Recombinant Yersinia pseudotuberculosis serotype IB Probable intracellular septation protein A (YPTS_2190)

What are the alternative names and identifiers for YPTS_2190?

The YPTS_2190 protein is known by several alternative names and identifiers:

• Gene Name: YPTS_2190

• Synonyms: yciB, Inner membrane-spanning protein YciB

• UniProt ID: B2K3V5

• Recommended Name: Probable intracellular septation protein A

These identifiers are essential for cross-referencing research data and accessing protein information in various databases .

What are the optimal storage conditions for recombinant YPTS_2190 protein?

For optimal preservation of protein activity, the following storage guidelines should be implemented:

• Long-term storage: Store at -20°C/-80°C

• Working aliquots: Maintain at 4°C for up to one week

• Storage buffer: Tris/PBS-based buffer with 6% Trehalose, pH 8.0 (or Tris-based buffer with 50% glycerol)

• Important precaution: Avoid repeated freeze-thaw cycles as this can significantly degrade protein quality

For reconstitution, briefly centrifuge the vial before opening to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (with 50% being standard) is recommended for aliquoting and long-term storage .

How does the membrane topology of YPTS_2190 influence its functional properties in bacterial cell division?

The membrane topology of YPTS_2190 is characterized by multiple transmembrane domains, as evidenced by its amino acid sequence motifs (MKQLLDFLPLVVFFIFYKMYDIFVASGALIVATLVALAFTW...). Analysis reveals hydrophobic regions consistent with membrane-spanning segments and charged residues that likely reside in cytoplasmic or periplasmic domains.

This topology is critical for its presumed role in septation, where it may participate in:

• Membrane remodeling during cell division

• Protein-protein interactions with other divisome components

• Coordination of peptidoglycan synthesis with membrane invagination

Research examining this protein in relation to other bacterial septation models suggests its position in the inner membrane allows it to function as a bridging element between cytoskeletal division machinery and membrane reorganization events. This interaction network makes it a potential target for understanding basic bacterial cell division mechanisms .

What experimental approaches would best elucidate the interaction of YPTS_2190 with other proteins in the septation complex?

Multiple complementary approaches should be employed to comprehensively map the protein-protein interactions of YPTS_2190:

• Bimolecular Fluorescence Complementation (BiFC): This technique can identify direct protein interactions in their native cellular environment. Following the approach demonstrated with Atg11 and Ypt1 interactions, researchers can fuse YPTS_2190 and potential binding partners with split fluorescent protein fragments to visualize interactions in vivo .

• Co-immunoprecipitation with His-tag leverage: The His-tagged recombinant YPTS_2190 enables efficient pull-down assays to identify binding partners. Following isolation of protein complexes, mass spectrometry analysis can identify unknown interactors.

• Yeast two-hybrid screening: This can serve as an initial high-throughput approach to identify potential interactors from a genomic library.

• Fluorescence co-localization studies: Using fluorescently tagged YPTS_2190 in conjunction with other labeled septal proteins would reveal spatial and temporal patterns of co-localization during the cell division cycle.

These approaches, particularly when combined, provide mechanistic insights into how YPTS_2190 participates in the bacterial cell division process .

How can researchers effectively compare structure-function relationships between YPTS_2190 and homologous proteins in other bacterial species?

A systematic approach to structure-function analysis should include:

• Multiple sequence alignment: Generate comprehensive alignments of YPTS_2190 with homologs from diverse bacterial species to identify conserved domains and species-specific variations.

• Evolutionary analysis: Construct phylogenetic trees to understand evolutionary relationships and functional divergence among homologs.

• Domain swapping experiments: Create chimeric proteins by exchanging domains between YPTS_2190 and homologs to determine which regions confer species-specific functions.

• Site-directed mutagenesis: Target highly conserved residues for mutation to assess their contribution to protein function.

• Complementation studies: Express YPTS_2190 in strains lacking homologous proteins to evaluate functional conservation and divergence.

This integrated approach allows researchers to map functional domains to specific structural elements and understand how variations in protein architecture influence cellular processes across bacterial species .

What is the recommended protocol for expression and purification of recombinant YPTS_2190?

