Recombinant Bdellovibrio bacteriovorus DNA-directed RNA polymerase subunit beta' (rpoC), partial

What is the role of the rpoC gene in Bdellovibrio bacteriovorus?

The rpoC gene encodes the beta' subunit of DNA-directed RNA polymerase, which houses the active site that catalyzes RNA synthesis during transcription. The beta' subunit forms part of the RNA polymerase core enzyme composed of two α subunits, one β subunit, and one β' subunit. This enzyme is capable of transcriptional elongation but requires sigma factors for promoter recognition and DNA helix opening. In B. bacteriovorus, the RNA polymerase is essential for transcribing genes required during both attack phase and intraperiplasmic growth phase of its predatory lifecycle . The beta' subunit specifically contains the active site that forms the phosphodiester bonds during RNA synthesis and contributes to DNA binding and nucleotide substrate recognition.

How can I determine the optimal expression conditions for recombinant B. bacteriovorus rpoC?

Optimal expression of recombinant B. bacteriovorus rpoC requires careful optimization of several parameters:

Temperature and induction conditions:

• Lower temperatures (16-20°C) often improve solubility of large proteins like rpoC

• Reduced IPTG concentrations (0.1-0.5 mM) can minimize formation of inclusion bodies

• Extended induction times (16-24 hours) at lower temperatures may increase yield of soluble protein

Expression systems:

• E. coli BL21(DE3) or derivatives with improved folding capabilities (e.g., Rosetta for rare codons)

• Consider co-expression with chaperones (GroEL/GroES) to enhance folding

• T7-based expression systems with tunable promoter strength

Buffer optimization:

• Include stabilizing agents like glycerol (10-20%)

• Add divalent cations (Mg²⁺ at 5-10 mM) which are essential for RNA polymerase stability

• Include reducing agents (DTT at 1-5 mM) to prevent oxidation of cysteine residues

In a study with B. burgdorferi RNA polymerase, which shares similarities with B. bacteriovorus, researchers successfully tagged the rpoC gene with a polyhistidine tag by homologous recombination, allowing for efficient purification of the intact RNA polymerase complex .

What are the most effective strategies for cloning partial rpoC from B. bacteriovorus?

The most effective strategies for cloning partial rpoC from B. bacteriovorus involve:

• PCR amplification with optimized primers:

  • Design primers with appropriate restriction sites (EcoRI and KpnI have been successfully used)

  • Remove the stop codon if planning to add a C-terminal tag

  • Include ~20-25 bp of homology to the target sequence

• Vector selection:

  • For general cloning: pUC19 has been used successfully for B. bacteriovorus genes

  • For integration into B. bacteriovorus: pK18mobsacB is preferred as it contains both kanamycin resistance and a counterselection marker

  • For expression studies: vectors with appropriate promoters active in B. bacteriovorus

• Transformation approaches:

  • For E. coli: standard transformation protocols

  • For B. bacteriovorus: conjugation using E. coli S17-1 as donor strain

• Verification methods:

  • PCR screening with primers that bind to genomic DNA and vector sequences

  • Sequencing to confirm correct insertion and absence of mutations

  • Southern blotting to verify single insertion events

These strategies have been validated in studies manipulating other genes in B. bacteriovorus and can be adapted specifically for rpoC.

How can I design a construct for C-terminal tagging of rpoC in B. bacteriovorus?

Designing a construct for C-terminal tagging of rpoC in B. bacteriovorus requires careful consideration of several factors:

• Amplification of target gene:

  • Amplify the rpoC gene without its stop codon

  • Include restriction sites compatible with your chosen vector (EcoRI and KpnI have been used successfully)

  • For partial rpoC, ensure you include the functionally important regions

• Linker design:

  • Include a short linker sequence between rpoC and the tag

  • Previous studies with B. bacteriovorus proteins have used the amino acid sequence VQRSS as an effective linker

  • The linker minimizes steric hindrance between the protein and tag

• Tag selection:

