Recombinant Staphylococcus aureus Probable nitronate monooxygenase (SAR0883)

What is the predicted function of SAR0883 in Staphylococcus aureus?

SAR0883 is annotated as a putative organic hydroperoxide resistance protein in Staphylococcus aureus with probable nitronate monooxygenase activity . Based on homology studies with other bacterial nitronate monooxygenases (NMOs), SAR0883 likely catalyzes the oxidation of alkyl nitronates to their corresponding carbonyl compounds with the release of nitrite. This enzymatic activity plays a potential role in S. aureus response to oxidative stress, particularly against toxic nitro compounds that may be encountered during host-pathogen interactions .

Similar to characterized NMOs from other organisms, SAR0883 would function by converting toxic nitronate compounds to less harmful aldehydes while producing nitrite as a byproduct. The protein appears to be upregulated during oxidative stress conditions, with expression studies showing a 3.82-fold increase in SAR0883 transcript levels during exposure to certain stress conditions .

How does SAR0883 relate to Staphylococcus aureus pathogenicity?

SAR0883's role as a putative organic hydroperoxide resistance protein suggests its involvement in S. aureus defense against oxidative stress, which is a key challenge faced by the bacterium during infection . The host immune system generates reactive oxygen species (ROS) and reactive nitrogen species (RNS) as antimicrobial mechanisms, and bacterial enzymes that neutralize these compounds contribute to pathogen survival and persistence.

When S. aureus encounters oxidative stress from host immune cells, proteins like SAR0883 are upregulated as part of a coordinated stress response. This response involves multiple systems, including superoxide dismutases (SODs), catalases, and peroxide reduction systems . The ability of S. aureus to resist oxidative killing mechanisms is crucial for its success as a pathogen, allowing it to evade or defend against the host immune response . SAR0883's potential role in detoxifying nitro compounds would complement other defensive mechanisms such as catalase (KatA) and alkyl hydroperoxide reductase, which collectively enhance bacterial survival during infection.

What structural features characterize SAR0883 as a nitronate monooxygenase?

Based on comparative analysis with characterized nitronate monooxygenases, SAR0883 likely contains the following key structural features:

• A conserved catalytic histidine residue essential for enzymatic activity

• A flavin mononucleotide (FMN) binding domain

• A substrate-binding pocket accommodating nitroalkanes/nitronates

Similar to other characterized NMOs, such as the well-studied Rv1894c from Mycobacterium tuberculosis, SAR0883 is expected to have a structure that binds FMN as a cofactor, which provides the yellow color observed in purified recombinant protein preparations . The catalytic mechanism involves a highly conserved histidine residue that serves as the active site for the oxidation reaction. Structural modeling would likely reveal equivalent positions between SAR0883 and other NMOs that reconstitute the cofactor (FMN) and substrate binding sites .

What is the recommended approach for expressing and purifying recombinant SAR0883?

For optimal expression and purification of recombinant SAR0883, the following protocol is recommended based on successful approaches with similar enzymes:

• Cloning strategy:

  • Amplify the SAR0883 gene from S. aureus genomic DNA using high-fidelity PCR

  • Clone into a pET-based expression vector with an N-terminal or C-terminal 6×His tag

  • Verify sequence integrity before proceeding to expression

• Expression conditions:

  • Transform into E. coli BL21(DE3) or Rosetta(DE3) strain for optimal expression

  • Culture in LB media supplemented with the appropriate antibiotic

  • Induce at OD600 of 0.6-0.8 with 0.5-1 mM IPTG

  • Grow at 18-25°C for 16-20 hours to maximize soluble protein yield

• Purification steps:

  • Harvest cells by centrifugation and resuspend in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole)

  • Lyse cells by sonication or pressure homogenization

  • Clarify lysate by centrifugation (18,000 × g, 30 min, 4°C)

  • Purify using Ni-NTA affinity chromatography

  • Apply imidazole gradient (10-250 mM) for elution

  • Further purify by size exclusion chromatography if higher purity is required

During purification, the presence of bound FMN cofactor can be observed as a yellow color in the protein fraction, with characteristic absorption peaks at approximately 370 and 445 nm in the visible spectrum . Protein purity should be assessed by SDS-PAGE, and concentration determined by Bradford assay or UV absorption at 280 nm with the appropriate extinction coefficient.

How can I reliably assay the enzymatic activity of recombinant SAR0883?

The enzymatic activity of recombinant SAR0883 can be reliably assessed using a nitrite detection assay following the conversion of model substrates such as 2-nitropropane. The following assay protocol is recommended:

Nitrite detection assay:

• Reaction setup:

  • Prepare reaction buffer (50 mM potassium phosphate, pH 7.4)

  • Add purified recombinant SAR0883 (1-5 μM final concentration)

  • Initiate reaction with 10 mM 2-nitropropane (or other suitable substrate)

  • Incubate at 37°C for various time points (6, 12, 24 hours)

• Nitrite detection:

  • At each time point, remove aliquots and terminate reactions

  • Measure nitrite production using Griess reagent

  • Quantify against a sodium nitrite standard curve

  • Calculate specific activity as μmol nitrite produced per minute per mg enzyme

Based on similar enzymes, the expected nitrite production for active SAR0883 would show a time-dependent increase. For reference, recombinant Rv1894c NMO from M. tuberculosis produced approximately 91.0 ± 33.5 μM nitrite after 6 hours, increasing to 515.7 ± 48.3 μM after 21 hours, and 587.4 ± 111.6 μM after 24 hours of incubation with 10 mM 2-nitropropane .

Table 1: Expected nitrite production by recombinant nitronate monooxygenase

Note: Actual values for SAR0883 may differ but should follow a similar pattern of time-dependent increase.

