Recombinant Chlorokybus atmophyticus Peptidoglycan synthase ftsI homolog (ftsI)

What is Chlorokybus atmophyticus peptidoglycan synthase ftsI homolog?

Chlorokybus atmophyticus peptidoglycan synthase ftsI homolog (ftsI) is a protein involved in cell wall synthesis in this charophytic alga. According to UniProt classification (A2CI41), this protein shares homology with bacterial FtsI, which functions as a peptidoglycan synthase . The ftsI homolog in C. atmophyticus represents an evolutionary bridge between bacterial cell wall synthesis machinery and the more complex cell wall structures found in land plants. While bacterial FtsI is well-characterized as penicillin-binding protein 3 (PBP3) involved in septum formation during cell division, the C. atmophyticus homolog likely has adapted functions related to the unique "pseudo-pectin" cell wall components found in this alga . This adaptation reflects the evolutionary transition from bacterial-type cell walls to the more complex polysaccharide structures characteristic of plants.

How is the ftsI gene conserved across different species?

The ftsI gene shows varying degrees of conservation with evidence of both vertical inheritance and horizontal gene transfer across species. In bacterial systems like Haemophilus influenzae, the ftsI gene undergoes horizontal gene transfer between different strains and even between different species such as H. influenzae and H. haemolyticus . Sequence analysis revealed that the divergence between H. haemolyticus ATCC 33390 and H. influenzae Rd was 12.7% (233 bp of 1,833 bp) . This transfer is facilitated by the presence of uptake signal sequences (USSs) located both within and downstream of the ftsI gene. The mosaic structure observed in some ftsI genes provides direct evidence of recombination events. For C. atmophyticus, while specific conservation data is not provided in the search results, its ftsI homolog likely represents a more distant evolutionary relationship to bacterial ftsI genes, reflecting the divergence of charophytic algae from the bacterial lineage.

What is the function of FtsI in cellular processes?

FtsI plays a crucial role in bacterial cell division processes. In bacteria like Escherichia coli, FtsI (also called penicillin-binding protein 3 or PBP3) is essential for the assembly of the division septum . It localizes to the division site where it contributes to the synthesis of septal peptidoglycan during cell division. Studies using green fluorescent protein (GFP) fusions have demonstrated that FtsI requires both FtsZ and FtsA for proper localization to the division site in bacteria . The transpeptidase activity of FtsI is critical for its function, as evidenced by studies showing that treatment with furazlocillin (a β-lactam antibiotic that inactivates this activity) prevented FtsI localization . In C. atmophyticus, while the protein is homologous to bacterial FtsI, its precise function has likely evolved to accommodate the unique cell wall composition of this charophytic alga, which contains "pseudo-pectin" structures rather than typical bacterial peptidoglycan .

What is the relationship between FtsI and penicillin-binding proteins?

FtsI is classified as a penicillin-binding protein (PBP), specifically identified as PBP3 in bacteria such as Escherichia coli and Haemophilus influenzae . PBPs are enzymes that catalyze the final stages of peptidoglycan synthesis and are the primary targets of β-lactam antibiotics. The relationship between FtsI and other PBPs is characterized by shared structural features, particularly the transpeptidase domain that cross-links peptidoglycan strands. According to research on H. influenzae, mutations in the ftsI gene can lead to resistance to β-lactam antibiotics, resulting in β-lactamase-nonproducing ampicillin-resistant (BLNAR) strains . The essential nature of FtsI's transpeptidase activity was demonstrated when treatment with furazlocillin (a β-lactam antibiotic that specifically inactivates this activity) prevented proper FtsI localization in division sites . In the evolutionary context, the C. atmophyticus FtsI homolog represents how PBP-like proteins have been maintained even as cell wall composition has drastically changed from bacterial peptidoglycan to plant-like polysaccharide structures.

How does Chlorokybus atmophyticus FtsI compare to bacterial FtsI proteins?