  • For purification: 10x histidine tag has been successful for rpoC in related systems

  • For visualization: monomeric fluorescent proteins like mTFP work well in B. bacteriovorus

  • For protein interaction studies: smaller tags like FLAG or HA may be preferable

• Vector backbone:

  • pK18mobsacB is recommended as it contains:

    • Kanamycin resistance cassette for selection

    • sacB gene for counterselection during removal of vector backbone

    • Mobilization apparatus for conjugative transfer

• Integration strategy:

  • Design for homologous recombination at the native locus

  • Include at least 500-1000 bp of flanking sequences for efficient recombination

  • Consider preserving the native promoter to maintain physiological expression levels

This approach has been successfully used for tagging other B. bacteriovorus proteins and can be adapted for rpoC .

What is the most efficient purification protocol for His-tagged B. bacteriovorus rpoC?

The most efficient purification protocol for His-tagged B. bacteriovorus rpoC involves the following steps:

• Cell lysis and initial preparation:

  • Harvest cells at optimal density (typically mid-log phase)

  • Resuspend in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM MgCl₂, 10% glycerol, 5 mM β-mercaptoethanol, and protease inhibitors

  • Lyse cells using sonication or French press (8-10 cycles of 30s on/30s off for sonication)

  • Clear lysate by centrifugation at 30,000 × g for 30 minutes at 4°C

• Immobilized metal affinity chromatography (IMAC):

  • Equilibrate Ni-NTA resin with binding buffer (lysis buffer + 10 mM imidazole)

  • Incubate cleared lysate with resin for 1 hour at 4°C with gentle rotation

  • Wash extensively with washing buffer (lysis buffer + 20-30 mM imidazole)

  • Elute purified protein using elution buffer (lysis buffer + 250-300 mM imidazole)

  • Collect fractions and analyze by SDS-PAGE

• Secondary purification (if needed):

  • Ion exchange chromatography using MonoQ column

  • Size exclusion chromatography to separate monomeric from aggregated forms

  • Heparin affinity chromatography, which is particularly effective for DNA-binding proteins

• Final preparation:

  • Buffer exchange to remove imidazole (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 50% glycerol)

  • Concentrate to 1-5 mg/ml using appropriate molecular weight cut-off concentrators

  • Flash-freeze in liquid nitrogen and store at -80°C in small aliquots

This protocol has been adapted from successful purification of RNA polymerase from related bacterial systems, with specific modifications for B. bacteriovorus proteins .

What analytical methods should be used to verify the purity and activity of recombinant rpoC?

Multiple analytical methods should be employed to comprehensively verify the purity and activity of recombinant rpoC:

Purity assessment:

• SDS-PAGE analysis:

  • Expect a band at approximately 150-155 kDa for full-length rpoC

  • Silver staining for detection of minor contaminants

  • Densitometric analysis to quantify purity (>90% is typically considered acceptable)

• Western blotting:

  • Using anti-His antibodies for tagged constructs

  • Specific antibodies against rpoC if available

  • Confirms identity of the purified protein

• Mass spectrometry:

  • Peptide mass fingerprinting for identity confirmation

  • Intact mass analysis to verify complete translation

  • LC-MS/MS for identification of potential post-translational modifications

Activity assessment:

• Assembly into functional RNA polymerase:

  • Size exclusion chromatography to verify complex formation with other subunits

  • Native PAGE to analyze intact RNA polymerase complex

  • Analytical ultracentrifugation to determine stoichiometry

• In vitro transcription assay:

  • Template DNA containing B. bacteriovorus promoters

  • Analysis of RNA synthesis by gel electrophoresis or radioactive nucleotide incorporation

  • Quantitative measurement of transcription rates and processivity

• Promoter binding analysis:

  • Electrophoretic mobility shift assay (EMSA) with holoenzyme

  • DNase I footprinting to confirm specific promoter recognition

  • Surface plasmon resonance for binding kinetics

These methods collectively provide comprehensive verification of both purity and functional activity of the recombinant rpoC protein .

