Recombinant Roseiflexus sp. Protein CrcB homolog (crcB)

Overview

This comprehensive FAQ document addresses key scientific questions about the Recombinant Roseiflexus sp. Protein CrcB homolog (crcB), focusing on experimental methodologies, research applications, and technical considerations for academic researchers. Content is derived from recent peer-reviewed literature as of April 2025 and organized from fundamental concepts to advanced research applications.

What is Roseiflexus castenholzii and why is it significant for photosynthesis research?

Roseiflexus castenholzii is a chlorosome-less filamentous anoxygenic phototrophic bacterium belonging to the Chloroflexota phylum. It represents one of the deepest branches of photosynthetic bacteria, making it particularly valuable for evolutionary studies of photosynthesis . Unlike other phototrophs, Roseiflexus contains only one light-harvesting (LH) complex, which forms an unusual reaction center (RC)-LH complex that structurally resembles RC-LH1 but exhibits spectroscopic characteristics similar to the peripheral LH2 of purple bacteria .

The significance of this organism lies in its unique photosynthetic apparatus, which provides critical insights into the evolution and diversity of photosynthetic systems. Recent high-resolution structures (2.85-2.86 Å) of the RC-LH complex have revealed previously unresolved details about its architecture and function, particularly regarding carotenoid assembly and its role in regulating quinone diffusion .

What is the CrcB homolog protein in Roseiflexus sp. and what methods are used to study it?

The CrcB homolog protein in Roseiflexus sp. (strain RS-1) is a membrane protein with a molecular weight of approximately 14 kDa, comprising 124 amino acids . The protein is characterized by its transmembrane structure and is believed to function in fluoride ion channel activity based on homology with other CrcB proteins, though specific functions in Roseiflexus require further investigation.

Research methodologies to study this protein include:

When working with the recombinant protein, it's recommended to store it at -20°C, and for extended storage at -80°C. Repeated freezing and thawing should be avoided, with working aliquots maintained at 4°C for up to one week .

What expression systems are most effective for producing recombinant Roseiflexus proteins?

For recombinant expression of Roseiflexus proteins, prokaryotic expression systems, particularly E. coli, have proven most effective due to the prokaryotic origin of Roseiflexus sp. . Key methodological considerations include:

• Codon optimization: Enhancing expression by adapting the codon usage to the prokaryotic expression system

• Expression vectors: Using vectors with strong promoters (typically T7) and appropriate fusion tags (often His-tags) for purification

• Expression conditions: Optimizing temperature (typically 16-30°C), induction parameters, and media composition

• Protein extraction: Employing membrane protein extraction protocols for membrane-associated proteins like CrcB

• Purification strategy: Implementing affinity chromatography followed by size exclusion chromatography

The expression and purification protocols have successfully yielded recombinant proteins with molecular weights of approximately 27 kDa (for chimeric constructs) and 14 kDa (for CrcB homolog) , as confirmed by Western blot analysis using anti-His antibodies.

For researchers encountering expression challenges, alternative approaches may include:

• Cell-free expression systems

• Membrane protein-specific expression hosts

• Fusion with solubility-enhancing partners (e.g., MBP, SUMO)

What regulatory considerations apply to research with recombinant Roseiflexus proteins?

Research involving recombinant Roseiflexus proteins falls under the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, which require institutional biosafety committee (IBC) approval prior to initiating work . Key regulatory considerations include:

• Registration requirements: All recombinant DNA research, including work with recombinant Roseiflexus proteins, must be registered with the institutional IBC

• Biosafety levels: Work typically falls under Biosafety Level 1 (BSL-1) for non-pathogenic organisms, though specific determinations should be made via risk assessment

• Principal Investigator responsibilities:

  • Ensuring compliance with NIH Guidelines

  • Maintaining proper safeguards and procedures

  • Selecting appropriate microbiological practices and laboratory techniques

  • Notifying the IBC of protocol changes or research location modifications

• Documentation requirements: Completion of the Biohazard Use Protocol form and submission through established administrative channels

• Shipping considerations: Compliance with shipping requirements for recombinant DNA molecules

Researchers should consult with their institutional IBC for specific guidance and approval before initiating experiments with recombinant Roseiflexus proteins .

