Recombinant Rhodospirillum centenum Protein CrcB homolog (crcB)

What is the Rhodospirillum centenum CrcB homolog protein and what is its primary function?

The CrcB homolog protein in Rhodospirillum centenum is part of a family of membrane proteins that have been identified in various bacterial species. Based on comparative genomic studies with other bacterial CrcB proteins, it likely functions as a fluoride ion channel/transporter that helps protect bacteria from fluoride toxicity. While not extensively characterized in R. centenum specifically, similar proteins such as the CrcB homolog in Bacillus cereus (as shown in search result ) consist of transmembrane domains that form channels for ion transport across bacterial membranes .

R. centenum is primarily studied for its photosynthetic capabilities and phototactic responses. The bacterium exhibits both scotophobic (movement away from darkness) responses in liquid media and phototactic colony movement on solid media, allowing for genetic screening of photosensory mutants . While CrcB is not directly implicated in these photo-responses based on available data, understanding its role may contribute to our comprehensive knowledge of R. centenum membrane biology.

How does Rhodospirillum centenum CrcB homolog compare to other bacterial CrcB proteins?

Bacterial CrcB proteins share conserved structural features across species. For comparison, the Bacillus cereus CrcB homolog 2 (crcB2) consists of 118 amino acids with a sequence of "MIEALLVATGGFFGAITRFAISNWFKKRNKTSFPIATFLINITGAFLLGYIIGSGVTTGWQLLLGTGFMGAFTTFSTFKLESVQLLNRKNFSTFLLYLSATYIVGILFAFLGMQLGGI" . Similarly, the Mycobacterium tuberculosis CrcB homolog 1 (Rv3069) consists of 132 amino acids .

The following table outlines key differences between known CrcB homologs:

While specific R. centenum CrcB sequence information is not provided in the search results, comparative genomic approaches would suggest structural and functional similarity to these other bacterial CrcB proteins.

What expression systems are optimal for producing recombinant R. centenum proteins?

Based on established protocols for other recombinant bacterial proteins, Escherichia coli remains the most widely used expression system for recombinant bacterial membrane proteins like CrcB. The search results indicate that for the Bacillus cereus CrcB homolog, E. coli was successfully used as an expression host with an N-terminal His tag fusion .

For R. centenum proteins specifically, genetic manipulation systems have been established that could potentially be adapted for recombinant protein production. The genetic systems described in search result include methods for generating mutations through ethyl methanesulfonate mutagenesis and Tn5 transposition mutagenesis using an IncP plasmid vector with a temperature-sensitive origin of replication . These established genetic manipulation techniques suggest that corresponding expression systems could be developed.

When expressing R. centenum membrane proteins like CrcB homologs, researchers should consider:

• Using low-copy number vectors to prevent toxicity

• Induction at lower temperatures (16-25°C) to improve proper folding

• Inclusion of appropriate chaperones to enhance proper membrane insertion

• Selection of suitable detergents for membrane protein extraction and purification

What are the optimal methods for site-directed mutagenesis of R. centenum CrcB homolog for structure-function studies?

For structure-function studies of R. centenum CrcB homolog, researchers can apply methodologies similar to those used for the ptr gene as described in search result . A comprehensive approach would include:

PCR-Based Site-Directed Mutagenesis:

• Design primers containing the desired mutation with 15-20 nucleotides of complementary sequence on either side

• Perform PCR amplification using a high-fidelity DNA polymerase

• Digest the parental template with DpnI (specific for methylated DNA)

• Transform the PCR product into a suitable E. coli strain

• Verify mutations by DNA sequencing

Chromosomal Gene Replacement:
The search results describe a method for targeted chromosomal disruption in R. centenum that could be adapted for CrcB studies. This involves:

• Construction of a deletion or mutation in the target gene using a suicide vector to promote allelic replacement through recombination

• Selection of transformed cells using appropriate antibiotic markers

• Confirmation of mutations using PCR analysis with relevant primers

For the ptr gene in R. centenum, researchers used pGmLacZ (described in ) as a suicide vector system, which could potentially be adapted for CrcB mutagenesis. Selection was performed on CENS plates with kanamycin, spectinomycin, and X-Gal as an indicator dye .

What assays can be used to evaluate the fluoride transport function of recombinant R. centenum CrcB homolog?