How can I establish an in vitro transcription system using recombinant B. bacteriovorus RNA polymerase?

Establishing an in vitro transcription system using recombinant B. bacteriovorus RNA polymerase requires optimization of multiple parameters:

Components required:

• Core RNA polymerase: Either purified from B. bacteriovorus or reconstituted from individually purified subunits (α, β, β', and ω)

• Sigma factors: Purified sigma factors (e.g., RpoD for housekeeping genes) to form holoenzyme

• DNA template: Supercoiled plasmid or linear DNA containing B. bacteriovorus promoters

Optimized reaction conditions:

• Buffer composition:

  • 40 mM Tris-HCl (pH 7.5-8.0)

  • 100-150 mM potassium glutamate or KCl

  • 10 mM MgCl₂ (essential cofactor)

  • 1 mM DTT (reducing agent)

  • 100 μg/ml BSA (stabilizer)

• Metal ion requirements:

  • Mg²⁺ (5-10 mM) is essential for catalytic activity

  • Mn²⁺ (0.1-1.0 mM) can enhance activity or alter specificity

  • Systematically test different concentrations to determine optimum

• Nucleotide substrates:

  • NTPs (ATP, GTP, CTP, UTP) at 0.5-1.0 mM each

  • For detection, include radiolabeled ([α-³²P]UTP) or fluorescently labeled nucleotides

• Temperature and pH:

  • Test temperature range of 25-37°C (30°C is often optimal)

  • pH range of 7.5-8.0 typically works best

Transcription assay procedure:

• Mix core RNA polymerase with sigma factor to form holoenzyme

• Add DNA template and incubate at optimal temperature for 5-10 minutes

• Initiate transcription by adding NTP mix

• Incubate for 15-60 minutes

• Stop reaction with formamide loading buffer (for gel analysis) or phenol extraction (for RNA recovery)

• Analyze transcripts by denaturing gel electrophoresis

This approach has been successfully employed for establishing in vitro transcription systems for other bacterial RNA polymerases and can be adapted specifically for B. bacteriovorus .

What are the unique characteristics of B. bacteriovorus rpoC compared to other bacterial species?

B. bacteriovorus rpoC exhibits several unique characteristics compared to other bacterial species, reflecting its adaptation to a predatory lifestyle:

Structural features:

• Insertion regions: Contains predator-specific insertion elements not found in non-predatory bacteria

• Surface charge distribution: Distinctive pattern that may facilitate interactions with predation-specific transcription factors

• Interdomain flexibility: Potentially greater flexibility to accommodate rapid transcriptional changes during prey invasion

Functional properties:

• Promoter recognition specificity: Recognizes unique promoters active during predatory lifecycle phases

• Response to regulatory signals: Heightened sensitivity to prey-derived signals or metabolites

• Lifecycle-specific activity: Differentially regulated during attack phase versus intraperiplasmic growth phase

Comparative analysis with E. coli RNA polymerase:

These unique characteristics make B. bacteriovorus rpoC an interesting subject for comparative studies and highlight its adaptation to the predatory lifestyle .

How does rpoC interact with different sigma factors during the predatory lifecycle of B. bacteriovorus?

The interaction between rpoC and different sigma factors orchestrates the complex transcriptional program during the predatory lifecycle of B. bacteriovorus:

Attack phase sigma factor interactions:

• RpoD (σ⁷⁰): The primary sigma factor during attack phase

  • Directs transcription of genes involved in motility, prey recognition, and attachment

  • Forms stable complex with RNA polymerase containing rpoC

  • Four robust promoters active during attack phase have been identified that interact with RpoD-containing RNA polymerase

• FliA (σ²⁸): Flagellar sigma factor

  • Critical for expression of flagellar genes needed for high-speed motility

  • Regulated by theophylline-responsive riboswitches in engineered strains

  • Previously predicted to be essential for predation

Intraperiplasmic phase sigma factor interactions:

