Recombinant Roseobacter denitrificans Large-conductance mechanosensitive channel (mscL)

What is the amino acid sequence of Recombinant Roseobacter denitrificans mscL protein?

The full-length Roseobacter denitrificans mscL protein consists of 142 amino acids with the following sequence: MLNEFKTFISKGNVMDMAVGIIIGAAFTAIVSSLVADLVNPFIALFTGGIDFSGWFYALDGETYASLAAATDAGAPVFAFGNFIMAVINFLIIAFVVFMLVKTVNRIKDAAEGEKEAVAEEPAGPTELDILKEIRDALAKQG . This sequence includes conserved domains characteristic of large-conductance mechanosensitive channels found across bacterial species, with structural features enabling its function in osmotic regulation.

How is the recombinant mscL protein typically expressed and purified?

The recombinant protein is typically expressed in E. coli expression systems with an N-terminal His tag to facilitate purification . The expression system allows for high yield production of the membrane protein, which is subsequently purified through affinity chromatography using the His tag. After expression, the protein undergoes purification steps that typically include cell lysis, membrane fraction isolation, solubilization with appropriate detergents, and affinity purification. The final product achieves greater than 90% purity as determined by SDS-PAGE analysis .

What are the optimal storage conditions for maintaining mscL protein stability?

For long-term storage, the protein is supplied as a lyophilized powder and should be stored at -20°C to -80°C upon receipt . Working aliquots can be maintained at 4°C for up to one week. Reconstituted protein is best stored with 5-50% glycerol (with 50% being the default final concentration) and aliquoted to avoid repeated freeze-thaw cycles, which can significantly compromise protein integrity and function . The recommended storage buffer is Tris/PBS-based with 6% trehalose at pH 8.0 to maintain proper folding and activity .

How should the lyophilized mscL protein be reconstituted for experimental use?

The protein should be reconstituted by first briefly centrifuging the vial to bring contents to the bottom. Reconstitution should be performed in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . For optimal stability during storage after reconstitution, the addition of glycerol to a final concentration of 5-50% is recommended. The solution should be mixed gently to ensure complete solubilization without denaturing the protein structure. Aliquoting the reconstituted protein for single-use applications is strongly recommended to avoid repeated freeze-thaw cycles .

What methodologies are most effective for studying Roseobacter species in environmental samples?

For quantifying Roseobacter species in environmental samples, two complementary approaches have proven effective:

• CARD-FISH (Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization): This technique uses the horseradish peroxidase-labeled Roseobacter-specific probe Roseo536 (5′-CAA CGC TAA CCC CCT CCG-3′ plus competitor) with 35% formamide at 46°C for hybridization . The method allows for direct visualization and counting of Roseobacter cells in sediment samples.

• Quantitative PCR (qPCR): For molecular quantification, the Roseobacter group can be targeted using specific primers Roseo536f (reverse complementary to Roseo536r) and GRb735R . This approach provides more precise quantification of Roseobacter abundance relative to total bacterial populations.

These methodologies have revealed that Roseobacter group members typically comprise between 1.7% (by CARD-FISH) and 6.3% (by qPCR) of bacterial communities in marine sediments .

What experimental approaches can be used to assess mscL channel activity in reconstituted systems?

For functional studies of the mscL channel, several approaches are recommended:

• Patch-clamp electrophysiology: This technique allows direct measurement of channel conductance and gating properties when the protein is reconstituted into liposomes or planar lipid bilayers.

• Fluorescence-based osmotic shock assays: By loading liposomes containing reconstituted mscL with self-quenching fluorescent dyes, researchers can monitor channel opening in response to osmotic stress through measuring fluorescence dequenching.

• Molecular dynamics simulations: Computational approaches based on the protein sequence can provide insights into structural changes during channel gating.

• Site-directed mutagenesis studies: Systematic alteration of specific amino acids can help identify residues critical for channel function, particularly those in the transmembrane domains that respond to membrane tension.

What is the ecological significance of Roseobacter denitrificans in marine ecosystems?

Roseobacter denitrificans belongs to the Roseobacter clade, a numerically abundant and biogeochemically active group of heterotrophic marine bacteria . These organisms play crucial roles in marine carbon and sulfur cycling through their metabolic versatility. R. denitrificans specifically contains genes for dimethylsulfoniopropionate demethylase (dmdA), sulfur oxidation protein complex (soxB), and carbon monoxide dehydrogenase (coxL) . Unlike some other Roseobacter species, R. denitrificans lacks certain nitrogen metabolism genes (nasA, nirB, napA, narG, and nirS) , suggesting specialized ecological adaptations.

The Roseobacter group exhibits distinct biogeographical distribution patterns linked to nutrient availability and water column productivity. Studies have shown lowest cell numbers at the edges of ocean gyres with low nutrient content, and highest abundances in productive regions like the Bering Sea . This distribution pattern indicates their importance in nutrient cycling in specific marine niches.

How does mscL contribute to the adaptation of Roseobacter denitrificans to changing marine environments?