How do carotenoids affect the assembly and function of the RC-LH complex in Roseiflexus castenholzii?

Recent high-resolution cryo-EM structures have provided critical insights into the role of carotenoids (Cars) in the assembly and function of the RC-LH complex from Roseiflexus castenholzii . The following methodology-focused findings are particularly relevant for researchers:

Experimental approach:

• Generation of carotenoidless (Crt-less) RC-LH complexes by growing cells in the presence of diphenylamine (DPA), which inhibits carotenogenesis but not phototrophic growth

• Comparative cryo-EM analysis of native (2.86 Å) and Crt-less (2.85 Å) RC-LH complexes

• Mass spectrometry analysis for protein identification and component characterization

Key findings on carotenoid function:

• Architectural role: Native RC-LH complexes contain ~30 all-trans carotenoids, with approximately two carotenoids per αβ heterodimer in the LH ring

• Carotenoid organization: Carotenoids are divided into two groups (A and B) based on biochemistry, orientation, and position:

  • Group A: Deeply embedded in the transmembrane region between α- and β-polypeptides with β-rings pointing toward the cytoplasmic side

  • Group B: Located at the interface between adjacent LH αβ heterodimers

• Quinone channel regulation: Exterior carotenoids function with bacteriochlorophyll B800 to block the proposed quinone channel between LHαβ subunits, forming a sealed LH ring that is disrupted by transmembrane helices from cytochrome c and subunit X to allow quinone shuttling

• Structural consequences of carotenoid depletion:

  • Carotenoid-depleted complexes lack subunit X

  • DPA treatment results in only five remaining carotenoids

  • Exposed LH ring with larger opening, accelerating quinone exchange rate

This research demonstrates that carotenoids are not merely accessory pigments but play critical structural roles in organizing the RC-LH complex and regulating its function through quinone exchange modulation .

What methodologies are most effective for studying the quinone exchange mechanism in Roseiflexus RC-LH complexes?

The quinone exchange mechanism in Roseiflexus castenholzii RC-LH complexes represents a unique system for understanding electron transport processes in anoxygenic photosynthesis. Recent research has employed the following methodological approaches to study this process:

Structural characterization approaches:

• High-resolution cryo-EM: Achieving 2.85-2.86 Å resolution to visualize the quinone channel architecture and protein subunits involved in forming the channel

• Carotenoid depletion studies: Using diphenylamine (DPA) to create carotenoid-depleted complexes that exhibit altered quinone exchange properties, providing a comparative system

• Protein subunit identification: Combining mass spectrometry with high-quality electron density maps to assign previously unresolved subunits that participate in quinone channel formation

Functional assays:

• Quinone exchange rate measurements: Comparing native and carotenoid-depleted complexes to quantify differences in quinone mobility

• Site-directed mutagenesis: Modifying amino acids in subunits that form the quinone channel to assess their role in regulating quinone exchange

Key discoveries about the quinone shuttling mechanism:

• The quinone channel in Roseiflexus castenholzii is formed by:

  • A gap in the LH ring

  • The cytochrome c transmembrane helix (c-TM)

  • Subunit X (TMx)

• Carotenoids and bacteriochlorophyll B800 function to block potential quinone channels between LHαβ subunits, creating a sealed LH ring

• Carotenoid depletion results in:

  • Loss of subunit X

  • Larger opening in the LH ring

  • Accelerated quinone exchange rate

These findings reveal the structural basis by which carotenoid assembly regulates quinone exchange in bacterial RC-LH complexes, providing insights into the evolution and diversity of prokaryotic photosynthetic apparatus .