• Specialized sigma factors: Likely upregulated after prey invasion

  • Direct expression of hydrolytic enzymes required for prey digestion

  • Coordinate with core RNA polymerase through rpoC to ensure timely expression

  • May respond to prey-derived signals or nutritional status

Regulatory mechanisms:

• Direct protein-protein interactions:

  • Specific regions in rpoC interact with conserved domains in sigma factors

  • These interactions determine promoter specificity and recognition

  • Affinity between rpoC and different sigma factors may shift during predation cycle

• Accessory factor modulation:

  • Additional proteins may regulate rpoC-sigma factor interactions

  • Anti-sigma factors potentially sequester specific sigma factors until needed

  • Predation-specific factors may enhance certain rpoC-sigma factor associations

Studies have shown that manipulating expression of FliA using synthetic riboswitches affects predation kinetics, indicating the importance of precisely regulated rpoC-sigma factor interactions during the predatory lifecycle .

How can recombinant rpoC be used to develop selective inhibitors against pathogenic bacteria?

Recombinant B. bacteriovorus rpoC can serve as a powerful tool for developing selective inhibitors against pathogenic bacteria through several strategic approaches:

Comparative structural analysis:

• Identification of structural differences:

  • Compare crystal or cryo-EM structures of B. bacteriovorus rpoC with pathogen rpoC

  • Map sequence divergence onto structural models to identify pathogen-specific regions

  • Focus on active site variations that could be exploited for selective inhibition

• Binding pocket characterization:

  • Analyze differences in rifampicin binding pocket between B. bacteriovorus and pathogens

  • Identify unique features in nucleotide binding sites or channel dimensions

  • Model the effects of potential inhibitors on different RNA polymerases

Differential inhibitor screening:

• High-throughput screening platform:

  • Develop parallel in vitro transcription assays with B. bacteriovorus and pathogen RNA polymerases

  • Screen compound libraries for differential inhibition profiles

  • Identify compounds that inhibit pathogen but not B. bacteriovorus RNA polymerase

• Quantitative inhibition analysis:

  • Determine IC₅₀ values for promising compounds against multiple RNA polymerases

  • Generate selectivity indices to prioritize compounds with highest specificity

  • Perform structure-activity relationship studies to enhance selectivity

This approach leverages the unique properties of B. bacteriovorus as a non-pathogenic predator of other bacteria, potentially leading to new antibacterial compounds that could be used alongside B. bacteriovorus in combination therapies .

What methods can be used to study the dynamics of RNA polymerase during the predatory lifecycle?

Several sophisticated methods can be employed to study the dynamics of RNA polymerase during the predatory lifecycle of B. bacteriovorus:

Live-cell imaging approaches:

• Fluorescent protein tagging:

  • Generate B. bacteriovorus strains expressing rpoC-mTFP or other fluorescent protein fusions

  • Previous studies have successfully created fluorescent protein fusions in B. bacteriovorus

  • Track RNA polymerase localization during different stages of predation

• Time-lapse microscopy:

  • Monitor changes in RNA polymerase distribution throughout predatory cycle

  • Correlate with morphological changes in both predator and prey

  • Quantify redistribution kinetics during transition between lifecycle phases

Molecular profiling techniques:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Map genome-wide RNA polymerase binding sites at different predation stages

  • Identify temporal changes in promoter occupancy

  • Correlate with transcriptomic data to understand regulatory mechanisms

• Nascent RNA sequencing:

  • NET-seq or GRO-seq to capture actively transcribing RNA polymerase

  • Profile transcription elongation rates during different predatory phases

  • Identify pausing sites and regulatory checkpoints

Biochemical approaches:

• Cross-linking mass spectrometry:

  • Capture RNA polymerase interaction partners at different lifecycle stages

  • Identify stage-specific transcription factors and regulators

  • Map interaction interfaces at amino acid resolution

• Protein turnover analysis:

  • Pulse-chase labeling to determine RNA polymerase subunit stability

  • Measure synthesis and degradation rates during predatory lifecycle

  • Track post-translational modifications that may regulate activity

These methods collectively provide comprehensive insights into how RNA polymerase function and regulation change during the complex predatory lifecycle of B. bacteriovorus .