The comparison between C. atmophyticus FtsI and bacterial FtsI proteins reveals important evolutionary adaptations. While both share homology and are classified as peptidoglycan synthases, they function in fundamentally different cell wall environments. Bacterial FtsI works with peptidoglycan, while C. atmophyticus has a unique cell wall composition featuring "pseudo-pectin" structures . This charophytic alga possesses anionic polysaccharides containing glucuronic acid (GlcA), galacturonic acid (GalA), and sulfate, plus neutral sugars (Ara≈Glc>Gal>Xyl) but notably lacks rhamnose which is common in land plant pectins . A distinctive feature is the presence of a relatively acid-resistant β-d-GlcA-(1→4)-l-Gal disaccharide . These substantial differences in substrate composition suggest that while C. atmophyticus FtsI may retain core catalytic mechanisms from its bacterial counterparts, it has likely evolved specialized domains for interacting with these unique polysaccharides. This comparison illustrates the remarkable plasticity of these enzymes through evolution, adapting from bacterial cell division to the more complex cell wall synthesis requirements of algae on the evolutionary path to land plants.

What techniques are most effective for studying FtsI localization in cellular models?

For studying FtsI localization, two complementary approaches have proven particularly effective, each with specific methodological considerations. Immunofluorescence microscopy (IFM) has been utilized to track FtsI localization, though sensitivity issues have been reported. According to research by Wang et al., IFM signals for FtsI were "extremely weak," potentially causing localization patterns to be overlooked in certain cellular structures . A more robust approach involves green fluorescent protein (GFP) fusion techniques, which provide significantly improved sensitivity and specificity. The optimal methodology includes fusing FtsI to a bright variant of GFP from Aequorea victoria and placing this construct under the control of a regulatable promoter (such as an IPTG-inducible system) . To ensure physiologically relevant expression levels and avoid variability from plasmid copy number fluctuations, the fusion construct should be chromosomally integrated rather than expressed from a plasmid . For C. atmophyticus studies specifically, these techniques would require adaptation to algal cellular systems, potentially using algal-optimized fluorescent proteins and appropriate promoters. Super-resolution microscopy techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) could provide enhanced visualization of FtsI localization patterns, especially in the context of the unique cell wall architecture of charophytic algae.

What are the implications of ftsI homologs in the evolution of cell wall biosynthesis?

The presence of ftsI homologs across diverse organisms from bacteria to algae provides profound insights into the evolution of cell wall biosynthesis. In evolutionary context, C. atmophyticus represents a crucial transitional form between bacterial ancestors and land plants. The co-existence of a peptidoglycan synthase ftsI homolog with the unique "pseudo-pectin" cell wall components in this alga suggests a fascinating evolutionary trajectory in which ancestral cell wall synthesis machinery was repurposed during the transition to more complex cell walls. The C. atmophyticus cell wall contains anionic polysaccharides with glucuronic acid (GlcA), galacturonic acid (GalA), and sulfate, plus neutral sugars (Ara≈Glc>Gal>Xyl), but notably lacks rhamnose which is common in land plant pectins . This composition represents an intermediate form between bacterial peptidoglycan and land plant cell walls. The evolution from C. atmophyticus to land plants involved the loss of sulfate, most L-Gal and most D-GlcA; reconfiguration of Ara, Xyl, and GalA; and the gain of Rha . These compositional changes would have required corresponding adaptations in the biosynthetic machinery, including ftsI-like proteins. This evolutionary perspective suggests that rather than developing entirely new biosynthetic pathways, early land plants may have repurposed and modified existing algal enzymes, with ftsI homologs potentially transitioning from peptidoglycan synthesis to roles in more complex polysaccharide assembly.

How does the structure of Chlorokybus atmophyticus FtsI influence its function compared to bacterial counterparts?

The structural adaptations of C. atmophyticus FtsI reflect its evolutionary divergence from bacterial counterparts and its specialized function in this charophytic alga. While detailed structural studies of C. atmophyticus FtsI are not provided in the search results, functional inferences can be made based on its cellular context. Bacterial FtsI (PBP3) contains a well-characterized transpeptidase domain that cross-links peptidoglycan strands during septum formation . In contrast, C. atmophyticus FtsI likely features structural modifications to interact with the alga's distinctive cell wall components. The presence of a β-d-GlcA-(1→4)-l-Gal disaccharide as a relatively acid-resistant component suggests that the binding pocket of C. atmophyticus FtsI may be optimized for recognition of this structure. Additionally, the sulfated nature of C. atmophyticus cell wall polysaccharides would require positively charged binding regions within the enzyme to facilitate interaction with these negatively charged substrates. The catalytic mechanism may also be adapted for the formation of different types of linkages than those found in bacterial peptidoglycan. These structural adaptations highlight the remarkable plasticity of this enzyme family through evolution, maintaining core catalytic functions while accommodating drastically different substrates. Detailed structural studies using X-ray crystallography or cryo-electron microscopy would be necessary to fully characterize these adaptations and their functional implications.