Based on the putative function of CrcB as a fluoride transporter (as indicated for other bacterial CrcB homologs ), several approaches can be implemented:

Fluoride Ion-Selective Electrode Measurements:

• Express the recombinant CrcB in liposomes or bacterial membrane vesicles

• Monitor fluoride ion flux across membranes using a fluoride-selective electrode

• Compare transport rates between wild-type and mutant versions of the protein

Fluorescence-Based Transport Assays:

• Load bacterial cells or proteoliposomes with fluoride-sensitive fluorescent dyes

• Monitor changes in fluorescence intensity upon addition of fluoride ions

• Calculate transport kinetics based on fluorescence changes

Growth Inhibition Assays:

• Express CrcB variants in CrcB-knockout bacterial strains

• Assess growth in media containing varying concentrations of NaF

• Determine the minimum inhibitory concentration of fluoride for each variant

• Compare with appropriate controls (vector-only, wild-type CrcB)

These functional assays should be complemented with protein localization studies using fluorescent protein fusions or immunolocalization to confirm membrane integration.

How can protein-protein interactions of R. centenum CrcB homolog be investigated?

To investigate protein-protein interactions of R. centenum CrcB homolog:

Co-Immunoprecipitation (Co-IP):

• Express epitope-tagged CrcB in R. centenum or a heterologous host

• Solubilize membranes using mild detergents (e.g., n-Dodecyl β-D-maltoside)

• Perform immunoprecipitation using antibodies against the epitope tag

• Identify co-precipitated proteins by mass spectrometry

Bacterial Two-Hybrid Systems:

• Clone CrcB homolog into appropriate bacterial two-hybrid vectors

• Co-transform with a library of R. centenum proteins or candidate interactors

• Screen for positive interactions based on reporter gene expression

• Confirm interactions using orthogonal methods

Chemical Cross-Linking Coupled with Mass Spectrometry:

• Treat intact cells or isolated membranes containing CrcB with membrane-permeable cross-linkers

• Isolate CrcB using affinity purification

• Analyze cross-linked protein complexes by LC-MS/MS

• Identify cross-linked peptides to map interaction interfaces

When investigating membrane protein interactions like CrcB, it's critical to maintain native-like membrane environments using appropriate detergents or nanodiscs to preserve physiologically relevant interactions.

What are common challenges in expressing and purifying recombinant R. centenum CrcB homolog?

Membrane proteins like CrcB present several challenges during recombinant expression and purification:

Expression Challenges and Solutions:

• Toxicity to host cells: Use tightly controlled inducible promoters and lower growth temperatures (16-25°C)

• Improper membrane insertion: Co-express with appropriate chaperones and optimize signal sequences

• Protein aggregation: Include osmolytes (glycerol, trehalose) in growth media and buffers

Purification Considerations:
Based on approaches used for the Bacillus cereus CrcB homolog , consider:

• N-terminal His-tag for affinity purification

• Storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Reconstitution in deionized sterile water to 0.1-1.0 mg/mL

• Addition of 5-50% glycerol for long-term storage at -20°C/-80°C

Quality Control Metrics:

• SDS-PAGE analysis for purity assessment (aim for >90% purity)

• Western blotting for identity confirmation

• Size-exclusion chromatography for aggregation assessment

• Circular dichroism for secondary structure verification

For CrcB homologs specifically, avoid repeated freeze-thaw cycles as this can compromise protein integrity and function .

How can issues with protein misfolding of recombinant R. centenum CrcB homolog be addressed?

Addressing protein misfolding requires multiple strategies:

Optimization of Expression Conditions:

• Reduce expression rate by lowering inducer concentration and temperature

• Test multiple E. coli strains (BL21(DE3), C41(DE3), C43(DE3), Lemo21(DE3))

• Co-express with molecular chaperones (GroEL/ES, DnaK/J/GrpE)

Membrane Mimetic Environments:

• Optimize detergent selection through systematic screening (DDM, LMNG, CHAPS)

• Consider native-like environments (nanodiscs, liposomes, amphipols)

• Include lipids that match R. centenum membrane composition

Refolding Strategies:

• Purify inclusion bodies under denaturing conditions

• Perform step-wise dialysis to remove denaturants

• Add lipids during refolding to facilitate proper membrane insertion

Validation of Proper Folding:

• Functional assays (ion transport activity)

• Biophysical characterization (CD spectroscopy, fluorescence spectroscopy)

• Thermal stability assays (differential scanning fluorimetry)

How can genomic and transcriptomic data be integrated to understand the regulation of CrcB homolog expression in R. centenum?