How does Roseiflexus castenholzii's RC-LH complex differ from those of other anoxygenic phototrophs, and what methods best reveal these differences?

Roseiflexus castenholzii occupies a unique evolutionary position, representing one of the deepest branches of photosynthetic bacteria . Its RC-LH complex exhibits distinctive characteristics that can be effectively studied through several methodological approaches:

Comparative structural analysis methods:

• High-resolution cryo-EM: Comparing the 2.85-2.86 Å structures of Roseiflexus RC-LH with those of other phototrophs reveals unique architectural features

• Spectroscopic characterization: Absorption spectra, particularly examining peaks at 740-750 nm (chlorosome-associated), 798 nm and 867 nm (RC-associated, present in Chloroflexus aurantiacus but absent in Roseiflexus)

• Pigment analysis: HPLC determination of bacteriochlorophyll and carotenoid composition

Key structural and functional distinctions:

These differences suggest that Roseiflexus represents a unique evolutionary pathway in the development of anoxygenic photosynthetic systems, with its RC-LH complex showing characteristics of both RC-LH1 (structural) and LH2 (spectroscopic) complexes of purple bacteria .

The recent discovery of "Candidatus Chlorohelix allophototropha" in the Chloroflexota phylum, which uses a type I reaction center (RCI) rather than the type II reaction center (RCII) used by other Chloroflexota members like Roseiflexus, further highlights the evolutionary significance of this bacterial group in understanding photosynthesis evolution .

Lambda Red recombineering system:

This system, which has revolutionized genetic engineering in E. coli, offers potential for adaptation to Roseiflexus :

• Key components:

  • Beta protein: Promotes single-strand annealing

  • Exo protein: Creates 3' overhangs through 5'→3' exonuclease activity

  • Gam protein: Inhibits host RecBCD nuclease to protect linear DNA

• Methodological approach:

  • Design PCR products with ~50 bp homology arms targeting Roseiflexus genomic regions

  • Express Lambda Red proteins from a plasmid with a temperature-sensitive origin

  • Transform linear DNA containing the desired modification

  • Select for recombinants using appropriate markers

• Optimization considerations:

  • Promoter selection for Lambda Red expression in Roseiflexus

  • Protection against restriction systems

  • Efficiency of DNA delivery methods

CRISPR-Cas9 based recombination:

The CRISPR-Cas9 system can enhance homologous recombination efficiency:

• Implementation strategy:

  • Design sgRNAs targeting the Roseiflexus genome region of interest

  • Provide repair template with homology arms

  • Express Cas9 and sgRNA from suitable vectors

  • Screen for desired recombinants

• Technical considerations:

  • PAM site availability in AT-rich regions

  • Cas9 codon optimization for Roseiflexus

  • Temperature sensitivity of CRISPR components

Special considerations for Roseiflexus:

When designing homologous recombination experiments for Roseiflexus, researchers should account for:

• Recombination efficiency factors:

  • Length of homology (longer homology arms may be required)

  • GC content and sequence context effects

  • Selection markers suitable for Roseiflexus

• Regulatory requirements:

  • Experiments require IBC approval prior to initiation

  • Risk assessment should consider potential horizontal gene transfer

  • Proper containment based on biosafety level determination

These methods represent promising approaches for genetic manipulation of Roseiflexus, though optimization for this specific organism will be necessary.

How can copy number alteration (CNA) techniques be applied to study homologous recombination in Roseiflexus?

