Recombinant Rhodopirellula baltica ATP-dependent zinc metalloprotease FtsH 1 (ftsH1)

What is Rhodopirellula baltica ATP-dependent zinc metalloprotease FtsH 1?

Rhodopirellula baltica ATP-dependent zinc metalloprotease FtsH 1 (ftsH1) is a full-length protein (672 amino acids) encoded by the ftsH1 gene (locus tag RB2966) in the marine bacterium Rhodopirellula baltica strain SH1. It belongs to the FtsH family of ATP-dependent zinc metalloproteases with Enzyme Commission number 3.4.24.-. The protein has a UniProt accession number Q7UUZ7 . Its amino acid sequence begins with "MKKDSESNSSDK..." and contains characteristic domains including an ATPase region and zinc-binding motifs essential for its proteolytic activity .

Why is Rhodopirellula baltica significant as a model organism?

Rhodopirellula baltica serves as a model organism for several unique biological processes and has significant ecological importance. It is a representative of the globally distributed phylum Planctomycetes, which plays a substantial role in carbon cycling in aquatic ecosystems . R. baltica exhibits several distinctive characteristics, including:

• Peptidoglycan-free proteinaceous cell walls

• Intracellular compartmentalization

• Reproduction via budding resulting in a life cycle with motile and sessile morphotypes

• Salt resistance and adhesion potential in the adult phase of the cell cycle

• A unique set of sulfatase genes and C1-metabolism pathways

These features make R. baltica particularly valuable for studying alternative cell biology and specialized metabolic pathways . Additionally, it has been identified as a model organism for aerobic carbohydrate degradation in marine systems .

What are the key structural features of R. baltica FtsH1?

R. baltica FtsH1 shares structural characteristics with other FtsH family proteins while possessing unique features. Key structural elements include:

• N-terminal region containing a transmembrane domain (membrane anchoring)

• ATPase domain with Walker A and B motifs (GPPGTGKTLLARAVAGE sequence region) responsible for ATP binding and hydrolysis

• Proteolytic domain with zinc-binding motifs that catalyzes peptide bond hydrolysis

• Full protein length spanning 672 amino acids with an expression region of 1-672

The protein likely forms a hexameric ring structure typical of AAA+ (ATPases Associated with diverse cellular Activities) family proteins, with the proteolytic sites facing the central pore through which substrates are threaded during degradation.

How does FtsH1 expression change during different growth phases in R. baltica?

While specific data on FtsH1 expression throughout the R. baltica growth cycle wasn't directly provided in the search results, related gene expression patterns during growth transitions provide valuable context. During the transition from exponential to stationary phase, R. baltica exhibits:

• Upregulation of glutamate dehydrogenase (RB6930), involved in the biosynthesis of amino acids including proline, a major component of R. baltica's cell wall

• Induction of stress-response genes including glutathione peroxidase (RB2244), thioredoxin (RB12160), bacterioferritin comigratory protein (RB12362), and universal stress protein (uspE, RB4742)

• Differential regulation of various dehydrogenases, hydrolases, and reductases for metabolic adaptation to nutrient limitation

As FtsH proteases typically function in protein quality control and stress response, FtsH1 expression likely increases during stress conditions to manage damaged proteins, though specific experimental verification is needed.

What are the optimal conditions for storing and handling recombinant R. baltica FtsH1?

Based on product specifications for recombinant R. baltica FtsH1, the following storage and handling conditions are recommended:

• Storage buffer: Tris-based buffer with 50% glycerol, optimized for protein stability

• Storage temperature: -20°C for regular use; -80°C for extended storage

• Working aliquots: Can be kept at 4°C for up to one week

• Stability considerations: Repeated freezing and thawing is not recommended as it may lead to protein denaturation and loss of activity

For experimental use, the protein should be thawed gently on ice and centrifuged briefly before opening to collect any condensation. Working dilutions should be prepared in appropriate buffers containing stabilizing agents such as BSA or glycerol.

How can CRISPR-Cas9 be used to modify FtsH1 for functional studies?

CRISPR-Cas9 technology offers powerful approaches for targeted genetic modification of FtsH1. Based on methodologies described for other FtsH proteins, the following strategy can be adapted for R. baltica FtsH1:

• Guide RNA design and construct preparation:

  • Design a guide RNA targeting a specific region of the ftsH1 gene

  • Create a plasmid containing Cas9 and the guide RNA sequence

  • Verify the specificity of the guide using sequence analysis tools

• Homology-directed repair template construction:

  • Amplify approximately 800 bp sequences upstream and downstream of the target site as homology regions

  • Insert desired modifications in the FtsH1 coding sequence (e.g., point mutations in the active site)

  • Include a selectable marker for screening transformants

• Transformation and screening:

  • Introduce both CRISPR-Cas9 and repair template into R. baltica

  • Select transformants on appropriate media

  • Verify successful modification using PCR and sequencing

This approach enables precise genome editing for creating point mutations, domain deletions, or fluorescent protein fusions to study structure-function relationships in FtsH1.

What methods are most effective for analyzing FtsH1 enzymatic activity?