Integration of genomic and transcriptomic data requires:

Regulatory Element Analysis:
Drawing from approaches used for other R. centenum genes , researchers should:

• Identify putative promoter regions and transcription factor binding sites

• Analyze the genomic context surrounding the CrcB homolog gene

• Look for cis-regulatory motifs similar to those identified for other genes (like those described for Rv3069 with e-values of 0.01 and 0.27)

Transcriptomic Analysis Under Various Conditions:

• Perform RNA-Seq under conditions relevant to CrcB function (varying fluoride concentrations, pH changes, etc.)

• Identify co-regulated genes that may form functional modules with CrcB

• Analyze expression patterns in response to environmental stressors

Integration with Protein-Level Data:

• Correlate transcriptomic data with proteomics data

• Identify post-transcriptional regulatory mechanisms

• Map protein abundance changes to transcript-level changes

For example, the Mycobacterium tuberculosis CrcB homolog (Rv3069) was predicted to be co-regulated in modules bicluster_0256 with residual 0.48 and bicluster_0471 with residual 0.52 . Similar approaches could be applied to R. centenum CrcB.

What are the implications of R. centenum CrcB homolog in photosynthetic electron transport and phototaxis?

While direct evidence linking CrcB to photosynthesis is not present in the search results, we can formulate research questions based on R. centenum's known phototactic properties:

Potential Interactions with Photosynthetic Machinery:

• Investigate whether CrcB expression is regulated by light conditions

• Assess whether CrcB knockout affects photosynthetic efficiency

• Examine potential interactions between CrcB and photosynthetic electron transport components

Phototaxis Connections:
R. centenum exhibits unique photosensory behaviors including scotophobic responses in liquid medium and phototactic colony movement on solid medium . Research should explore:

• Whether CrcB mutants affect phototactic responses similar to ptr mutants

• Potential interactions between CrcB and known photosensory components like Ptr

• The effect of fluoride concentration on phototactic behavior and whether this is mediated by CrcB

Integration with Known Photosensory Pathways:
The ptr gene in R. centenum transmits signals from the photosynthesis-driven electron transport chain to the chemotaxis signal transduction cascade . Researchers should investigate if CrcB plays a role in:

• Modulating membrane potential during photosynthesis

• Regulating ion homeostasis in response to light-induced metabolic changes

• Protecting photosynthetic machinery from fluoride toxicity

How do mutations in the R. centenum CrcB homolog affect bacterial stress responses?

To investigate the role of CrcB in stress responses:

Targeted Mutagenesis Approaches:
Following methodologies used for other R. centenum genes :

• Generate CrcB knockout mutants using suicide vectors and allelic replacement

• Create point mutations in conserved regions of the CrcB protein

• Develop conditional expression systems to study CrcB depletion effects

Stress Response Phenotyping:

• Assess growth under various stressors (fluoride, pH extremes, oxidative stress)

• Measure survival rates following acute stress exposure

• Monitor morphological changes using microscopy

Comparative Transcriptomics:

• Compare gene expression profiles between wild-type and CrcB mutant strains

• Identify stress response pathways that are dysregulated in CrcB mutants

• Correlate with phenotypic observations

R. centenum has established genetic systems for generating and characterizing mutations , which could be applied to study CrcB function in stress responses. The approaches used to study photosynthetic mutants (including those affecting electron transport) could be particularly relevant for investigating CrcB's role in membrane homeostasis during stress.

What computational approaches can predict the structure and function of R. centenum CrcB homolog?