Expression and Purification Protocol:

• Expression System Selection:

  • Recommended host: E. coli (as successfully used in available recombinant forms)

  • Expression vector: containing N-terminal His-tag for purification

  • Culture conditions: Optimize temperature (typically 18-25°C post-induction) to enhance soluble protein yield

• Protein Induction:

  • Grow culture to mid-log phase (OD600 = 0.6-0.8)

  • Induce with IPTG (0.1-1.0 mM)

  • Continue expression for 4-16 hours (optimize time for maximum yield)

• Cell Lysis:

  • Harvest cells by centrifugation (5,000 × g, 10 min, 4°C)

  • Resuspend in lysis buffer containing appropriate protease inhibitors

  • Lyse cells using sonication or pressure-based methods

• Purification:

  • Ni-NTA affinity chromatography using the N-terminal His-tag

  • Wash extensively to remove non-specific binding

  • Elute with imidazole gradient

• Post-purification Processing:

  • Dialyze against Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • Concentrate to desired concentration

  • Flash-freeze aliquots for long-term storage

This protocol yields greater than 90% purity as determined by SDS-PAGE analysis .

How should researchers design experiments to investigate YPTS_2190 function in intracellular septation?

A comprehensive experimental design should include:

• Gene Knockout and Complementation:

  • Create YPTS_2190 deletion mutants

  • Observe phenotypic changes in cell division, morphology, and growth rate

  • Complement with wild-type and mutant variants to confirm phenotype specificity

• Fluorescent Tagging for Localization:

  • Generate C- or N-terminal fluorescent protein fusions (carefully considering topology)

  • Perform time-lapse microscopy to track protein localization during cell division

  • Co-localize with known septation markers (FtsZ, FtsA)

• Interaction Studies:

  • Apply BiFC assays as described in research on other septation proteins

  • Use pull-down assays with the His-tagged recombinant protein

• Environmental Variables Testing:

  • Assess function under varying conditions (temperature, pH, osmotic stress)

  • Examine how environmental factors affect localization and interaction patterns

• Data Collection and Analysis:

  • Record quantitative measurements of division timing, septum formation, and localization dynamics

  • Organize data in properly formatted tables following scientific reporting standards

This multifaceted approach provides both qualitative and quantitative insights into YPTS_2190 function .

What are the critical quality control measures for verifying recombinant YPTS_2190 integrity before experimental use?

Essential quality control procedures include:

• Purity Assessment:

  • SDS-PAGE analysis (standard should exceed 90% purity)

  • Densitometry analysis of protein bands

• Identity Verification:

  • Western blot using antibodies against the His-tag and/or YPTS_2190

  • Mass spectrometry to confirm protein identity and detect modifications

  • N-terminal sequencing of at least 10 amino acids

• Folding Verification:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Limited proteolysis to evaluate structural integrity

  • Size exclusion chromatography to detect aggregation

• Functional Testing:

  • Binding assays with known interaction partners

  • Activity assays if enzymatic function is established

• Contamination Screening:

  • Endotoxin testing if intended for cellular assays

  • Nucleic acid contamination assessment

These quality control measures ensure experimental reproducibility and reliability of subsequent functional studies .

How can researchers effectively incorporate YPTS_2190 into multicellular experimental models to study bacterial pathogenesis?

When incorporating YPTS_2190 into pathogenesis studies using multicellular models, researchers should:

• Select Appropriate Models:

  • Multicellular tumor spheroids offer three-dimensional tissue architecture that mimics avascular regions

  • Infection models should incorporate both normoxic and hypoxic conditions to replicate in vivo environments

• Develop Tagged Protein Variants:

  • Create fluorescently tagged YPTS_2190 constructs to track localization

  • Develop antibodies against YPTS_2190 for immunohistochemistry in tissue sections

• Design Cellular Uptake Experiments:

  • Similar to evaluating doxorubicin delivery, assess how YPTS_2190 interacts with host cells using an individual-cell-based mathematical model

  • Track protein distribution through tissue layers to understand diffusion parameters

• Measure Host Response:

  • Evaluate host cell transcriptional and proteomic responses to YPTS_2190 exposure

  • Assess cytokine production and inflammatory signaling pathways

• Data Collection Framework:

  • Implement systematic data collection that captures both spatial and temporal dynamics

  • Utilize quantitative image analysis for protein distribution studies

This integrated approach draws on established methodologies for investigating bacterial protein interactions with host tissues while specifically addressing the unique properties of YPTS_2190 .

What statistical approaches are most appropriate for analyzing YPTS_2190-related experimental data?

When analyzing experimental data related to YPTS_2190, researchers should employ the following statistical approaches:

• For Localization Studies:

  • Pearson's correlation coefficient for co-localization analysis

  • Spatial distribution analysis using Ripley's K-function

  • Time-series analysis for dynamic localization studies

• For Protein-Protein Interaction Data:

  • ANOVA with post-hoc tests for comparing multiple interaction partners

  • Non-parametric tests (Mann-Whitney U) when normality cannot be established

  • Multiple testing correction (FDR) for high-throughput interaction screens

• For Functional Impact Studies:

  • Survival analysis techniques for assessing effects on bacterial viability

  • Mixed-effects models for experiments with nested design structures

  • Power analysis to determine appropriate sample sizes (minimum n=3 biological replicates)

• For Imaging Data:

  • Proper background subtraction and normalization procedures

  • Automated object identification and tracking algorithms

  • Machine learning approaches for pattern recognition in complex datasets

• Data Presentation:

  • Properly formatted data tables following scientific standards

  • Appropriate error representation (standard deviation vs. standard error)

How should data tables be structured to effectively present YPTS_2190 research findings?

