Recombinant Pseudomonas stutzeri Protein CrcB homolog (crcB)

What is the function of CrcB protein in Pseudomonas stutzeri?

CrcB in P. stutzeri functions as a camphor resistance protein that is part of the bacterial defense system. While initially characterized for camphor resistance, recent studies suggest it may play a broader role in ion transport and resistance to environmental stresses. The protein belongs to a highly conserved family found in 440 different genera, indicating its fundamental importance across bacterial species . The conservation pattern suggests it plays a crucial role in bacterial survival under various environmental conditions.

How is the crcB gene organized in the Pseudomonas stutzeri genome?

The crcB gene in P. stutzeri A1501 (locus tag PST_2290) is located on the negative strand of the chromosome at position 2499937-2500311. It is not part of any known operons, suggesting independent regulation . Unlike other regulatory elements such as the carbon catabolite repression system in P. stutzeri (which includes genes like crcZ, hfq, rpoN, crc, and crcY that belong to different clusters), crcB appears to function independently . This genomic organization differs from some other resistance genes in P. stutzeri that are often clustered together.

What is the evolutionary relationship of CrcB across Pseudomonas species?

CrcB belongs to a highly conserved protein family (POG016646) with 224 members across various Pseudomonas species . Comparative genomic analyses reveal that the P. stutzeri complex can be divided into 27 genomovars (likely representing distinct species to some extent), comprising 16 known and 11 unknown genomovars . The crcB gene is part of the core genome rather than the accessory genome, suggesting its essential role in bacterial physiology. Phylogenetic analysis indicates that CrcB has been maintained through extensive genetic gain and loss events that have driven diversification within the P. stutzeri species complex .

What are the optimal conditions for expressing recombinant CrcB in Pseudomonas stutzeri?

For optimal expression of recombinant CrcB in P. stutzeri, the pL2020 expression vector system has shown high success rates. This system utilizes an L-arabinose inducible promoter with the following recommended conditions:

Studies indicate that P. stutzeri can properly fold certain proteins that form inclusion bodies in E. coli, potentially making it an excellent host for CrcB expression if folding issues are encountered .

How can I optimize purification of recombinant CrcB from Pseudomonas stutzeri?

Purification of recombinant CrcB from P. stutzeri can be achieved using the following optimized protocol based on successful purification of other recombinant proteins from this organism:

• Membrane preparation: Harvest cells and lyse using a French press (15,000 psi) in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and protease inhibitors.

• Solubilization: Due to CrcB's hydrophobicity value (1.068), mild detergents like n-dodecyl β-D-maltoside (β-DDM) at 1% concentration are recommended for efficient extraction from membranes .

• Affinity chromatography: Use Ni-NTA affinity chromatography with the following buffers:

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% β-DDM, 20 mM imidazole

  • Wash buffer: Same as binding buffer with 50 mM imidazole

  • Elution buffer: Same as binding buffer with 250 mM imidazole

• Size exclusion chromatography: Further purify using Superdex 200 in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.05% β-DDM .

For optimal results, maintain all buffers at 4°C throughout the purification process and include 1 mM DTT if the protein contains cysteine residues.

What are the advantages of using Pseudomonas stutzeri over E. coli for CrcB expression?

P. stutzeri offers several advantages over E. coli for recombinant protein expression, particularly for challenging membrane proteins:

Research shows that some proteins that form aggregates in E. coli maintain proper folding in P. stutzeri, potentially making it an excellent alternative host for challenging proteins like CrcB .

How can I assess the functional activity of recombinant CrcB?

Functional assessment of recombinant CrcB can be accomplished through multiple complementary approaches:

• Camphor resistance assay: As CrcB is annotated as a camphor resistance protein, measuring growth inhibition in the presence of increasing camphor concentrations can provide functional validation. Compare wild-type, crcB deletion mutant, and complemented strains to determine the protective effect of CrcB.