What are the current challenges in expressing and purifying functional recombinant FtsI?

Expression and purification of functional recombinant FtsI present several technical challenges that require methodological solutions. Based on information about the commercially available recombinant C. atmophyticus FtsI , specific buffer conditions including Tris-based buffer with 50% glycerol are necessary for stability, highlighting the protein's sensitivity to storage conditions. As a membrane-associated protein involved in cell wall synthesis, FtsI poses common challenges including: maintaining native conformation in heterologous expression systems, preventing aggregation and inclusion body formation, and preserving enzymatic activity during purification. Bacterial expression systems may struggle with proper folding of the algal protein due to differences in membrane composition and chaperone availability. Expression strategies should consider using low induction temperatures (16-20°C) to slow protein production and enhance folding. Purification challenges include the need for detergents or membrane mimetics to maintain the native structure of membrane-associated domains. For activity assays, the unique substrates of C. atmophyticus FtsI (related to its "pseudo-pectin" cell wall) may not be commercially available, requiring custom synthesis of substrate analogs. Additionally, the potential presence of post-translational modifications in the native algal protein may necessitate eukaryotic expression systems rather than bacterial ones. These challenges explain why specialized storage conditions (like the 50% glycerol mentioned in the product description ) are necessary for maintaining stability of the purified protein.

What expression systems are optimal for producing recombinant Chlorokybus atmophyticus FtsI?

Selecting the optimal expression system for C. atmophyticus FtsI requires careful consideration of protein characteristics and experimental goals. While prokaryotic systems offer simplicity and high yields, eukaryotic systems may better accommodate the folding requirements of this algal protein. Escherichia coli remains a first-line option, particularly with specialized strains like BL21(DE3) Rosetta that supply rare codons or C41/C43 designed for membrane protein expression. Codon optimization of the algal gene for E. coli usage is essential, as is fusion to solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO. For challenging cases where E. coli yields improperly folded protein, yeast systems (Pichia pastoris or Saccharomyces cerevisiae) offer eukaryotic folding machinery while maintaining reasonable yields and cost-effectiveness. For highest fidelity to the native protein, algal expression systems like Chlamydomonas reinhardtii might be considered, though with lower yields and more complex protocols. Expression conditions require careful optimization, with lower temperatures (16-20°C) generally favoring proper folding over expression rate. Initial expression trials should test multiple constructs with different purification tags (His, GST, MBP) positioned at either terminus to identify configurations that minimize interference with folding and function. The product description indicating storage in Tris-based buffer with 50% glycerol suggests that stability is a significant concern, highlighting the importance of rapid purification protocols and appropriate buffer composition to maintain functional integrity.

What protocols are recommended for assessing the enzymatic activity of recombinant FtsI?

Assessing the enzymatic activity of recombinant C. atmophyticus FtsI requires specialized protocols that account for its unique substrate specificity and evolutionary context. Transpeptidase activity assays represent the foundation of functional characterization, requiring synthetic substrates that mimic the native cell wall components. Given the distinctive "pseudo-pectin" composition in C. atmophyticus—containing GlcA, GalA, sulfate, and neutral sugars (Ara≈Glc>Gal>Xyl) —custom synthesis of substrate analogs containing the characteristic β-d-GlcA-(1→4)-l-Gal disaccharide would be necessary. Activity can be monitored through various analytical techniques including high-performance liquid chromatography (HPLC), mass spectrometry, or colorimetric assays using modified substrates with chromogenic or fluorogenic leaving groups. β-lactam binding assays provide an alternative approach, utilizing fluorescent or biotinylated β-lactams to quantify binding affinity through techniques like fluorescence polarization or surface plasmon resonance. These assays can determine whether the C. atmophyticus FtsI maintains the penicillin-binding characteristic of its bacterial homologs despite evolutionary divergence. For validating physiological relevance, heterologous complementation studies in bacterial systems with temperature-sensitive ftsI mutations could assess whether the algal homolog retains any functional conservation. Inhibition studies using various classes of β-lactams and other cell wall synthesis inhibitors would establish the pharmacological profile of the enzyme and potentially reveal mechanistic insights. Throughout these assays, careful attention to buffer composition is essential, with inclusion of appropriate stabilizing agents as suggested by the storage conditions (Tris-based buffer, 50% glycerol) mentioned in the product description .