Data tables for YPTS_2190 research should follow these guidelines for maximum clarity and scientific rigor:

• Table Title and Structure:

  • Clear, descriptive title indicating the experimental purpose

  • Independent variables in the left column

  • Dependent variables in subsequent columns

  • Derived calculations (averages, ratios) in the rightmost columns

• Example Table Structure:

Table 1: Effects of Environmental Conditions on YPTS_2190 Localization

• Essential Elements:

  • Include units of measurement in column headers

  • Present mean values with appropriate error measurements (±SD or ±SEM)

  • Maintain consistent significant figures

  • Include sample size (n) in table footnotes or headers

• Additional Considerations:

  • Use clear row and column dividers

  • Apply consistent formatting throughout

  • Include explanatory footnotes for abbreviations or statistical methods

  • Number tables sequentially and refer to them explicitly in text

Following these guidelines ensures that data is presented in a standardized format that facilitates interpretation and comparisons across studies .

How can contradictory findings in YPTS_2190 functional studies be reconciled and addressed methodologically?

When facing contradictory results in YPTS_2190 research, scientists should implement the following systematic approach:

• Methodological Reconciliation Strategy:

  • Perform side-by-side comparison of contradictory protocols using identical biological materials

  • Systematically vary individual experimental parameters to identify sources of variation

  • Implement blinded analysis to minimize unconscious bias in data interpretation

• Common Sources of Contradiction to Investigate:

  • Expression system differences (E. coli strain variations, induction conditions)

  • Tag position effects (N-terminal vs. C-terminal tags may differently affect function)

  • Buffer composition variations that may alter protein folding or activity

  • Cell growth phase differences affecting septation protein function

  • Antibody specificity issues in detection methods

• Resolution Approach:

  • Create a standardized experimental pipeline with defined quality control checkpoints

  • Utilize multiple complementary techniques to verify each finding

  • Perform collaborative cross-laboratory validation studies

  • Document and share detailed protocols including "silent variables" often omitted from methods sections

• Reporting Recommendations:

  • Explicitly acknowledge contradictions in the literature

  • Present both supporting and contradicting evidence

  • Provide specific hypotheses for observed differences

This systematic approach establishes a framework for reconciling contradictory findings while advancing understanding of YPTS_2190 function .

What are the most promising research directions for understanding YPTS_2190 role in bacterial pathogenesis?

Several high-potential research directions emerge from current understanding of YPTS_2190:

• Infection Dynamics Studies:

  • Investigate how YPTS_2190 influences Yersinia pseudotuberculosis invasion processes

  • Determine if YPTS_2190 functions independently or cooperatively with YadA, a major adhesin that promotes tight adhesion to mammalian cells

  • Examine potential competitive or synergistic interactions with invasin, the major invasive factor

• Host-Pathogen Interface:

  • Explore whether YPTS_2190 participates in host membrane interaction similar to YadA's binding to extracellular matrix proteins

  • Determine if YPTS_2190 contributes to bacterial adhesion and invasion efficiency

  • Investigate potential roles in immune evasion mechanisms

• Structural Biology Approaches:

  • Determine high-resolution crystal structure to identify functional domains

  • Map interaction interfaces with host and bacterial proteins

  • Develop structure-based inhibitor design targeting conserved functional motifs

• Systems Biology Integration:

  • Map YPTS_2190 within the broader network of virulence factors

  • Identify regulatory mechanisms controlling its expression during infection

  • Develop mathematical models predicting infection dynamics based on YPTS_2190 expression levels

These research directions offer potential for significant advances in understanding how this membrane protein contributes to bacterial pathogenesis and could identify novel therapeutic targets .

How might advancements in imaging and computational techniques enhance YPTS_2190 functional studies?