• Ion flux measurements: Recent studies suggest CrcB may function in ion transport. Use fluorescent ion indicators or electrophysiology to measure changes in ion flux (particularly fluoride) across membranes in response to CrcB expression.

• Protein-protein interaction studies: Employ microscale thermophoresis (MST) to identify potential binding partners. This technique has been successfully used with other P. stutzeri proteins, such as Hfq and Crc .

• Structural analysis: Circular dichroism (CD) spectroscopy can determine secondary structure elements, while electron paramagnetic resonance (EPR) can investigate potential metal binding, as demonstrated with other P. stutzeri proteins .

• Subcellular localization: Use GFP fusion constructs to determine the cellular localization of CrcB, providing insights into its potential function .

What experimental approaches can determine CrcB's role in metal resistance?

Several experimental approaches can elucidate CrcB's potential role in metal resistance:

• Metal sensitivity assays: Compare growth of wild-type, ΔcrcB mutant, and complemented strains in media containing various concentrations of metal ions (Cu²⁺, Zn²⁺, etc.). Determine minimal inhibitory concentrations (MICs) as shown in this table adapted from P. stutzeri antibiotic resistance studies:

• Transporter activity: Use radioactive or fluorescently labeled metal ions to measure transport across membranes in proteoliposomes containing purified CrcB.

• Mutant fitness analysis: Similar to approaches used for other P. stutzeri metal resistance genes, measure fitness values (w) of ΔcrcB mutants under metal stress conditions. Negative fitness values would indicate importance for metal resistance .

• Gene expression analysis: Use droplet digital PCR (ddPCR) to measure crcB expression levels under various metal stress conditions, as this technique has been successfully applied to other P. stutzeri genes .

How does CrcB interact with other stress response systems in Pseudomonas stutzeri?

CrcB may interact with several stress response systems in P. stutzeri:

• Oxidative stress response: Investigate potential interactions with the oxidative stress response pathway, which in P. stutzeri involves proteins like KatB (catalase) and the regulatory ncRNA NfiS. Measure expression levels of crcB in response to H₂O₂ exposure and compare oxidative stress resistance between wild-type and ΔcrcB strains .

• Carbon catabolite repression: Examine whether crcB is subject to carbon catabolite repression, which in P. stutzeri is mediated by Hfq and Crc proteins. Measure crcB expression levels during growth on different carbon sources .

• Nitrogen fixation: As P. stutzeri is a nitrogen-fixing bacterium, investigate if crcB expression changes under nitrogen-fixing conditions or if nitrogen fixation efficiency is affected in ΔcrcB mutants .

• Denitrification pathway: Determine if crcB expression is affected by growth under denitrifying conditions by measuring transcript levels using ddPCR in media with different nitrogen sources (NH₄Cl, NaNO₂, NaNO₃) .

• Metal homeostasis: Investigate potential interactions with known metal resistance systems in P. stutzeri, such as the CzcCBA transporter system for zinc resistance .

How conserved is CrcB across different Pseudomonas stutzeri genomovars?

CrcB is highly conserved across the P. stutzeri complex, which comprises 27 genomovars as identified by comparative genomics studies. Analysis shows:

• The crcB gene is part of the core genome rather than the accessory genome within the P. stutzeri complex .

• ANI (Average Nucleotide Identity) and dDDH (digital DNA-DNA hybridization) analyses consistently support the division of P. stutzeri strains into 27 genomovars (16 known and 11 unknown), all of which contain crcB homologs .

• The conservation of crcB suggests its fundamental importance for P. stutzeri physiology, despite the extensive genetic gain and loss events that have driven diversification within this species complex.

• Protein sequence identity of CrcB typically ranges from 95-100% within genomovars and 85-95% between different genomovars, based on patterns observed with other highly conserved proteins in P. stutzeri.

• Unlike some other resistance genes that show evidence of horizontal gene transfer, crcB appears to have evolved primarily through vertical inheritance within the P. stutzeri complex.

What structural features distinguish CrcB from other ion transport proteins?

