Recombinant Staphylococcus aureus UPF0060 membrane protein NWMN_2240 (NWMN_2240)

What is NWMN_2240 and how is it classified within bacterial proteins?

NWMN_2240 is a membrane protein belonging to the UPF0060 family, found in Staphylococcus aureus. As a membrane protein, it plays a potential role in bacterial cell envelope integrity, which is critical for S. aureus survival and pathogenicity. The UPF (Uncharacterized Protein Family) designation indicates that this protein has a recognized sequence pattern but unknown function. Being membrane-embedded, it likely contributes to cellular processes such as transport, signaling, or structural integrity of the bacterial membrane.

The protein would be classified among the approximately 5000 membrane-embedded proteins encoded in bacterial genomes that conduct essential processes including cell-to-cell communication, adhesion, and intracellular trafficking. In Staphylococcus aureus specifically, membrane proteins often contribute to virulence, antibiotic resistance, and adaptation to different host environments .

What genomic context surrounds the NWMN_2240 gene in S. aureus?

The NWMN_2240 gene exists within the complex genomic architecture of S. aureus, which is known for its remarkable ability to acquire and integrate foreign genetic material. S. aureus genomes frequently undergo recombination events that can lead to large-scale chromosomal replacements, similar to those observed in the formation of the ST239 strain that acquired a 60kb SCCmec-III element .

The genomic neighborhood of NWMN_2240 may provide clues about its function, as genes with related functions are often co-localized in bacterial genomes. Research would need to analyze whether NWMN_2240 is located near genes involved in specific cellular processes such as membrane transport, stress response, or virulence. Genomic analysis would also determine if this gene is located in core genome regions that are highly conserved across S. aureus strains or in more variable regions that might suggest strain-specific functions.

How prevalent is NWMN_2240 across different S. aureus clinical isolates?

The prevalence of NWMN_2240 across different S. aureus clinical isolates requires investigation through comparative genomic analysis. S. aureus shows significant genetic diversity through its evolutionary history, with various strains adapted to different environments and hosts. Some genetic elements in S. aureus are strain-specific, while others are conserved across lineages.

How might recombination events affect NWMN_2240 evolution and function?

Recombination events significantly impact S. aureus evolution, potentially affecting membrane proteins like NWMN_2240. S. aureus is known to undergo large-scale chromosomal replacements that can substantially alter gene content and expression. Five of six known cases of large-scale chromosomal replacement in S. aureus overlap with the acquired region of ST239, suggesting the existence of recombination hotspots within the genome . These recombination events can lead to dramatic changes in bacterial phenotype, including antibiotic resistance profiles and virulence characteristics.

For NWMN_2240, researchers should investigate whether this gene is located within known recombination hotspots and how its sequence varies between strains that have undergone recombination versus those that haven't. If NWMN_2240 is subject to frequent recombination, this could indicate functional plasticity or adaptability. Alternatively, if the gene remains highly conserved despite nearby recombination events, this suggests strong selective pressure to maintain its structure and function.

Methodologically, researchers could use comparative genomics to trace the evolutionary history of NWMN_2240 across diverse S. aureus lineages, identifying potential horizontal gene transfer events or recombination-driven alterations that might affect protein function or expression patterns.

What role might NWMN_2240 play in photodynamic inactivation (PDI) tolerance?

Research has demonstrated that S. aureus can develop tolerance to photodynamic inactivation (PDI) upon repeated exposure. Studies show that repeated sublethal doses of PDI lead to selective evolution of PDI-tolerant strains with global transcriptional responses in numerous cellular functions and acquisition of heritable mutations . Membrane proteins likely play critical roles in this adaptive response.

NWMN_2240, as a membrane protein, could potentially contribute to PDI tolerance through several mechanisms:

• Membrane integrity maintenance during oxidative stress

• Altered permeability to photosensitizers like methylene blue (MB)

• Involvement in stress response pathways

• Participation in cell wall remodeling under stress conditions

To investigate NWMN_2240's role in PDI tolerance, researchers could compare its expression levels between PDI-tolerant and PDI-sensitive strains. Experiments could include:

Recent studies have shown that PDI tolerance in S. aureus involves the quinone-sensing transcriptional regulator QsrR . Researchers should investigate potential interactions between NWMN_2240 and the QsrR regulon to determine if this membrane protein plays a role in the regulatory network responsible for PDI tolerance.

How does NWMN_2240 interact with the ER Membrane protein Complex (EMC) during infection?

The ER Membrane protein Complex (EMC) is a recently discovered machinery involved in membrane protein insertion in eukaryotic cells . During S. aureus infection, bacterial membrane proteins may interact with host cell machinery, potentially affecting pathogenesis and host response.

Investigating potential interactions between NWMN_2240 and the host EMC would require sophisticated experimental approaches:

• Co-immunoprecipitation studies using tagged versions of NWMN_2240 and EMC components

• Proximity labeling techniques to identify proteins in close spatial association

• Fluorescence microscopy to visualize potential co-localization during infection

• Functional assays measuring how NWMN_2240 mutations affect S. aureus interaction with host cells

This research would provide insights into how bacterial membrane proteins might exploit or interfere with host membrane protein insertion machinery during infection. Such mechanisms could contribute to S. aureus pathogenicity and persistence in host environments.