A comprehensive approach to characterizing R. baltica FtsH1 enzymatic activity should include:

• ATPase activity assays:

  • Malachite green phosphate detection method

  • Coupled enzyme assays using pyruvate kinase and lactate dehydrogenase

  • Analysis of ATP hydrolysis kinetics (Km, Vmax, effect of inhibitors)

• Proteolytic activity assays:

  • Fluorogenic peptide substrates with FRET-based detection

  • SDS-PAGE analysis of model substrate degradation

  • Mass spectrometry to identify cleavage sites and products

• Optimal conditions determination:

  Parameter	Methodology	Expected Range
  pH optimum	Activity assays in different buffers	pH 7.0-8.5
  Temperature optimum	Activity at various temperatures	25-37°C
  Metal dependence	Activity with various metal ions	Zn²⁺ requirement
  Salt tolerance	Activity at different salt concentrations	0-500 mM NaCl

• Substrate specificity analysis:

  • Peptide library screening

  • Comparison with known substrates from other organisms

  • Computational prediction of recognition motifs

These approaches provide complementary data on the biochemical properties and functional characteristics of FtsH1.

How can I design experiments to investigate FtsH1 regulation during stress conditions?

To investigate FtsH1 regulation under stress conditions in R. baltica, implement this experimental design:

• Stress condition parameters:

  • Heat shock (4-8°C above optimal growth temperature)

  • Oxidative stress (H₂O₂ or paraquat exposure)

  • Nutrient limitation (carbon, nitrogen, phosphorus)

  • Salt stress (elevated NaCl concentrations)

  • Stationary phase transition

• Transcriptional analysis:

  • RT-qPCR targeting ftsH1 mRNA levels

  • Promoter analysis using reporter constructs

  • RNA-seq to place FtsH1 regulation in the context of global stress response

• Proteomic analysis:

  • Western blotting with anti-FtsH1 antibodies

  • 2D gel electrophoresis following protocols similar to those used for R. baltica carbohydrate metabolism studies

  • Mass spectrometry to quantify protein levels and identify post-translational modifications

• Physiological correlations:

  • Monitor growth parameters in wild-type versus FtsH1-modified strains

  • Analyze cell morphology changes using microscopy

  • Measure sensitivity to various stressors

This comprehensive approach would elucidate how FtsH1 is regulated as part of the stress response network in R. baltica, building on previous studies of differential protein expression during growth phase transitions .

What approaches can help resolve contradictory results in FtsH1 functional studies?

When facing contradictory results in FtsH1 research, consider the following analytical framework:

• Methodological variables analysis:

  Variable	Assessment Method	Resolution Approach
  Protein preparation	SDS-PAGE, mass spectrometry	Standardize purification protocol
  Assay conditions	Systematic parameter variation	Identify condition-dependent effects
  Strain differences	Genomic sequencing	Use isogenic strains or complementation
  Growth phase	Growth curve monitoring	Standardize harvesting points

• Experimental design modifications:

  • Increase biological and technical replicates

  • Include appropriate positive and negative controls

  • Implement blinding procedures where applicable

  • Use orthogonal techniques to verify key findings

• Data integration strategies:

  • Meta-analysis of multiple studies

  • Bayesian approaches to integrate prior knowledge

  • Systems biology modeling to contextualize contradictory results

  • Collaboration with groups reporting different outcomes

This systematic approach addresses the multifactorial nature of protein function and helps distinguish genuine biological variability from methodological artifacts.

How does R. baltica FtsH1 compare to FtsH proteins in other bacterial species?

Comparative analysis of FtsH proteins across bacterial species reveals both conserved features and specialized adaptations:

The comparative approach highlights several aspects:

• Core domains (ATPase, zinc-binding sites) are highly conserved

• N-terminal regions show greater variability, reflecting membrane organization differences

• Substrate specificity has evolved to match the specific proteome and stress conditions of each organism

• R. baltica FtsH1 likely has adaptations related to its unusual cell biology, including peptidoglycan-free cell walls and compartmentalized structure

This evolutionary context provides insights into both the fundamental mechanisms of FtsH proteases and the specialized functions that have evolved in different bacterial lineages.

What are the most promising directions for future research on R. baltica FtsH1?

Several high-priority research directions for R. baltica FtsH1 include:

• Structural biology:

  • Determination of crystal or cryo-EM structure

  • Analysis of hexamer formation and substrate binding sites

  • Investigation of conformational changes during ATP hydrolysis cycle

• Systems biology:

  • Integration of FtsH1 into R. baltica stress response networks

  • Identification of the complete substrate repertoire

  • Understanding the coordination between FtsH1 and other proteases

• Biotechnological applications:

  • Exploration of FtsH1 as a tool for controlled protein degradation

  • Investigation of potential applications in protein engineering

  • Development of inhibitors or activators for research tools

• Ecological significance:

  • Analysis of FtsH1 role in R. baltica's adaptation to marine environments

  • Comparative studies across Planctomycetes from different habitats

  • Investigation of FtsH1 contribution to R. baltica's role in carbon cycling

These research directions build upon the foundation of current knowledge while addressing significant gaps in our understanding of this important protein.

How can bioinformatic approaches enhance our understanding of FtsH1 function?

Bioinformatic approaches offer powerful tools for analyzing R. baltica FtsH1:

• Sequence analysis:

  • Multiple sequence alignment with FtsH proteins from diverse bacteria

  • Identification of conserved motifs and variable regions

  • Phylogenetic analysis to understand evolutionary relationships

• Structural prediction:

  • Homology modeling based on solved FtsH structures

  • Prediction of substrate binding sites and specificity determinants

  • Molecular dynamics simulations of protein dynamics

• Functional prediction:

  • Inference of substrate specificity from sequence features

  • Prediction of protein-protein interaction networks

  • Identification of potential regulatory mechanisms

• Integration with experimental data:

  • Mapping of experimental findings to structural models

  • Interpretation of mutant phenotypes based on predicted structural effects

  • Generation of testable hypotheses for experimental validation