While specific structural data for P. stutzeri CrcB is limited, comparative analysis with homologous proteins reveals several distinguishing features:

• Transmembrane topology: CrcB contains multiple predicted transmembrane helices, consistent with its role as a membrane protein involved in transport.

• Conserved domains: The protein contains the characteristic CrcB domain (Pfam: PF02537), which is associated with camphor resistance and potentially fluoride ion transport.

• Structural modeling: Using the crystal structure of related proteins as templates, homology modeling suggests that CrcB forms a dual-topology membrane protein that assembles as a homodimer.

• Active site residues: Key conserved residues likely form the ion selectivity filter, distinguishing CrcB from other transporters.

• Oligomeric state: Unlike many other transporters that function as monomers or heteromultimers, CrcB likely functions as a homodimer or homotetramer.

Experimental approaches including circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy could further elucidate these structural features, as they have been successfully employed with other P. stutzeri proteins .

How does the genetic context of crcB vary across different bacterial species?

The genetic context of crcB varies significantly across bacterial species, providing insights into its functional associations:

• In P. stutzeri A1501, crcB (PST_2290) is located on the negative strand at position 2499937-2500311 of the chromosome .

• Unlike some resistance genes that are organized in operons (such as the czcICBA zinc resistance operon in P. stutzeri), crcB appears to be independently transcribed in most Pseudomonas species .

• Comparative genomics across the 9,548 analyzed Pseudomonas genomes reveals varying genetic neighborhoods for crcB, suggesting different regulatory mechanisms across species .

• In some bacterial species, crcB is found in association with genes involved in fluoride resistance, supporting its proposed role in ion transport.

• The presence of crcB in the genome of both free-living and host-associated bacteria suggests its fundamental importance for bacterial physiology across diverse ecological niches.

Understanding this genetic context variability is crucial for interpreting the function and regulation of crcB in different species and for designing genetic manipulation experiments.

What gene deletion strategies are most effective for creating crcB knockout mutants in Pseudomonas stutzeri?

Several effective strategies for creating crcB knockout mutants in P. stutzeri have been developed:

• pK18 mobsacB system: This system has been successfully used for gene deletion in P. stutzeri A1501. The approach uses:

  • Homologous arms upstream and downstream of the target gene

  • Fusion of these fragments via recombinant PCR

  • Cloning into pK18 mobsacB vector

  • Tri-parental mating for plasmid transfer

  • Selection of double-crossover events using sucrose resistance

• Marker exchange method: This approach uses:

  • Construction of an unstable marker exchange plasmid

  • Assembly of PCR products via sequence- and ligation-independent cloning (SLIC)

  • Selection with appropriate antibiotics (kanamycin 50 μg/ml)

  • Screening for spectinomycin sensitivity (100 μg/ml) to identify true double-crossover events

• CRISPR-Cas9 system: Recently adapted for use in P. stutzeri, this approach offers:

  • Precise, marker-free deletions

  • Higher efficiency than traditional methods

  • Ability to create multiple knockouts simultaneously

For all approaches, confirmation of gene deletion should be performed using both PCR and Southern blotting to ensure complete removal of the target gene.

How can I determine the subcellular localization of CrcB in Pseudomonas stutzeri?

Multiple complementary approaches can determine the subcellular localization of CrcB:

• GFP fusion constructs: Create N- and C-terminal GFP fusions with CrcB and observe localization using fluorescence microscopy. This approach has been successful for other P. stutzeri membrane proteins . Consider the following:

  • Both N- and C-terminal fusions should be tested as one orientation may disrupt localization

  • Expression levels should be carefully controlled using varying concentrations of inducer (0.2%, 0.02%, 0.002%, and 0.0002% L-arabinose)

  • In-gel fluorescence can distinguish between properly folded (fluorescent) and misfolded (non-fluorescent) protein

• Subcellular fractionation: Separate bacterial cell components (cytoplasm, membrane, periplasm) and detect CrcB using immunoblotting with specific antibodies.

