Recombinant Gluconobacter oxydans DNA-directed RNA polymerase subunit beta' (rpoC), partial

What is the function of RNA polymerase subunit beta' (rpoC) in Gluconobacter oxydans?

RNA polymerase subunit beta' (rpoC) in G. oxydans functions as an essential component of the bacterial RNA polymerase complex, catalyzing RNA synthesis through DNA template binding and phosphodiester bond formation. Similar to rpoC in other bacterial species like Haemophilus ducreyi, it contains domains critical for enzymatic activity including the active site for binding initiating nucleotides and magnesium ions, clamp helices for stabilizing DNA templates during elongation, and a trigger loop that facilitates nucleotide addition and proofreading. The rpoC gene product is indispensable for executing transcriptional programs in G. oxydans, which is particularly important given this organism's unique metabolic pathways including periplasmic glucose oxidation systems .

How conserved is the rpoC gene across different bacterial species compared to Gluconobacter oxydans?

The rpoC gene is highly conserved across bacterial species, reflecting its essential role in transcription. Based on comparative analysis with other bacteria, the expected conservation patterns would be:

The high degree of conservation facilitates comparative studies and allows researchers to apply insights from model organisms to understand G. oxydans rpoC function. Despite this conservation, species-specific variations exist, particularly in regions that interact with regulatory factors.

Is rpoC expression affected by the unique metabolic pathways of Gluconobacter oxydans?

While rpoC expression is generally constitutive, it may be indirectly influenced by G. oxydans' unique metabolism. G. oxydans possesses an unusual glucose metabolism where most glucose is oxidized in the periplasm to gluconate and ketogluconates, with less than 10% metabolized in the cytoplasm . This metabolic configuration creates a distinct cellular environment that could affect transcriptional patterns.

When membrane-bound glucose dehydrogenase (mgdH) is inactivated, G. oxydans shifts to increased intracellular glucose metabolism, resulting in a 110% increase in growth yield and 39% improvement in growth rate . This metabolic reconfiguration likely alters the transcriptional landscape, potentially affecting the activity (though not necessarily expression) of RNA polymerase containing the rpoC-encoded β' subunit.

What methods are most effective for expressing and purifying recombinant G. oxydans rpoC for structural studies?

For optimal expression and purification of recombinant G. oxydans rpoC:

• Expression system selection: E. coli BL21(DE3) with pET-based vectors incorporating a His6-tag is recommended for initial attempts, based on successful experiences with other bacterial RNA polymerase subunits .

• Optimization protocol:

• Reconstitution strategy: For functional studies, purified rpoC should be reconstituted with other RNA polymerase subunits (α, β, ω) to form the holoenzyme. This can be achieved through:

  • Co-expression of all subunits

  • Sequential addition of purified subunits with verification by gel filtration

  • Native PAGE to confirm complex formation

This approach minimizes proteolytic degradation while maximizing yield of properly folded protein.

How can researchers effectively design CRISPR-Cas9 systems for targeted modification of rpoC in Gluconobacter oxydans?

The design of CRISPR-Cas9 systems for modifying rpoC in G. oxydans requires careful consideration of several factors:

• sgRNA design strategy:

  • Identify target sequences with NGG PAM sites in non-conserved regions

  • Design guideRNAs with minimal off-target effects

  • Test multiple sgRNAs targeting different regions to optimize editing efficiency

• Delivery method optimization:

• Verification and phenotyping protocol:

  • PCR verification with primers flanking the modified region

  • Sequencing to confirm precise edits

  • RNA polymerase activity assays

  • Growth curve analysis similar to methods used in mgdH knockout studies

  • Metabolic profiling to assess indirect effects

The methodology should build upon the approaches successfully used for gene deletion in G. oxydans, such as the overlap extension PCR and two-step recombination process used to create the mgdH deletion strain .

What experimental approaches can resolve contradictory data on rpoC mutation effects on transcription fidelity in Gluconobacter species?

To resolve contradictory data on rpoC mutation effects on transcription fidelity:

• Comprehensive mutational analysis:

  • Create a panel of site-directed mutations in conserved catalytic regions

  • Include mutations in trigger loop and bridge helix regions

  • Compare with equivalent mutations studied in model organisms

• Multi-level transcriptional fidelity assessment:

• Biochemical characterization:

  • Measure elongation rates of wild-type vs. mutant RNA polymerases

  • Assess pausing frequency and duration

  • Quantify abortive transcription products

  • Determine binding affinities for nucleotides and DNA templates

• Physiological impact analysis:

  • Evaluate changes in stress response pathways

  • Measure induction of SOS response genes

  • Assess impacts on G. oxydans-specific metabolic pathways, particularly periplasmic vs. cytoplasmic glucose oxidation

This multi-faceted approach enables disambiguation of direct effects on transcription fidelity from indirect metabolic consequences.

How can modifications to rpoC enhance glucose metabolism in Gluconobacter oxydans for improved growth yield?

