Recombinant Rhodopirellula baltica Flagellar hook-basal body complex protein FliE (fliE)
Description
Overview of Recombinant Rhodopirellula baltica Flagellar Hook-Basal Body Complex Protein FliE (FliE)
Rhodopirellula baltica Flagellar hook-basal body complex protein FliE (FliE) is a protein component of the flagellar apparatus in the bacterium Rhodopirellula baltica . R. baltica is a marine bacterium known for its unique cell structure and ecological significance . FliE is essential for flagellar motility, which depends on the sodium electrochemical gradient in R. baltica . The recombinant form of this protein is produced in a laboratory setting for research purposes .
Characteristics of Rhodopirellula baltica
Rhodopirellula baltica is a member of the Planctomycetes, a phylum of bacteria recognized for their unusual cell morphology and lifestyle . Key characteristics include:
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Habitat: Commonly found in terrestrial and marine environments .
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Cell Structure: Exhibits complex internal structures and partial compartmentalization, including a ribosome-containing pirellulosome with condensed nucleoid DNA .
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Genetics: The genome contains biotechnologically promising features, such as unique sulfatases and C1-metabolism genes .
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Stress Response: Adapts to nutrient limitation by upregulating genes coding for glutathione peroxidase, thioredoxin, and chaperones .
Function and Structure of FliE
FliE is a component of the flagellar hook-basal body complex, essential for bacterial motility. Research on FliE in Salmonella provides insights into its function :
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Structure: FliE in Salmonella consists of three α-helices (α1, α2, and α3) . The α1 helix binds to the inner wall of the MS-ring, while the α2 and α3 helices form domain D0, which interacts with other proteins like FliP, FliR, FlgB, and FlgC in the basal body .
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Functional Domains: Contains N- and C-terminal functional domains critical for FliE function .
Biotechnological and Evolutionary Significance
Rhodopirellula baltica and its proteins, including FliE, have significant biotechnological and evolutionary implications:
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Cardiolipin Biosynthesis: R. baltica utilizes a PTPMT1-like phosphatase in cardiolipin biosynthesis, similar to mammals, which supports the close relation of planctomycetes to eukaryotes .
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Unique Features: The unique, eukaryote-like features of planctomycetes challenge current hypotheses regarding the origin of eukaryotic organelles .
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Transport Proteins: R. baltica possesses a variety of transport proteins, including Na+-translocating NADH:quinone dehydrogenase and Na+:H+ antiporters, which are crucial for its physiology and adaptation to its environment .
Research Applications
FliE and other proteins from Rhodopirellula baltica are valuable in understanding bacterial physiology, evolution, and potential biotechnological applications . Some applications include:
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Gene Expression Analysis: Studying gene expression patterns to understand the functions and regulation mechanisms of potentially useful genes .
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Proteomic Analysis: Analyzing protein expression to understand the organism's response to different growth phases and environmental conditions .
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Structural Biology: Determining the crystal structure of proteins to understand their function (example: Crystal structure of conserved uncharacterized protein from Rhodopirellula baltica) .
Product Specs
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Note: All proteins are shipped with standard blue ice packs unless otherwise requested. Dry ice shipping requires prior arrangement and incurs additional charges.
Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized formulations have a 12-month shelf life at -20°C/-80°C.
Target Background
KEGG: rba:RB7444
STRING: 243090.RB7444
Q&A
What is Rhodopirellula baltica and what makes it significant for research?
Rhodopirellula baltica (R. baltica) is a marine bacterium belonging to the phylum Planctomycetes, which was initially isolated from the Kiel Fjord in the Baltic Sea . This organism has gained significant research attention due to several remarkable features:
R. baltica exhibits a complex life cycle with distinct morphological phases, shifting from motile swarmer cells to sessile cells with holdfast substances throughout its growth cycle . The organism possesses an intriguing cell structure including peptidoglycan-free proteinaceous cell walls and intracellular compartmentalization, which is unusual for bacteria . Its genome contains numerous biotechnologically promising features, including a unique set of sulfatases, carbohydrate-active enzymes (CAZymes), and specialized C1-metabolism pathways . These characteristics make R. baltica an excellent model organism for studying bacterial adaptation to marine environments and for potentially novel biotechnological applications.