Copy number alteration (CNA) analysis has emerged as a valuable technique for studying homologous recombination deficiency (HRD) in various systems . While not yet widely applied to Roseiflexus specifically, these methodologies can be adapted to investigate recombination mechanisms in this organism:

Methodological approaches for CNA analysis in Roseiflexus:

• Genomic data acquisition:

  • Whole genome sequencing (WGS) at sufficient coverage (≥30×)

  • SNP array analysis

  • Targeted panel sequencing focusing on recombination-related genes

• CNA feature extraction and analysis:

  • BP10MB: Number of breakpoints per 10MB of DNA

  • Segment size distribution analysis (SS features)

  • Telomeric allelic imbalance (TAI)

  • Large-scale state transitions (LST)

  • Loss of heterozygosity (LOH) patterns

• Machine learning implementation:

  • Gradient boosting machine models for predicting recombination efficiency

  • Feature importance analysis to identify key CNA signatures associated with successful homologous recombination in Roseiflexus

Experimental applications for Roseiflexus research:

• Characterization of natural recombination mechanisms:

  • Analyze CNA patterns in wild-type Roseiflexus genomes

  • Compare with other Chloroflexota to understand evolutionary adaptations in recombination machinery

• Optimization of genetic engineering approaches:

  • Use CNA signatures to predict likely success of homologous recombination experiments

  • Identify genomic regions more amenable to recombination-based editing

• Investigation of recombination protein roles:

  • Create knockout mutants of putative recombination genes

  • Characterize resulting CNA patterns to determine protein function

  • Compare with CNA signatures in other bacteria to identify unique features

Important considerations:

When applying CNA techniques to Roseiflexus, researchers should account for:

• The circular bacterial genome structure

• Lack of telomeres (requiring adaptation of TAI measures)

• Different ploidy considerations compared to eukaryotic systems

• GC content effects on recombination frequency and breakpoint distribution

These approaches may provide valuable insights into both the natural recombination mechanisms of Roseiflexus and optimize genetic engineering approaches for this important photosynthetic bacterium.

What are the implications of recent discoveries about type I and type II reaction centers in Chloroflexota for understanding Roseiflexus evolution?

Recent groundbreaking research has uncovered an anoxygenic phototroph from the Chloroflexota phylum that uses a type I reaction center (RCI) rather than the type II reaction center (RCII) used by Roseiflexus and other previously known Chloroflexota members . This discovery has profound implications for understanding the evolution of photosynthesis and Roseiflexus's position in this evolutionary history:

Methodological approaches that revealed this discovery:

• Cultivation-based discovery: Lake water incubations from Boreal Shield lakes led to the isolation of "Candidatus Chlorohelix allophototropha" strain L227-S17

• Spectroscopic characterization:

  • In vivo absorption spectra comparing the novel isolate with Chloroflexus aurantiacus and Chlorobium ferrooxidans

  • Analysis of bacteriochlorophyll c species and modifications

• Phylogenomic analysis:

  • Whole genome sequencing

  • Comparative analysis of photosynthetic gene clusters

  • Evolutionary reconstruction of photosynthetic pathways

• Environmental metatranscriptomics: Verification of active RCI-utilizing metabolism in environmental samples

Key findings and implications for Roseiflexus research:

• Evolutionary significance:

  • The Chloroflexota phylum now represents the only known bacterial group with members using contrasting modes of light energy conversion (RCI vs. RCII)

  • Strong phylogenomic evidence suggests RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor

• Functional implications for Roseiflexus:

  • The metabolic flexibility observed in RCII-utilizing Chloroflexota members like Roseiflexus may be linked to adaptations that allowed the phylum to support multiple phototrophic modes

  • Understanding the specific adaptations that allowed contrasting photosynthetic reaction center classes to function in related genetic backgrounds may reveal new insights into Roseiflexus biology

• Research directions for Roseiflexus studies:

  • Comparative analysis of quinone exchange mechanisms between RCI and RCII-utilizing Chloroflexota

  • Investigation of evolutionary constraints and adaptations in carotenoid biosynthesis pathways

  • Examination of the potential for ancestral hybrid photosystems that may have influenced Roseiflexus evolution

This discovery fundamentally changes our understanding of photosynthesis evolution and suggests that Roseiflexus represents one evolutionary path within a phylum that has demonstrated remarkable adaptability in photosynthetic mechanisms .

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