How can mutagenesis be used to investigate structure-function relationships in FtsI?

Mutagenesis offers powerful approaches for deciphering structure-function relationships in C. atmophyticus FtsI, particularly when guided by evolutionary and structural insights. A comprehensive mutagenesis strategy should begin with sequence alignment across bacterial FtsI proteins and available algal homologs to identify conserved residues likely essential for catalytic function versus divergent regions that may confer substrate specificity. Site-directed mutagenesis should target the predicted active site residues, particularly those in the transpeptidase domain, with mutations ranging from conservative substitutions to assess subtle functional requirements to complete replacements that abolish activity. Domain swapping experiments between bacterial and algal FtsI can identify regions responsible for the differing substrate specificities, particularly relevant given the unique "pseudo-pectin" components in C. atmophyticus cell walls . Alanine-scanning mutagenesis across putative substrate-binding regions can systematically map residues involved in recognizing the distinctive β-d-GlcA-(1→4)-l-Gal disaccharide and sulfated polysaccharides characteristic of this alga . For investigating protein-protein interactions, mutations in predicted surface-exposed regions can identify interfaces involved in localization or multiprotein complex formation. Each mutant should be characterized through multiple approaches, including in vitro activity assays, binding studies with substrate analogs, localization analysis if expressed in vivo, and ideally structural studies to confirm the predicted effects on protein conformation. This systematic mutagenesis strategy can elucidate how this ancient enzyme family adapted from bacterial peptidoglycan synthesis to function in the dramatically different cell wall environment of charophytic algae.

What considerations are important when designing antibodies against Chlorokybus atmophyticus FtsI?

Designing effective antibodies against C. atmophyticus FtsI requires strategic considerations across multiple dimensions to ensure specificity, sensitivity, and versatility for different applications. Epitope selection represents the critical first step and should be guided by both sequence and structural analysis. Bioinformatic tools should identify regions unique to C. atmophyticus FtsI with minimal homology to other proteins in the organism or common experimental systems, reducing cross-reactivity. Hydrophilicity analysis, secondary structure prediction, and surface accessibility calculations can identify epitopes likely to be exposed in the native protein. For maximum versatility, multiple antibodies targeting different epitopes should be developed—linear epitopes from hydrophilic loops for Western blotting and immunoprecipitation, and conformational epitopes for applications requiring recognition of the native protein such as immunofluorescence or chromatin immunoprecipitation. The immunization strategy should consider both peptide antigens for targeting specific regions and properly folded recombinant protein domains for generating antibodies against conformational epitopes. Validation represents a critical phase, including positive controls using the recombinant protein , negative controls with closely related proteins, and testing across multiple applications and experimental conditions. For monoclonal antibody development, screening should prioritize clones that recognize the native protein under non-denaturing conditions. Special consideration should be given to potential post-translational modifications in the native algal protein that might be absent in recombinant versions expressed in bacterial systems, as these could affect epitope recognition. Thorough validation and characterization of antibody specificity will ensure reliable results in the diverse applications needed for studying this evolutionarily significant protein.

What statistical approaches are appropriate for analyzing FtsI evolutionary conservation data?