Multiple computational approaches can be employed:

Homology Modeling:

• Use experimentally determined structures of related proteins as templates

• Align R. centenum CrcB sequence with structural templates

• Generate 3D models using software like MODELLER, I-TASSER, or AlphaFold

• Refine models with molecular dynamics simulations

Evolutionary Analysis:

• Perform multiple sequence alignments of CrcB homologs across species

• Identify conserved residues likely important for function

• Conduct evolutionary coupling analysis to predict interacting residues

• Use these predictions to guide mutagenesis studies

Molecular Dynamics Simulations:

• Embed the CrcB homology model in a lipid bilayer mimicking R. centenum membrane composition

• Perform all-atom MD simulations to study conformational dynamics

• Simulate ion permeation to characterize transport mechanism

• Calculate binding free energies for fluoride and other potential substrates

For reference, the Bacillus cereus CrcB homolog 2 consists of 118 amino acids , while the Mycobacterium tuberculosis Rv3069 CrcB homolog consists of 132 amino acids . Sequence comparison with these homologs would provide a starting point for R. centenum CrcB structural prediction.

What are the latest crystallization techniques suitable for determining the structure of R. centenum CrcB homolog?

For membrane proteins like CrcB, consider these advanced crystallization approaches:

Lipidic Cubic Phase (LCP) Crystallization:

• Reconstitute purified CrcB in monoolein or other suitable lipids

• Set up crystallization trials in LCP format

• Screen various precipitants, detergents, and additives

• Optimize hits using grid screens varying pH, salt, and precipitant concentration

Antibody-Mediated Crystallization:

• Generate or select antibody fragments (Fab, nanobodies) that bind CrcB

• Form stable CrcB-antibody complexes

• Use these complexes in crystallization trials to provide crystal contacts

Engineering Approaches:

• Create fusion constructs with crystallization chaperones (T4 lysozyme, BRIL)

• Perform surface entropy reduction by mutating flexible, charged residues

• Truncate N- and C-termini to identify minimal functional domains

Alternative Structure Determination Methods:

• Single-particle cryo-electron microscopy for larger complexes

• Micro-electron diffraction (MicroED) for small 3D crystals

• Solid-state NMR for specific structural questions

When preparing recombinant CrcB for structural studies, researchers should consider the storage and reconstitution protocols established for similar proteins, such as using Tris/PBS-based buffer with 6% trehalose at pH 8.0 and adding 5-50% glycerol for long-term storage .

How can electrophysiological techniques be applied to study R. centenum CrcB homolog ion transport properties?

Several electrophysiological approaches are suitable for characterizing CrcB:

Planar Lipid Bilayer Recordings:

• Reconstitute purified CrcB into liposomes

• Fuse proteoliposomes with planar lipid bilayers

• Record single-channel currents under voltage-clamp conditions

• Analyze conductance, ion selectivity, and gating properties

Patch-Clamp of Giant Liposomes:

• Generate giant unilamellar vesicles (GUVs) containing CrcB

• Perform patch-clamp recordings in various configurations

• Measure macroscopic currents and single-channel events

• Determine ion selectivity using ion substitution experiments

Solid-Supported Membrane Electrophysiology:

• Adsorb CrcB-containing proteoliposomes onto a solid-supported membrane

• Apply rapid solution exchange to trigger transport

• Record transient currents reflecting conformational changes

• Analyze kinetics of transport cycle

These approaches should be combined with site-directed mutagenesis of key residues to establish structure-function relationships. The amino acid sequence of Bacillus cereus CrcB homolog 2 (provided in search result ) could guide the identification of conserved residues likely involved in ion selectivity and permeation.

What approaches can be used to investigate the role of R. centenum CrcB homolog in fluoride resistance?

To investigate CrcB's role in fluoride resistance:

Genetic Approaches:

• Generate CrcB knockout strains using established genetic methods for R. centenum

• Complement knockouts with wild-type and mutant versions of CrcB

• Perform growth inhibition assays with varying fluoride concentrations

• Measure survival rates following fluoride exposure

Fluoride Accumulation Assays:

• Use fluoride-specific probes or electrodes to measure intracellular fluoride levels

• Compare accumulation rates between wild-type and CrcB mutant strains

• Monitor fluoride efflux after loading cells with fluoride

Physiological Impact Assessment:

• Analyze changes in membrane potential using voltage-sensitive dyes

• Measure cytoplasmic pH in response to fluoride exposure

• Assess metabolic activity using resazurin or ATP assays

• Examine morphological changes using electron microscopy

The genetic systems described for R. centenum in search result , including methods for generating mutations through ethyl methanesulfonate mutagenesis and Tn5 transposition mutagenesis, provide established frameworks for these investigations .

How can isotope labeling be used to track ion movements through R. centenum CrcB homolog channels?

