown	Unknown	-

  To study rpsF effects:

  1. Generate rpsF mutants or deletion strains using CRISPR/Cpf1-FokI system

  2. Analyze growth kinetics in bioreactors with controlled pH and aeration

  3. Measure biomass yield, substrate consumption, and product formation

  4. Analyze translation efficiency through polysome profiling and ribosome activity assays

  5. Assess industrial biotransformation potential with engineered strains

  Targeting ribosomal components represents a novel approach to strain improvement compared to traditional metabolic engineering of oxidation pathways.

Experimental Design Questions

• What are the optimal conditions for in vitro reconstitution of G. oxydans 30S ribosomal subunits containing recombinant S6?

  In vitro reconstitution of 30S ribosomal subunits provides a powerful approach to study the function of individual ribosomal proteins like S6.

  Methodological protocol:

  1. Component Preparation:

     • Purify 16S rRNA from G. oxydans or use in vitro transcribed 16S rRNA

     • Express and purify all 30S ribosomal proteins (S1-S21) individually using the SUMO fusion method

     • Ensure removal of His-SUMO tags using Ulp1 protease

  2. Reconstitution Conditions:

     • Conventional high-salt method: Incubate components in buffer containing 20 mM MgCl₂, 400 mM NH₄Cl, 20 mM Tris-HCl (pH 7.5) at 42°C for 20 minutes, followed by 37°C for 20 minutes

     • Physiological condition method: Use buffer containing 5 mM Mg²⁺, 150 mM K⁺, with addition of biogenesis factors like Era and YjeQ GTPases

  3. Activity Assessment:

     • Sedimentation analysis by sucrose density gradient centrifugation

     • Poly(U)-directed polyphenylalanine synthesis assay

     • Full-length protein synthesis using the PURE system

  Research shows that "reconstituted 30S subunits containing all 30S subunit proteins was successful using purified components" , with activity reaching approximately 30% of native 30S subunits, increasing to about 80% when S1 protein is added .

• How can the CRISPR/Cpf1-FokI system be optimized for rpsF editing in G. oxydans?

  G. oxydans presents challenges for genetic manipulation, but recent developments in CRISPR/Cpf1-FokI systems offer promising approaches .

  Optimization strategy:

  1. Vector Selection and Design:

     • Use pBBR1MCS-5-p114-FnCpf1 for FnCpf1 expression

     • Design pK18mobSacB-leader-crRNA targeting rpsF

     • Include homologous arms of 800-1000 bp for recombination

  2. Transformation Protocol:

     • Two-step electroporation: first introduce FnCpf1, then crRNA plasmid with homologous arms

     • Incubate in sorbitol medium with appropriate antibiotics (50 μg/mL cephalexin, gentamicin)

     • Plate on selective media and incubate for 48 hours

  3. Efficiency Optimization:

     • Target sites with minimal secondary structure

     • Avoid regions with anti-CRISPR proteins like AcrVA6

     • Use RecET protein from E. coli to enhance homologous recombination

  4. Screening and Verification:

     • Colony PCR for initial screening

     • Sequence verification of mutations

     • Phenotypic characterization

  Current CRISPR/Cpf1-FokI systems in G. oxydans achieve single-gene knockout efficiencies of up to 100% and double-gene editing efficiencies of 27.5-45% .

• What methods are most effective for analyzing the interaction between rpsF and other ribosomal components in G. oxydans?

  Understanding S6 interactions within the ribosome is critical for functional characterization.

  Methodological approaches:

  1. Cryo-Electron Microscopy:

     • Purify intact ribosomes or 30S subunits from G. oxydans

     • Perform cryo-EM to determine the structure at near-atomic resolution

     • Map the position of S6 relative to other components

  2. Cross-linking Mass Spectrometry (XL-MS):

     • Treat purified 30S subunits with crosslinking agents (DSS, BS3)

     • Digest and analyze by LC-MS/MS

     • Identify crosslinked peptides to map proximity relationships

  3. Ribosome Assembly Maps:

     • Generate assembly maps similar to Nomura's 30S assembly map

     • Determine the dependency relationships between S6 and other components

     • "According to Nomura's 30S subunit assembly map, S2 binds 16S rRNA in the final step" , providing context for understanding S6's position in assembly

  4. In vitro Binding Assays:

     • Surface Plasmon Resonance (SPR) to measure binding kinetics

     • Microscale Thermophoresis (MST) for interaction studies

     • Fluorescence techniques to monitor conformational changes

  These approaches help elucidate S6's role in ribosome assembly and function, particularly important since "the band corresponding to S2 was thin, and the proportion of fully reconstituted 30S was low" in some reconstitution experiments, suggesting interplay between various ribosomal proteins.