Analyzing evolutionary conservation of FtsI requires sophisticated statistical approaches that can detect patterns across multiple scales, from sequence-level conservation to broader evolutionary dynamics. For sequence-level analysis, pairwise and multiple sequence alignment tools establish basic conservation metrics, as demonstrated in the H. influenzae and H. haemolyticus comparison showing 12.7% divergence (233 bp of 1,833 bp) . Beyond simple identity metrics, position-specific scoring matrices can identify differentially conserved regions while accounting for biochemically similar substitutions. For detecting selection pressures, dN/dS ratio analysis (comparing nonsynonymous to synonymous substitution rates) can identify regions under purifying selection (conserved functional domains) versus positive selection (potentially indicating adaptation to new substrates). Sliding window analysis of sequence conservation can pinpoint domains evolving at different rates, particularly valuable for C. atmophyticus FtsI given its likely functional divergence from bacterial homologs. For detecting horizontal gene transfer events, statistical tests for recombination such as the phi test, MaxChi, or Geneconv can identify mosaic gene structures similar to those found in bacterial ftsI . Phylogenetic analysis should employ maximum likelihood or Bayesian approaches with appropriate substitution models, with hypothesis testing using likelihood ratio tests or Bayes factors to evaluate alternative evolutionary scenarios. Synteny analysis comparing gene neighborhood conservation provides additional evolutionary context beyond sequence-level metrics. These approaches collectively can reconstruct the evolutionary trajectory of FtsI from bacterial peptidoglycan synthesis to its role in the unique cell wall composition of charophytic algae, offering insights into the transition from bacterial to plant cell wall biosynthesis machinery.

What bioinformatic tools are most useful for studying FtsI protein domains?

A comprehensive bioinformatic analysis of FtsI protein domains requires an integrated toolkit that addresses structure, function, and evolutionary context. For primary domain identification, InterPro, PFAM, and SMART provide complementary approaches to recognizing conserved domains through different underlying algorithms and databases. For C. atmophyticus FtsI, these tools could identify both domains shared with bacterial homologs and potentially novel domains unique to algal proteins. Secondary structure prediction using PSIPRED, JPred, or SPIDER3 helps map structural elements like alpha-helices and beta-sheets that might be conserved despite sequence divergence. Transmembrane topology prediction is particularly relevant given FtsI's membrane association, with TMHMM, Phobius, and TOPCONS offering complementary approaches to mapping membrane-spanning regions. Active site prediction tools like ConSurf, which maps conservation onto structural models, can identify catalytic residues likely involved in peptidoglycan synthesis or interaction with the unique "pseudo-pectin" components of C. atmophyticus . Homology modeling platforms including SWISS-MODEL, I-TASSER, or AlphaFold2 can generate structural models based on related proteins with known structures, particularly valuable for regions sharing homology with crystallized bacterial PBPs. Protein-protein interaction site prediction using tools like SPPIDER can identify potential binding interfaces for interaction partners. Coiled-coil prediction tools like COILS or Paircoil2 may identify structural motifs involved in protein oligomerization. Signal peptide and subcellular localization prediction tools (SignalP, TargetP) can help determine how the protein is trafficked within the cell. Integration of these diverse analyses through platforms like Jalview or PyMOL allows visualization of multiple data types on sequence and structural models, facilitating holistic interpretation of domain architecture and functional predictions.

How can structural modeling help interpret experimental data on FtsI function?

Structural modeling provides a powerful framework for interpreting experimental data on FtsI function by connecting molecular-level details to functional observations. For C. atmophyticus FtsI, homology modeling using bacterial penicillin-binding proteins as templates can generate three-dimensional structural predictions despite potentially low sequence identity. These models should be validated using structure assessment tools and refined using molecular dynamics simulations to optimize conformations. Mutation mapping onto these structural models can provide mechanistic explanations for experimental phenotypes—for example, if mutations in a specific region affect activity against only certain substrates, this might indicate a substrate-specific binding pocket. Docking simulations can predict interactions with unique C. atmophyticus cell wall components like the β-d-GlcA-(1→4)-l-Gal disaccharide or sulfated polysaccharides , generating testable hypotheses about substrate specificity. Electrostatic surface analysis can identify positively charged patches potentially involved in interactions with negatively charged substrates like the anionic "pseudo-pectin" components . Conservation mapping onto the structural model can distinguish functionally critical regions (highly conserved) from more variable regions that might confer species-specific functions. Molecular dynamics simulations can predict conformational changes upon substrate binding or interactions with other proteins, particularly valuable for understanding complex assembly mechanisms. These structural insights can guide rational experimental design, including site-directed mutagenesis targeting predicted functional regions, construction of chimeric proteins to test domain functions, and design of specific inhibitors or activity probes. By integrating structural modeling with experimental data, researchers can develop a mechanistic understanding of how this ancient enzyme family has evolved from bacterial peptidoglycan synthesis to functioning in the unique cell wall environment of charophytic algae.