Isotope labeling offers powerful approaches to study ion transport:

18F Radioisotope Flux Assays:

• Load bacterial cells or proteoliposomes with 18F-labeled fluoride

• Monitor efflux/influx rates under various conditions

• Compare transport rates between wild-type and mutant CrcB variants

• Determine kinetic parameters (Km, Vmax) for fluoride transport

Neutron Diffraction with Deuterium Labeling:

• Replace specific hydrogen atoms with deuterium in the protein or transported ions

• Perform neutron diffraction on reconstituted systems

• Locate deuterated positions to map ion pathways through the channel

NMR Spectroscopy with 19F:

• Use 19F NMR to detect fluoride ion binding to purified CrcB

• Measure chemical shift changes upon fluoride binding

• Determine binding affinities for fluoride and potential inhibitors

• Identify key residues involved in fluoride coordination

For methodology inspiration, researchers could adapt the volatile methanol evolution assay described in search result that was used to measure 3H-labeled methanol release from R. centenum cells undergoing light intensity changes . This approach involves carefully washing and suspending cells, adding labeled compounds, and measuring release over time.

How has the CrcB protein family evolved across photosynthetic bacteria?

To understand the evolution of CrcB in photosynthetic bacteria:

Phylogenetic Analysis:

• Collect CrcB homolog sequences from diverse photosynthetic bacteria

• Align sequences using algorithms optimized for membrane proteins

• Construct phylogenetic trees using maximum likelihood or Bayesian approaches

• Compare CrcB evolution with the evolution of photosynthetic systems

Synteny Analysis:

• Examine genomic context of CrcB genes across species

• Identify conserved gene neighborhoods that might indicate functional relationships

• Determine if CrcB co-evolves with specific photosynthetic components

Selective Pressure Analysis:

• Calculate dN/dS ratios to identify sites under positive or purifying selection

• Compare evolutionary rates between photosynthetic and non-photosynthetic lineages

• Correlate evolutionary patterns with functional domains and environmental niches

From search result , we know that Mycobacterium tuberculosis Rv3069 (CrcB homolog 1) has been found to be important for growth on cholesterol , suggesting diverse functional adaptations of CrcB homologs across bacterial species.

What is known about the distribution and diversity of CrcB homologs across different Rhodospirillum species?

Although the search results don't provide comprehensive information on CrcB distribution across Rhodospirillum species, a systematic approach to this question would include:

Genome Mining:

• Search for CrcB homologs in all sequenced Rhodospirillum genomes

• Compare sequence conservation, copy number, and genomic context

• Identify species-specific adaptations in CrcB sequence or regulation

Sequence-Structure-Function Analysis:

• Align CrcB sequences from different Rhodospirillum species

• Map conserved and variable regions onto predicted structural models

• Correlate sequence variations with ecological niches and physiological adaptations

Functional Conservation Testing:

• Express CrcB homologs from different Rhodospirillum species in a model system

• Compare functional properties (ion selectivity, gating, regulation)

• Assess complementation capability in CrcB-deficient strains

This comparative approach would provide insights into how CrcB function may have specialized across Rhodospirillum species with different ecological adaptations and photosynthetic capabilities.

How do horizontal gene transfer events impact the evolution of CrcB homologs in bacterial genomes?

To investigate horizontal gene transfer (HGT) of CrcB homologs:

Phylogenetic Incongruence Analysis:

• Compare CrcB gene trees with species trees

• Identify discordant topologies suggesting HGT events

• Estimate the timing and frequency of transfer events

Compositional Bias Analysis:

• Analyze GC content, codon usage, and oligonucleotide frequencies of CrcB genes

• Compare with genomic averages to identify recently transferred genes

• Look for signatures of amelioration in older transfer events

Mobile Genetic Element Association:

• Examine proximity of CrcB genes to insertion sequences, transposons, or phage genes

• Identify potential vehicles for mobilization

• Assess conservation of these associations across species

Functional Consequences:

• Compare functional properties of horizontally transferred CrcB genes

• Assess selective advantages conferred by acquired CrcB variants

• Determine if transfers correlate with ecological transitions or stress adaptations

The established genetic manipulation systems for R. centenum described in search results and could potentially be used to experimentally test the functional consequences of introducing CrcB variants from other species.