• Immunogold electron microscopy: Use specific antibodies against CrcB followed by gold-conjugated secondary antibodies to visualize the protein at ultrastructural resolution.

• Protease accessibility assays: Determine the topology of membrane-embedded CrcB by assessing protease sensitivity of different protein regions.

• Computational prediction: Tools such as TMHMM, HMMTOP, and Phobius can predict membrane protein topology based on the amino acid sequence, providing initial insights into localization.

What are the best methods for studying protein-protein interactions involving CrcB in Pseudomonas stutzeri?

Several complementary methods can effectively study protein-protein interactions involving CrcB:

• Bacterial two-hybrid system: Adapted for membrane proteins, this system can identify interaction partners in vivo. Consider using the BACTH system which has been successfully applied to other bacterial membrane proteins.

• Microscale thermophoresis (MST): This technique has been successfully used with other P. stutzeri proteins like Hfq and can detect interactions with high sensitivity in solution . Benefits include:

  • Small sample requirements (nM-μM concentrations)

  • Ability to determine binding affinity (Kd values)

  • Compatibility with detergent-solubilized membrane proteins

• Co-immunoprecipitation (Co-IP): Using specific antibodies against CrcB or an epitope tag, pull down the protein complex and identify interaction partners by mass spectrometry.

• Cross-linking coupled with mass spectrometry: Chemical cross-linking can capture transient interactions, followed by identification of cross-linked peptides using MS. This approach has been successful in identifying interaction networks in P. stutzeri .

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics between purified CrcB and potential interaction partners.

• Split-GFP complementation: This technique can visualize protein interactions in living cells and has been adapted for membrane proteins.

For all these methods, appropriate controls should be included to distinguish specific from non-specific interactions, particularly important for membrane proteins that may aggregate in solution.

How can CrcB be used as a model system for studying protein evolution across the Pseudomonas genus?

CrcB represents an excellent model system for studying protein evolution due to several characteristics:

• High conservation: As a member of protein family POG016646 with 224 members across Pseudomonas species, CrcB shows sufficient conservation to track evolutionary relationships .

• Genomic context variation: The varying genetic neighborhoods of crcB across species provide insights into the evolution of gene organization and regulation.

• Functional adaptation: Comparing CrcB function across species inhabiting different ecological niches can reveal how protein function adapts to environmental pressures.

• Experimental system advantages: P. stutzeri can be readily manipulated genetically, allowing experimental validation of evolutionary hypotheses through:

  • Complementation experiments across species

  • Creation of chimeric proteins

  • Site-directed mutagenesis of conserved residues

• Practical methodology: Cross-species complementation experiments can be conducted using the pL2020 expression vector, which functions in multiple Pseudomonas species, to determine functional conservation .

This research can contribute to understanding how proteins evolve new functions while maintaining core activities, particularly important for proteins involved in environmental stress responses.

What is the relationship between CrcB and bacterial response to fluoride toxicity?

Recent research suggests CrcB may play a crucial role in fluoride resistance:

• Putative function: While annotated as a camphor resistance protein in P. stutzeri, CrcB homologs in other bacteria have been implicated in fluoride export.

• Mechanism hypothesis: CrcB likely functions as a fluoride ion channel or transporter, expelling toxic fluoride ions from the cell cytoplasm.

• Experimental approach: To investigate this function:

  • Measure growth of wild-type and ΔcrcB strains in media containing various fluoride concentrations

  • Use fluoride-specific electrodes to measure intracellular fluoride accumulation

  • Conduct fluoride efflux assays with membrane vesicles containing purified CrcB

• Regulatory context: Determine if crcB expression is induced by fluoride exposure using quantitative RT-PCR or RNA-seq approaches.

• Structural basis: Homology modeling suggests CrcB forms a dual-topology membrane protein with a central fluoride-selective pore, though experimental validation in P. stutzeri is needed.

This research direction could establish CrcB as a model system for studying bacterial resistance to naturally occurring antimicrobial compounds like fluoride.