Emerging technologies offer transformative approaches to studying YPTS_2190 function:

• Advanced Imaging Applications:

  • Super-resolution microscopy (PALM/STORM) to visualize YPTS_2190 nanoscale organization within bacterial membranes

  • Cryo-electron tomography to observe YPTS_2190 in near-native environments

  • Single-molecule tracking to monitor dynamic behavior during septation events

  • FRET-based biosensors to detect conformational changes during protein activation

• Computational Modeling Enhancements:

  • Molecular dynamics simulations to predict membrane protein behavior and interactions

  • Individual-cell-based mathematical models adapted from tumor spheroid research to simulate bacterial populations

  • Machine learning approaches to identify patterns in large-scale phenotypic screens

  • Network analysis to place YPTS_2190 within the context of bacterial protein interaction networks

• Implementation Strategy:

  • Combine experimental data with computational predictions in iterative cycles

  • Develop quantitative metrics for model validation

  • Create standardized data formats to facilitate integration across platforms

• Example Application:

  • Adapt the individual-cell-based mathematical model used for doxorubicin delivery to track YPTS_2190 distribution within bacterial populations

  • Incorporate variables for protein diffusion, membrane localization, and septation dynamics

  • Generate predictions for protein behavior under various environmental conditions

These technological applications provide unprecedented resolution and predictive power for understanding YPTS_2190 function in complex biological contexts .

What standardized protocols should be established to ensure reproducibility in YPTS_2190 research?

To enhance reproducibility in YPTS_2190 research, the following standardized protocols are recommended:

• Protein Production and Quality Control:

  • Standardized expression system (E. coli strain, vector, induction parameters)

  • Defined purification protocol with specific buffer compositions

  • Comprehensive quality control checklist including purity thresholds and functional verification

• Functional Assays:

  • Validated localization protocols with controls for tag interference

  • Standardized interaction assays with positive and negative controls

  • Normalized reporting formats for quantitative measurements

• Data Collection and Management:

  • Minimum dataset requirements for publication

  • Standardized data table formats following scientific guidelines

  • Required deposition of raw data in appropriate repositories

• Methodological Transparency:

  • Detailed reporting of "silent variables" (laboratory temperature, plastic consumable brands)

  • Explicit description of randomization and blinding procedures

  • Comprehensive statistical analysis plans established before data collection

• Strain and Reagent Verification:

  • Sequence verification of all constructs

  • Mycoplasma and contamination testing

  • Antibody validation requirements

Implementing these standardized protocols will significantly enhance data reliability and cross-laboratory reproducibility, accelerating progress in understanding YPTS_2190 function .

How can researchers effectively integrate structural, genetic, and functional data to build comprehensive models of YPTS_2190 activity?

A multi-omics integration approach offers the most complete understanding of YPTS_2190:

• Data Integration Framework:

  • Establish centralized databases for YPTS_2190-related data across methodologies

  • Develop common ontologies and metadata standards

  • Implement computational pipelines for cross-platform data analysis

• Integration Methods:

  • Structure-function correlation through combined crystallography and mutagenesis

  • Network analysis linking genetic interactions with protein-protein interactions

  • Temporal integration mapping expression dynamics to functional outcomes

  • Spatial integration correlating subcellular localization with interaction partners

• Workflow Example:

  • Begin with structural determination (X-ray crystallography or cryo-EM)

  • Map conserved domains through comparative genomics

  • Validate functional predictions through targeted mutagenesis

  • Place findings in cellular context through localization studies

  • Integrate with systems-level data on bacterial pathogenesis

• Visualization and Analysis:

  • Develop interactive visualization tools for multi-dimensional data exploration

  • Implement machine learning approaches to identify patterns across datasets

  • Create mathematical models incorporating data from multiple experimental approaches

This integrated approach transforms disparate data points into cohesive models with greater explanatory and predictive power than any individual methodology could provide .

What ethical considerations should guide research involving YPTS_2190 and other virulence-associated proteins?

Researchers studying YPTS_2190 and similar virulence factors should adhere to the following ethical framework:

• Biosafety Considerations:

  • Implement appropriate biosafety level protocols (BSL-2 minimum for Yersinia pseudotuberculosis)

  • Develop risk mitigation strategies for recombinant proteins with potential virulence functions

  • Ensure proper training and oversight for all laboratory personnel

• Dual-Use Research Assessment:

  • Evaluate potential for misuse of research findings

  • Implement the "Do No Harm" principle in experimental design

  • Balance scientific transparency with security considerations

• Collaborative Ethics:

  • Establish clear material transfer agreements for sharing strains and reagents

  • Define authorship and credit allocation in advance of multi-institution studies

  • Create frameworks for resolving disputes over intellectual property

• Publication Responsibility:

  • Consider security implications of methodological details

  • Practice responsible communication of findings to public audiences

  • Ensure appropriate contextual framing to prevent misinterpretation

• Environmental Impact:

  • Assess ecological risks of genetically modified organisms

  • Implement proper waste disposal procedures

  • Consider sustainability in research design