What is the function of FliE in bacterial flagellar assembly?
The FliE protein serves as a critical junction between the MS (membrane-supramembrane) ring and the rod component of the bacterial flagellum . Based on research primarily conducted with Salmonella, FliE appears to constitute a specialized zone connecting these two major structural elements of the flagellar basal body . This junction role makes FliE essential for proper flagellar assembly and function.
FliE demonstrates strong interactions with FlgB, which is a proximal rod component, suggesting these proteins work together during flagellar construction . Importantly, FliE possesses the unique property of being necessary for the efficient export of other flagellar proteins, positioning it as a gatekeeper in the flagellar assembly process . This makes it not only structurally important but also functionally critical for the flagellar export pathway.
What growth conditions are optimal for R. baltica cultivation prior to FliE studies?
Cultivating R. baltica requires specific conditions for optimal growth:
Table 1: Optimal Growth Parameters for R. baltica
R. baltica can survive in carbon-free medium but requires carbon source supplementation for growth and division . Interestingly, different carbon sources affect the growth characteristics – for example, N-acetylglucosamine (NAG) and dextran can trigger biofilm formation rather than planktonic growth . For large-scale cultivation, R. baltica has been successfully grown in bench-top bioreactors (10L), with protocols available for both batch and fed-batch cultivation that allow seamless scale-up to 100L .
What methods are most effective for studying FliE expression during R. baltica's life cycle?
Based on research approaches used for studying gene expression in R. baltica:
A whole genome microarray approach has been successfully employed to monitor gene expression throughout R. baltica's growth curve, providing insights into the regulation of numerous genes during different growth phases . Transcriptional profiling can reveal how genes (potentially including fliE) are regulated during the transition between different cell morphologies – from swarmer cells to sessile cells with holdfast substances .
Combining transcriptomic data with microscopic examination of cell morphology allows researchers to correlate gene expression patterns with specific phases of the life cycle. The early exponential growth phase is dominated by swarmer and budding cells, the transition phase shows single and budding cells as well as rosettes, while the stationary phase is dominated by rosette formations .
How can recombinant FliE from R. baltica be effectively produced and purified?
While the search results don't provide specific protocols for R. baltica FliE production, search result indicates that recombinant R. baltica FliE can be produced in both yeast and E. coli expression systems:
Table 2: Expression Systems for Recombinant R. baltica FliE Production
| Expression Host | Product Identifier | Special Features |
|---|---|---|
| Yeast | CSB-YP763320RDR | Standard expression |
| E. coli | CSB-EP763320RDR | Standard expression |
| E. coli | CSB-EP763320RDR-B | Avi-tag Biotinylated variant |
The Avi-tag biotinylated variant is particularly noteworthy for research applications, as it involves in vivo biotinylation via AviTag-BirA technology . This method catalyzes an amide linkage between biotin and a specific lysine within the AviTag sequence, providing a site-specific biotin label that can be valuable for protein interaction studies, immobilization, or detection purposes.
How do mutations in FliE affect flagellar assembly and function?
Studies in Salmonella provide insights into how FliE mutations affect flagellar assembly:
A mutant allele of fliE caused extremely poor flagellation and swarming, demonstrating its essential role in flagellar assembly . Extragenic suppressors of this mutation were found in flgB, suggesting a functional interaction between these two proteins . The specific mutation V99G (close to the C-terminus of the 104-amino-acid FliE sequence) severely impaired the export of flagellar protein FlgD, confirming FliE's role in the flagellar export pathway .
Overproduction of mutant FliE protein improved motility but not to wild-type levels, indicating that the mutant protein had reduced assembly probability but retained some functionality when assembled . These findings from Salmonella suggest experimental approaches that could be applied to studying structure-function relationships in R. baltica FliE.
What protein-protein interactions does FliE form within the flagellar basal body complex?
Based on affinity blotting experiments with Salmonella FliE:
Strong interactions exist between FliE and FlgB, consistent with their proposed adjacent locations in the flagellar structure . Weaker interactions were observed between FliE and other rod proteins, which might be attributed to structural similarities among these proteins . FliE also interacts with components of the type III flagellar export apparatus and with FlgJ, a periplasmic muramidase .