Strategic modifications to rpoC could potentially enhance glucose metabolism in G. oxydans by altering transcriptional patterns. Based on metabolic engineering principles:

• Target design for rpoC modification:

  • Introduce mutations that favor expression of genes involved in cytoplasmic glucose metabolism

  • Modify regions that interact with regulators controlling the pentose phosphate and Entner-Doudoroff pathways

  • Engineer variants with altered promoter recognition properties

• Synergistic engineering approach:

• Metabolic outcome analysis:

  • Monitor changes in carbon flux through glycolytic pathways

  • Measure CO2 production (which increased 4-5.5 fold in dehydrogenase mutants)

  • Quantify acetate production as an indicator of enhanced pyruvate metabolism

  • Assess impact on incomplete oxidation products like 2-ketogluconate and 2,5-diketogluconate

These approaches leverage insights from the dramatic growth improvements achieved through dehydrogenase gene inactivation (39-78% improved growth rates) while targeting the transcriptional machinery to further optimize metabolism.

What role does rpoC play in regulating oxidative versus non-oxidative pathways in Gluconobacter oxydans?

The rpoC-encoded β' subunit plays a central role in the transcriptional regulation that balances oxidative and non-oxidative pathways in G. oxydans:

• Pathway competition analysis:

  • In wild-type G. oxydans, periplasmic oxidation dominates with less than 10% of glucose metabolized through cytoplasmic pathways

  • This balance is regulated at the transcriptional level, with rpoC-containing RNA polymerase controlling expression of key enzymes

• Regulatory interaction network:

• Experimental evidence from pathway rewiring:

  • Deletion of mgdH redirects glucose metabolism from periplasmic oxidation to cytoplasmic pathways

  • This redirection increases CO2 production by 4-fold, indicating enhanced complete oxidation

  • The transcriptional adaptation to this metabolic shift necessarily involves RNA polymerase containing the rpoC product

Understanding the role of rpoC in this regulation could provide insights for engineering strains with optimized pathway balance for specific biotechnological applications.

How do post-translational modifications of rpoC influence RNA polymerase activity in Gluconobacter oxydans?

Post-translational modifications (PTMs) of rpoC likely play significant roles in regulating RNA polymerase activity in G. oxydans:

• Predicted PTM landscape:

• Methodological approaches for PTM characterization:

  • Mass spectrometry analysis of purified RNA polymerase

  • Phosphoproteomic analysis under different growth conditions

  • Site-directed mutagenesis of putative modification sites

  • Activity assays comparing native and recombinant (potentially lacking PTMs) enzyme

• Functional implications in G. oxydans metabolism:

  • Given G. oxydans' highly oxidative metabolism , PTMs may provide mechanisms to adapt transcription to varying oxygen tensions

  • PTMs could facilitate rapid switching between periplasmic and cytoplasmic glucose metabolism

  • The significantly different growth phenotypes observed in metabolically engineered strains may partly reflect altered PTM patterns on transcriptional machinery

What are the key differences in rifampicin binding sites between G. oxydans rpoC and clinically relevant bacterial species?

Rifampicin resistance is mediated primarily through mutations in the β subunit (rpoB), but the β' subunit (rpoC) contributes to the binding pocket and can harbor compensatory mutations. Key differences include:

• Binding pocket architecture comparison:

• Methodological approach for comparative analysis:

  • Homology modeling of G. oxydans RNA polymerase based on crystal structures

  • Molecular docking simulations with rifampicin

  • Alanine scanning mutagenesis of predicted interface residues

  • Minimum inhibitory concentration (MIC) determination for rifampicin derivatives

• Potential biotechnological applications:

  • Development of selective inhibitors for bacterial contamination control

  • Engineering rifampicin-resistant G. oxydans strains for specific industrial applications

  • Design of RNA polymerase variants with altered regulatory properties

How can rpoC variants be utilized to enhance production of industrially valuable compounds in engineered G. oxydans strains?

Strategic modifications to rpoC could be leveraged to enhance production of industrially valuable compounds through targeted transcriptional engineering:

• Production pathway optimization strategy:

• Integration with existing metabolic engineering approaches:

  • Combine with mgdH/sgdH deletion strategies that have demonstrated substantial growth improvements

  • Layer transcriptional engineering with pathway gene overexpression

  • Implement conditional expression systems for dynamic regulation

• Performance metrics and characterization:

  • Measure product titers, rates, and yields

  • Conduct transcriptomic and metabolomic analyses to verify expected expression changes

  • Compare growth characteristics with the established benchmarks (e.g., 271% improved growth yield in double deletion mutant)

  • Quantify carbon flux distribution through central metabolic pathways

This approach leverages fundamental understanding of rpoC function to design transcriptional landscapes optimized for specific biotechnological applications.

What methodological approaches can determine if rpoC mutations contribute to the improved growth phenotypes observed in engineered G. oxydans strains?

To determine if spontaneous rpoC mutations contribute to improved growth phenotypes in engineered strains:

• Comprehensive mutation screening protocol:

  • Whole genome sequencing of adapted strains showing enhanced growth

  • Targeted sequencing of rpoC in multiple independent adaptation lines

  • Computational prediction of functional effects of identified mutations

• Experimental validation strategy:

• Phenotypic characterization matrix:

  • Growth rates in different carbon sources

  • Metabolic flexibility assessment

  • Product formation profiles

  • Stress tolerance characteristics

  • Comparison to established improvements (39-78% growth rate improvement in dehydrogenase mutants)

This methodological framework would establish whether transcriptional machinery modifications contribute to the remarkable growth improvements observed in metabolically engineered G. oxydans strains, potentially revealing new principles for strain optimization.