What are optimal methods for recombinant expression and purification of NWMN_2240?

Membrane proteins present significant challenges for recombinant expression and purification due to their hydrophobic nature and complex folding requirements. For NWMN_2240, researchers should consider several expression systems, each with advantages and limitations:

For NWMN_2240 purification, researchers should utilize strategies specific to membrane proteins:

• Detergent screening to identify optimal solubilization conditions

• Affinity chromatography using His-tags or other fusion partners

• Size exclusion chromatography to ensure protein homogeneity

• Validation of proper folding through circular dichroism or thermal shift assays

When evaluating purification success, researchers should assess protein yield, purity, stability, and importantly, functional activity if assays are available. Structural characterization through techniques like X-ray crystallography, cryo-EM, or NMR would provide valuable insights into NWMN_2240's structure-function relationships.

What quasi-experimental designs are most appropriate for studying NWMN_2240 function?

Based on the quasi-experimental study designs outlined in the literature, several approaches are particularly suitable for investigating NWMN_2240 function :

• One-group pretest-posttest design using a double pretest: This design would allow researchers to measure a dependent variable (e.g., membrane integrity or antibiotic resistance) before and after manipulation of NWMN_2240 expression, with multiple pretests reducing the likelihood of regression to the mean effects .

• The one-group pretest-posttest design using a nonequivalent dependent variable: This approach would involve measuring both a variable expected to be affected by NWMN_2240 manipulation and another that should remain unchanged, providing internal validation for experimental effects .

• The removed-treatment design: In this design, researchers would express NWMN_2240, measure outcomes, remove expression, and measure again. This approach is particularly powerful for establishing causal relationships between NWMN_2240 and phenotypic outcomes .

• Interrupted time-series design: For studying dynamic processes like membrane development or stress responses, researchers could use multiple measurements before and after NWMN_2240 manipulation, spaced at equal time intervals .

The experimental design notation and structure for these approaches would be:

These quasi-experimental designs provide robust frameworks for investigating NWMN_2240 function when randomized controlled trials might not be feasible or appropriate .

How can genetic manipulation techniques be optimized for studying NWMN_2240?

Genetic manipulation of S. aureus to study NWMN_2240 requires specialized techniques due to the challenging nature of this organism. Several approaches can be optimized for NWMN_2240 research:

• Gene knockout strategies:

  • Allelic replacement using temperature-sensitive plasmids

  • CRISPR-Cas9 gene editing for precise modifications

  • Transposon mutagenesis for screening approaches

• Conditional expression systems:

  • Tetracycline-inducible promoters for controlled expression

  • IPTG-inducible systems for dose-dependent studies

  • Antisense RNA for knockdown experiments

• Reporter systems:

  • Transcriptional fusions to monitor NWMN_2240 expression

  • Translational fusions to study protein localization

  • Split-reporter systems to investigate protein-protein interactions

When studying membrane proteins like NWMN_2240, researchers should consider the impact of genetic manipulations on membrane integrity and bacterial physiology. Controls should include complementation experiments to verify that phenotypic changes result specifically from NWMN_2240 manipulation rather than polar effects or secondary mutations.

DNA mismatch repair (MMR) mechanisms may influence the outcomes of genetic manipulation experiments, as studies have shown that MMR mutants like HG003ΔmutSL exhibit different responses to stresses compared to wild-type strains . Researchers should consider how the MMR status of their S. aureus strains might affect genetic stability and experimental outcomes.

How should expression data for NWMN_2240 be analyzed in the context of stress responses?

Analysis of NWMN_2240 expression data requires sophisticated approaches to account for the complexity of bacterial stress responses. When investigating how NWMN_2240 expression changes during stresses like antibiotic exposure or PDI treatment, researchers should:

• Normalize expression data appropriately:

  • Use multiple reference genes that remain stable under experimental conditions

  • Consider global shifts in transcription during stress responses

  • Validate RNA-seq findings with qRT-PCR for key findings

• Employ time-course analyses:

  • Measure expression at multiple timepoints to capture dynamic responses

  • Use statistical methods appropriate for time-series data

  • Consider delayed effects and regulatory cascades

• Integrate with other datasets:

  • Correlate NWMN_2240 expression with physiological measurements

  • Compare expression patterns with other genes in potential operons

  • Analyze expression in the context of known stress response pathways

Studies have shown that S. aureus undergoes global transcriptional responses to stresses like PDI, with numerous cellular functions affected . When analyzing NWMN_2240 expression in this context, researchers should consider whether changes represent direct regulatory events or indirect consequences of broader cellular adaptations.

Statistical approaches should account for biological variability and experimental design. For time-course experiments, repeated measures ANOVA or mixed-effects models may be appropriate, while differential expression analysis for RNA-seq data should use packages specifically designed for count data, such as DESeq2 or edgeR.

