Recombinant Haemophilus influenzae Fumarate reductase subunit C (frdC)

Basic Research Questions

• What is Fumarate Reductase Subunit C (frdC) in Haemophilus influenzae and what is its function?

  Fumarate Reductase Subunit C (frdC) in Haemophilus influenzae is a 132-amino acid membrane protein that serves as an anchor component of the fumarate reductase complex. It appears to be primarily involved in anchoring the catalytic components of the fumarate reductase complex to the cytoplasmic membrane . The full amino acid sequence of H. influenzae frdC is: MSKRKKYVRPMTATWWQKLDFYKAYMLREATSVFAVWFCIVLLYGVLCFASNPMPGLGILSFIEFLRNPIVVFLNIITLIATLYHTVTYFLMTPKVMNIIVKNERLPHTVVRNALWAVTALVSVIALVLVYI . This membrane-bound protein is part of the FrdC protein family and plays a crucial role in the organism's respiratory and metabolic processes.

• How is the Fumarate Reductase complex structured in H. influenzae?

  While the exact structure of H. influenzae's Fumarate Reductase complex hasn't been fully characterized in the provided literature, comparative genomics suggests similarity to other bacterial systems. The complex likely contains three primary subunits: FrdC, FrdA, and FrdB, similar to the arrangement observed in Campylobacter jejuni . Based on studies of related bacterial systems, FrdC (Cj0408 in C. jejuni) functions as the membrane anchor and may contain diheme cytochrome b, FrdA (Cj0409) is the flavoprotein where the reduction of fumarate to succinate occurs, and FrdB (Cj0410) contains iron-sulfur clusters essential for electron transfer . The gene order in H. influenzae's frd operon appears to be conserved with that of Wolinella succinogenes and Helicobacter pylori, suggesting functional and structural similarities in the enzyme complex .

• What expression systems are used for recombinant H. influenzae frdC production?

  Recombinant H. influenzae frdC is typically expressed in E. coli expression systems. Based on commercial production approaches, the recombinant protein is often produced with an N-terminal His tag to facilitate purification . The full-length protein (amino acids 1-132) has been successfully expressed, suggesting that the entire protein can be produced in a heterologous system without significant toxicity issues that are sometimes encountered with membrane proteins . The expression of this recombinant protein allows researchers to study its properties independent of other fumarate reductase components and provides a tool for investigating its specific functions.

Experimental Methodologies

• How can the activity of fumarate reductase be measured in experimental systems?

  Fumarate reductase activity can be measured through spectrophotometric assays that monitor electron transfer. A specific methodology employed for related enzymes involves:

  1. Preparing reaction mixtures in 1-ml quartz cuvettes containing:

     • 75 mM sodium phosphate buffer (pH 6.8)

     • 0.2 mM benzyl viologen (as an electron donor)

     • 1-5 μg of cell extract containing the enzyme

  2. Under anaerobic conditions (N₂ gas flushing), adding freshly prepared 20 mM sodium dithionite until the absorbance at 585 nm reaches 0.8-0.9, representing half-reduced benzyl viologen

  3. Adding an anaerobic solution of sodium fumarate (5 mM final concentration)

  4. Measuring the oxidation kinetics of benzyl viologen spectrophotometrically at 585 nm

  Activity is typically expressed as nmol of benzyl viologen oxidized min⁻¹ mg⁻¹ of protein, using the extinction coefficient of 8.65 cm⁻¹ mM⁻¹ for benzyl viologen .

• What purification strategies are recommended for His-tagged recombinant frdC?

  For His-tagged recombinant frdC purification, the following protocol is recommended:

  1. Initial preparation: Briefly centrifuge the expression culture to pellet cells

  2. Cell lysis: Use appropriate buffer systems containing mild detergents to solubilize the membrane protein without denaturing it

  3. Affinity chromatography: Purify using Ni-NTA or similar metal affinity resins that bind the His-tag

  4. Storage preparation: Based on commercial preparations, the purified protein is often stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  5. Long-term storage: Lyophilization or storage with 5-50% glycerol at -20°C/-80°C, with aliquoting recommended to avoid repeated freeze-thaw cycles

  The purified protein should exhibit greater than 90% purity as determined by SDS-PAGE analysis .

Genetic and Molecular Analysis

• How can the frdC gene be cloned and manipulated for research purposes?

  For cloning and manipulation of the frdC gene from H. influenzae, researchers can employ the following approach:

  1. PCR amplification: Design primers flanking the complete frdC coding sequence (396 bp encoding 132 amino acids) and amplify from H. influenzae chromosomal DNA using high-fidelity polymerase

  2. Cloning options:

     • For basic cloning, insert the PCR product into pCR2.1-TOPO vectors or similar general-purpose vectors

     • For expression, clone into appropriate expression vectors with His-tag or other affinity tags

  3. Mutagenesis approaches:

     • For disruption studies, the gene can be interrupted with antibiotic resistance markers such as chloramphenicol resistance genes

     • For site-directed mutagenesis, use appropriate restriction sites or PCR-based mutagenesis techniques

  4. Verification: Confirm constructs by restriction analysis and DNA sequencing before proceeding with expression or transformation

  5. Transformation: For reintroducing manipulated genes into H. influenzae, use competent cell preparation and transformation protocols specific to this organism

• What transformation techniques are effective for introducing recombinant frdC DNA into H. influenzae?

  Effective transformation of H. influenzae with recombinant DNA requires attention to several key factors:

  1. Competent cell preparation: Use MIV medium to induce competence in H. influenzae cells

  2. DNA uptake considerations: H. influenzae preferentially takes up DNA containing specific eleven base-pair "uptake sequences" of which approximately 600 copies are distributed throughout the H. influenzae genome

  3. Transformation protocol:

     • Incubate competent cells with DNA (1-2 μg) for 15-30 minutes at 37°C

     • Plate on appropriate selective media depending on the marker used

     • Incubate plates at 37°C for 16-24 hours (48 hours if selecting for chloramphenicol resistance)

  4. Recombination considerations: Be aware that the rec-1 gene function is necessary for obtaining higher transformation frequencies with recombinant DNA

  5. Efficiency measurement: Calculate transformation efficiency by dividing the number of transformants per ml by the amount of DNA used, or transformation frequency by dividing transformants per ml by the total number of cells per ml

Advanced Research Applications