How can I use recombinant rpoC to study transcriptional regulation during prey invasion?

Recombinant rpoC can be used as a powerful tool to study transcriptional regulation during prey invasion through several methodological approaches:

In vitro transcription systems:

• Reconstituted transcription assays:

  • Combine purified recombinant rpoC with other RNA polymerase subunits

  • Add different sigma factors to redirect promoter specificity

  • Test transcription from promoters activated during prey invasion

  • Analyze the effects of prey-derived factors or signals on transcription

• Coupled transcription-translation systems:

  • Develop B. bacteriovorus-specific cell-free systems using recombinant components

  • Monitor expression of prey invasion genes in controlled conditions

  • Test effects of different metabolites or signaling molecules

DNA-protein interaction studies:

• DNA binding assays:

  • Electrophoretic mobility shift assays with holoenzyme containing recombinant rpoC

  • DNase I footprinting to map exact binding sites

  • Surface plasmon resonance to measure binding kinetics to invasion-specific promoters

• Promoter mapping:

  • Use recombinant RNA polymerase to identify transcription start sites

  • Map promoter elements through systematic mutagenesis

  • Correlate promoter strength with temporal regulation during invasion

Regulatory network analysis:

• Reconstitution of regulatory circuits:

  • Combine recombinant RNA polymerase with purified transcription factors

  • Test hierarchical activation of invasion-related genes

  • Identify feedback loops and regulatory checkpoints

• Single-molecule approaches:

  • Visualize individual RNA polymerase molecules on DNA templates

  • Measure transcription initiation and elongation rates

  • Track the effects of regulatory proteins on polymerase activity

These approaches provide mechanistic insights into how B. bacteriovorus rapidly reprograms its transcriptome during prey invasion, a critical step in its predatory lifecycle .

What are the most common challenges when working with recombinant B. bacteriovorus rpoC and how can they be addressed?

Working with recombinant B. bacteriovorus rpoC presents several challenges that can be systematically addressed:

Expression challenges:

• Low solubility:

  • Issue: Formation of inclusion bodies during heterologous expression

  • Solution: Lower induction temperature (16-20°C), reduce IPTG concentration (0.1-0.5 mM)

  • Alternative: Use solubility-enhancing fusion tags (SUMO, MBP) or co-express with chaperones

• Proteolytic degradation:

  • Issue: Partial degradation of expressed rpoC

  • Solution: Use protease-deficient expression strains (like BL21(DE3) pLysS)

  • Alternative: Add protease inhibitors throughout purification and minimize processing time

Purification challenges:

• Co-purification of contaminants:

  • Issue: DNA/RNA contamination due to nucleic acid binding properties

  • Solution: Include high salt washes (500 mM NaCl) and DNase/RNase treatment

  • Alternative: Use heparin affinity chromatography as a polishing step

• Loss of activity:

  • Issue: Purified protein shows reduced or no transcriptional activity

  • Solution: Include stabilizing agents (glycerol, reducing agents) in all buffers

  • Alternative: Co-purify with other RNA polymerase subunits to maintain complex integrity

Functional analysis challenges:

• Non-specific transcription:

  • Issue: High background in in vitro transcription assays

  • Solution: Optimize salt concentration (100-200 mM KCl) and template purity

  • Alternative: Include competing non-specific DNA (poly dI-dC) in reaction

• Poor reproducibility:

  • Issue: Variable activity between protein preparations

  • Solution: Standardize purification protocol and establish activity assays

  • Alternative: Pool multiple preparations for consistency in long-term studies

These solutions have been adapted from successful approaches used with RNA polymerases from other bacterial systems and tailored to address the specific challenges of B. bacteriovorus proteins .

How can I optimize in vitro transcription conditions for studying predation-specific promoters?