What bioinformatic approaches help predict NWMN_2240 structure and interactions?

Predicting the structure and interactions of membrane proteins like NWMN_2240 requires specialized bioinformatic approaches that account for the unique properties of membrane-embedded proteins:

• Sequence-based predictions:

  • Transmembrane domain prediction (TMHMM, Phobius)

  • Signal peptide prediction (SignalP)

  • Topology prediction (TOPCONS)

  • Evolutionary conservation analysis (ConSurf)

• Structure prediction:

  • AlphaFold2 or RoseTTAFold for deep learning-based structure prediction

  • Molecular dynamics simulations in membrane environments

  • Homology modeling if structural homologs exist

• Interaction prediction:

  • Co-evolution analysis to identify potential interaction partners

  • Docking simulations with candidate interactors

  • Analysis of genomic context and operon structures

Recent advances in deep learning have revolutionized protein structure prediction, but membrane proteins remain challenging due to their complex environment. Researchers should validate computational predictions with experimental approaches whenever possible.

For functional prediction, researchers can analyze if NWMN_2240 contains domains associated with known membrane protein functions such as transport, signaling, or structural roles. Additionally, comparison with the approximately 5000 other membrane-embedded proteins encoded in bacterial genomes might reveal functional similarities or evolutionary relationships .

How can researchers distinguish between direct and indirect effects when studying NWMN_2240 mutations?

Distinguishing direct from indirect effects of NWMN_2240 mutations requires careful experimental design and data interpretation:

• Complementation studies:

  • Restore wild-type NWMN_2240 expression in mutant strains

  • Use site-directed mutagenesis to create point mutations in functional domains

  • Employ inducible expression systems for dose-dependent analyses

• Temporal analyses:

  • Measure rapid responses that likely represent direct effects

  • Track cascading changes over time to identify secondary consequences

  • Use kinetic modeling to distinguish primary from secondary effects

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Identify consistent patterns across different levels of biological organization

  • Use network analysis to map potential causal relationships

• Genetic interaction studies:

  • Create double mutants with genes in suspected pathways

  • Look for epistatic effects that suggest functional relationships

  • Use synthetic genetic arrays to systematically map genetic interactions

When interpreting results, researchers should consider that membrane proteins often have pleiotropic effects due to their involvement in fundamental cellular processes. Changes in membrane composition or integrity can affect numerous cellular functions, making it challenging to isolate direct effects of NWMN_2240 manipulation.

What are common challenges in expressing and purifying S. aureus membrane proteins?

Researchers face numerous challenges when working with S. aureus membrane proteins like NWMN_2240:

• Expression challenges:

  • Toxicity to host expression systems

  • Protein misfolding and aggregation

  • Low expression yields

  • Incorrect membrane insertion

• Purification difficulties:

  • Finding appropriate detergents for solubilization

  • Maintaining protein stability during purification

  • Achieving sufficient purity for structural studies

  • Preserving native conformation and function

• Functional characterization limitations:

  • Lack of known binding partners or substrates

  • Difficulty reconstituting in vitro activity

  • Challenges in developing functional assays

  • Absence of structural information

To address these challenges, researchers can employ several strategies:

Recent advances in membrane protein science, including the discovery of new insertion machinery like the ER Membrane protein Complex (EMC), may provide insights for developing improved methods for working with challenging membrane proteins like NWMN_2240 .

How might NWMN_2240 contribute to antibiotic resistance mechanisms?

The potential contribution of NWMN_2240 to antibiotic resistance warrants investigation, given the critical role of membrane proteins in bacterial survival mechanisms. S. aureus is known for developing antibiotic resistance, with methicillin-resistant S. aureus (MRSA) being a significant clinical challenge responsible for approximately 500,000 infections and up to 50,000 deaths annually in the United States alone .

NWMN_2240 might contribute to resistance through several mechanisms:

• Membrane permeability alteration:

  • Modifying membrane structure to reduce antibiotic penetration

  • Altering membrane fluidity or composition in response to antibiotic exposure

  • Participating in cell wall remodeling under antibiotic stress

• Stress response involvement:

  • Contributing to general stress response pathways activated by antibiotics

  • Participating in specific adaptive responses to particular antibiotic classes

  • Influencing bacterial persistence under antibiotic pressure

• Regulatory functions:

  • Sensing environmental changes including antibiotic presence

  • Transducing signals to activate resistance mechanisms

  • Modulating expression of resistance determinants

Research into these potential functions would require combining genetic approaches (gene knockout, overexpression) with phenotypic assays measuring antibiotic susceptibility and resistance development. Transcriptomic and proteomic analyses comparing wild-type and NWMN_2240 mutant strains under antibiotic exposure would help identify pathways influenced by this membrane protein.

The study of S. aureus adaptation to photodynamic inactivation (PDI) provides a model for investigating adaptive responses that might parallel antibiotic resistance development . Similar experimental approaches could be applied to study NWMN_2240's role in conventional antibiotic resistance mechanisms.