For R. baltica FliE, similar interaction studies could be performed using the recombinant protein, particularly the biotinylated variant mentioned in source , which would be well-suited for protein-protein interaction assays.
How does R. baltica's FliE compare to homologs in other Planctomycetes species?
While specific comparative data for FliE across Planctomycetes is not provided in the search results, the following approach could be used:
Researchers could compare the genomic context of fliE in R. baltica with other sequenced Planctomycetes genomes, looking at gene neighborhoods and potential operonic structures. Using bioinformatic tools, sequence analysis of FliE proteins from various Planctomycetes could reveal conserved domains and species-specific adaptations. This is particularly relevant given that Planctomycetes have unique cell biology features compared to other bacteria .
What techniques are available for studying FliE localization within R. baltica cells?
Based on approaches used in bacterial flagellar research:
Fluorescent protein fusions could be created by genetically fusing fluorescent proteins like GFP to FliE, allowing visualization of the protein's localization in living cells. For higher resolution studies, immunogold electron microscopy using antibodies against FliE would provide detailed information about its precise location within the flagellar structure.
The biotinylated recombinant FliE variant mentioned in source could potentially be used to develop detection reagents for localization studies. Advanced microscopy techniques like super-resolution microscopy or cryo-electron tomography could provide structural insights at the nanometer scale.
How can the effects of environmental conditions on FliE expression be measured?
Drawing from approaches used to study R. baltica gene expression:
Whole genome microarray analysis has been successfully used to monitor gene expression changes throughout R. baltica's growth cycle and could be applied to study fliE expression specifically . Gene expression studies under varying salt concentrations could be particularly relevant, as R. baltica exhibits salt resistance as part of its adaptation to marine environments .
Researchers have observed that R. baltica modifies its cell wall composition in response to nutrient limitation and other stressors . Similar studies could examine whether flagellar gene expression, including fliE, is affected by these environmental shifts.
What controls should be included when performing functional studies on recombinant R. baltica FliE?
For rigorous experimental design:
Table 3: Recommended Controls for R. baltica FliE Functional Studies
| Control Type | Description | Purpose |
|---|---|---|
| Wild-type FliE | Non-mutated R. baltica FliE | Baseline for normal function |
| Heterologous FliE | FliE from well-studied organisms (e.g., Salmonella) | Comparative functional analysis |
| Tagged vs. Untagged | Comparison of tagged recombinant FliE with untagged version | Assess impact of tags on function |
| Deletion Controls | R. baltica strains with fliE deletion (if available) | Confirm phenotypes attributable to FliE |
| Complementation | Restoration of fliE in deletion strains | Verify functional restoration |
Additional controls might include testing FliE function under various temperature and pH conditions that reflect R. baltica's natural environment, as growth parameters like pH 7.5 have been established as optimal for this organism .
How does FliE expression correlate with the morphological changes in R. baltica's life cycle?
R. baltica undergoes significant morphological transitions throughout its life cycle:
The early exponential growth phase is dominated by motile swarmer cells and budding cells . As the culture transitions to later growth phases, the population shifts toward sessile cells and rosette formations . Gene expression studies have revealed differential regulation of numerous genes during these transitions .
While the provided search results don't specifically address FliE expression patterns during these transitions, the whole genome microarray approach described could be applied to examine whether fliE expression correlates with specific morphological states, particularly the transition between motile and sessile forms.
What role might FliE play in R. baltica's adaptation to marine environments?
Considering R. baltica's environmental niche:
R. baltica exhibits salt resistance and forms biofilms through the production of holdfast substances, particularly in the adult phase of its cell cycle . The organism's cell wall composition is modified in response to changing environmental conditions . These adaptations suggest specialized mechanisms for surviving in marine environments.
The flagellar apparatus, including FliE as a critical component, likely plays a role in R. baltica's ability to navigate its environment and transition between planktonic and biofilm lifestyles. Understanding the regulation and function of flagellar proteins like FliE in response to environmental stimuli could provide insights into the organism's ecological strategies.