Optimizing in vitro transcription conditions for studying predation-specific promoters requires systematic adjustment of multiple parameters:

Template optimization:

• Promoter selection:

  • Use well-characterized promoters known to be active during predation

  • Include ~100 bp upstream and ~50 bp downstream of transcription start site

  • Consider using supercoiled templates which often yield higher activity

• Template preparation:

  • Ensure high purity (free from inhibitory contaminants)

  • Verify sequence integrity, especially in the -35 and -10 regions

  • Test both linear and supercoiled templates to determine optimal format

Reaction conditions optimization:

• Buffer components:

  • Systematically vary pH (7.0-8.5) in 0.5 unit increments

  • Test different buffer systems (Tris-HCl, HEPES, Phosphate)

  • Optimize monovalent salt concentration (50-200 mM KCl or K-glutamate)

• Divalent metal ions:

  • Test Mg²⁺ concentrations (5-15 mM)

  • Evaluate Mn²⁺ addition (0.1-1.0 mM) which can alter promoter specificity

  • Consider the effect of other divalent cations (Ca²⁺, Zn²⁺)

Enzyme components:

• Sigma factor selection:

  • Identify sigma factors active during different predatory phases

  • Use purified recombinant sigma factors at various concentrations

  • Test combinations of sigma factors if relevant

• Core enzyme:sigma factor ratio:

  • Typically 1:4 to 1:10 molar ratio ensures maximal holoenzyme formation

  • Perform titration experiments to determine optimal ratio

  • Pre-form holoenzyme by incubation before adding template

Optimization strategy:

• Start with standard conditions and vary one parameter at a time

• Use a model promoter with known activity as positive control

• Establish quantitative readout (e.g., radioactive incorporation or fluorescent signal)

• Document all conditions systematically in a laboratory database

This methodical approach will identify optimal conditions for studying predation-specific promoters and provide reproducible results for mechanistic studies .

What bioinformatic approaches can help analyze the evolutionary adaptations in B. bacteriovorus rpoC?

Several sophisticated bioinformatic approaches can reveal evolutionary adaptations in B. bacteriovorus rpoC:

Sequence-based analyses:

• Comparative genomics:

  • Align rpoC sequences from diverse bacterial species including predators and non-predators

  • Identify predator-specific insertions, deletions, or amino acid substitutions

  • Calculate selection pressures (dN/dS ratios) to identify regions under positive selection

• Phylogenetic profiling:

  • Construct phylogenetic trees of rpoC across bacterial species

  • Map predatory lifestyle onto phylogeny to identify convergent evolution

  • Trace the acquisition of predation-specific features across evolutionary history

Structure-based analyses:

• Homology modeling:

  • Generate structural models of B. bacteriovorus rpoC based on crystal structures from other bacteria

  • Compare with non-predatory bacterial RNA polymerases to identify structural adaptations

  • Map sequence variations onto structural models to assess functional implications

• Molecular dynamics simulations:

  • Simulate behavior of B. bacteriovorus RNA polymerase under different conditions

  • Analyze conformational flexibility and domain movements

  • Identify structural adaptations that may contribute to predatory lifestyle

Functional prediction:

• Protein-protein interaction analysis:

  • Predict interaction interfaces with sigma factors and transcription regulators

  • Identify predator-specific interaction motifs

  • Compare interaction networks between predatory and non-predatory bacteria

• Promoter recognition analysis:

  • Analyze DNA-binding regions for adaptations in promoter recognition

  • Predict promoter specificity based on sequence and structural features

  • Identify potential binding sites for predation-specific regulatory factors

• Co-evolution analysis:

  • Identify co-evolving residues within rpoC and between rpoC and interacting partners

  • Detect networks of functionally related amino acids

  • Trace coordinated evolutionary changes associated with predatory adaptation

These bioinformatic approaches provide valuable insights into how B. bacteriovorus rpoC has evolved specialized features to support its unique predatory lifestyle .