The mscL protein functions as a pressure-sensitive "emergency release valve" that opens in response to hypoosmotic shock, preventing cell lysis. In marine environments where salinity fluctuations occur frequently, especially in coastal and estuarine regions, this adaptation is crucial for Roseobacter denitrificans survival. The mechanosensitive channel allows for rapid response to changing osmotic conditions by:

This adaptation likely contributes to the ecological success of Roseobacter species across various marine environments with different salinity regimes and osmotic conditions.

What genomic and phylogenetic analyses have been conducted on Roseobacter denitrificans?

Phylogenetic analyses of Roseobacter species have utilized both 16S rRNA gene sequences and whole-genome approaches such as Genome Blast Distance Phylogeny . These analyses reveal that the Roseobacter group represents a diverse clade with complex evolutionary relationships. Whole-genome sequencing has identified several gene clusters involved in secondary metabolite synthesis, including a terpene cluster encoding the synthesis of spheroidenone, the main light-harvesting carotenoid in Roseobacter group members .

Illumina tag sequencing of 16S rRNA genes and transcripts has shown that Roseobacter-affiliated OTUs comprise approximately 0.7-0.9% of bacterial communities in marine sediments, with most OTUs assigned to uncultured members . Among cultured representatives, genera such as Sedimentitalea and Sulfitobacter often make up the largest proportions in environmental samples .

How do membrane composition and lipid interactions affect mscL channel gating properties?

The gating mechanism of mscL channels is intrinsically linked to membrane properties and lipid-protein interactions. Research considerations should include:

• Membrane thickness effects: The hydrophobic mismatch between the protein's transmembrane domains and the lipid bilayer thickness can significantly affect channel sensitivity and gating threshold.

• Lipid composition: The presence of specific phospholipids, sterols, or other membrane components can modulate channel function through direct interactions with the protein or by altering membrane physical properties.

• Lateral pressure profile: The distribution of forces within the membrane bilayer affects the energetics of channel opening and closing.

• Charge interactions: Electrostatic interactions between charged amino acid residues and lipid headgroups can influence channel conformation and gating properties.

Experimental approaches should control membrane composition when reconstituting the protein into artificial systems to ensure reproducible functional assessments.

What insights can comparative genomics provide about mscL evolution and function across Roseobacter species?

Comparative genomic analyses between different Roseobacter species can reveal:

• Conservation patterns of mscL sequences, indicating functional constraints and evolutionary pressure

• Potential gene duplication events leading to functional diversification

• Horizontal gene transfer patterns that might have contributed to the distribution of mscL variants

• Co-evolution with other membrane proteins or osmotic stress response systems

When designing comparative studies, researchers should consider that while R. denitrificans is well-characterized, many environmental Roseobacter group members remain uncultured, with their genetic diversity yet to be fully explored . This gap highlights the importance of metagenomic approaches in conjunction with culture-based studies.

What are the recommended approaches for functional expression of recombinant mscL in heterologous systems?

For successful functional expression of Roseobacter denitrificans mscL in heterologous systems, researchers should consider:

• Expression system selection: E. coli has been successfully used for recombinant production , but alternative systems such as yeast or insect cells might provide advantages for specific applications.

• Codon optimization: Adapting the coding sequence to the preferred codon usage of the expression host can improve yields.

• Fusion partners: The N-terminal His tag has proven effective , but other fusion partners might enhance solubility or facilitate membrane insertion.

• Membrane targeting strategies: Inclusion of appropriate signal sequences or membrane-targeting domains can improve proper localization.

• Induction conditions: Optimizing temperature, inducer concentration, and induction timing can significantly impact the yield of functional protein.

• Detergent selection: For membrane protein extraction and purification, detergent choice critically affects protein stability and functionality.

What typical yield and purity can be expected when expressing Roseobacter denitrificans mscL?

What is the relative abundance of Roseobacter species across different marine environments?

What are the current limitations in Roseobacter denitrificans mscL research?

Current limitations include:

• Limited structural data: High-resolution structural information specific to Roseobacter denitrificans mscL is not yet available, constraining structure-function relationship studies.

• Dominance of uncultured species: The predominance of uncultured Roseobacter group members in environmental samples limits our understanding of the full diversity and ecological roles of these organisms .

• Integration challenges: Connecting molecular mechanisms of mscL function to ecological adaptations and biogeochemical processes requires interdisciplinary approaches that are methodologically challenging.

• Standardization issues: Varied methodologies across studies make comparative analyses difficult, particularly when relating protein function to ecological observations.

What promising research directions might advance our understanding of Roseobacter denitrificans mscL?

Future research directions could include:

• Cryo-electron microscopy studies to resolve the high-resolution structure of Roseobacter denitrificans mscL in different conformational states.

• Integration of omics approaches (transcriptomics, proteomics, metabolomics) to understand mscL regulation in response to environmental stressors.

• Development of genetic tools specifically for Roseobacter species to enable in vivo functional studies.

• Ecological studies examining the correlation between mscL variants and adaptation to specific marine niches.

• Comparative analyses of mscL function across marine bacteria adapted to different osmotic regimes to understand evolutionary convergence and divergence in mechanosensitive channel mechanisms.

• Application of microfluidic techniques to study single-cell responses to osmotic challenges in real-time.