What are the key controls needed for SAR0883 enzymatic activity studies?

When conducting enzymatic activity studies with recombinant SAR0883, the following controls are essential to ensure reliable and interpretable results:

• Negative controls:

  • Enzyme-free reaction (substrate + buffer only)

  • Heat-inactivated enzyme (boiled SAR0883 + substrate)

  • Catalytically inactive mutant (e.g., SAR0883 with alanine substitution at the conserved histidine residue)

• Positive controls:

  • Well-characterized NMO from another organism (if available)

  • Commercial NMO enzyme (if available)

• Substrate specificity controls:

  • Different nitroalkane substrates (e.g., 2-nitropropane, nitroethane, 3-nitropropionate)

  • Non-nitro compounds to confirm substrate specificity

• Cofactor controls:

  • Enzyme with exogenously added FMN to ensure saturating cofactor conditions

  • Enzyme treated with charcoal to remove bound flavin, followed by reconstitution experiments

A particularly informative control is to create a site-directed mutant where the conserved catalytic histidine is replaced with alanine (H→A). Based on similar studies with Rv1894c, this mutation would be expected to abolish enzymatic activity while maintaining protein folding and flavin binding . This control helps confirm that the observed activity is specific to the catalytic mechanism of SAR0883 rather than a contaminating enzymatic activity or non-enzymatic reaction.

How does SAR0883 activity correlate with S. aureus response to oxidative stress?

The expression and activity of SAR0883 should be analyzed in the context of the broader oxidative stress response network in S. aureus. Evidence suggests that SAR0883, annotated as a putative organic hydroperoxide resistance protein, shows increased expression (3.82-fold) under stress conditions , indicating its role in the bacteria's defense mechanisms.

To investigate the correlation between SAR0883 activity and oxidative stress response:

• Gene expression analysis:

  • Measure SAR0883 transcript levels using qRT-PCR under various oxidative stress conditions (H₂O₂, paraquat, nitric oxide donors)

  • Compare with known oxidative stress response genes (katA, sodA, sodM)

• Protein level assessment:

  • Quantify SAR0883 protein levels using Western blot or targeted proteomics

  • Monitor changes in protein abundance following exposure to oxidants

• Physiological relevance:

  • Create and characterize SAR0883 deletion mutants

  • Assess mutant susceptibility to various oxidative stressors

  • Complement the mutation to confirm phenotype specificity

What is the substrate specificity profile of SAR0883 compared to other bacterial nitronate monooxygenases?

Understanding the substrate specificity of SAR0883 relative to other bacterial NMOs is crucial for determining its precise physiological role. A comprehensive substrate profiling approach is recommended:

• Substrate screening protocol:

  • Test a panel of potential substrates (C2-C6 nitroalkanes, primary and secondary)

  • Measure activity using standardized conditions for each substrate

  • Calculate kinetic parameters (Km, kcat, kcat/Km) for each active substrate

• Comparative analysis:

  • Create a substrate specificity profile comparing SAR0883 with characterized NMOs

  • Identify structural features that correlate with substrate preferences

  • Use molecular docking to predict substrate binding modes

Based on studies of other NMOs, SAR0883 would likely show activity against primary and secondary nitroalkanes, with potential variation in efficiency. For example, the well-characterized Rv1894c from M. tuberculosis demonstrates activity with 2-nitropropane , but the relative activities across different substrates could reveal important insights into SAR0883's biological role.

Table 2: Predicted substrate specificity comparison between bacterial nitronate monooxygenases

Note: +++ = high activity, ++ = moderate activity, + = low activity, ? = activity to be determined This table is predictive based on similar enzymes and would need experimental verification.

How do environmental factors influence SAR0883 expression and activity in S. aureus?

The expression and activity of SAR0883 are likely influenced by various environmental factors encountered during S. aureus infection and colonization. To investigate these influences:

• Environmental condition testing:

  • pH variations (5.5-8.0)

  • Oxygen tension (aerobic, microaerobic, anaerobic)

  • Nutrient limitation (carbon, nitrogen, phosphate starvation)

  • Temperature shifts (30°C, 37°C, 42°C)

  • Osmotic stress (NaCl concentration)

  • Host-derived factors (fatty acids, antimicrobial peptides)

• Regulatory mechanisms:

  • Identify transcription factors controlling SAR0883 expression

  • Map promoter elements using reporter constructs

  • Determine if regulation occurs via stress-responsive sigma factors (σᴮ)

Research has shown that S. aureus gene expression is significantly altered during exposure to host-derived antimicrobial fatty acids, with increased expression of stress response genes . SAR0883, being part of the stress response network, is likely regulated by transcription factors that respond to these environmental cues. The data suggest coordination with other stress responses, including increased expression of genes involved in staphyloxanthin synthesis, capsule formation, and peptidoglycan regulation .

What are the best approaches for creating a SAR0883 knockout in S. aureus for functional studies?

Creating a clean SAR0883 knockout strain is essential for functional characterization. Several methodological approaches are recommended based on successful S. aureus genetic manipulation techniques:

• Allelic replacement approach:

  • Design primers to amplify 500-1000 bp homology arms flanking SAR0883

  • Clone homology arms into a temperature-sensitive plasmid (e.g., pIMAY)

  • Insert antibiotic resistance cassette between homology arms

  • Perform two-step selection process (integration/excision)

  • Confirm deletion by PCR and sequencing

• CRISPR-Cas9 approach:

  • Design guide RNA targeting SAR0883

  • Provide repair template with homology arms

  • Transform into S. aureus expressing Cas9

  • Select transformants and screen for successful editing

• Transposon mutagenesis approach:

  • Screen existing transposon libraries for SAR0883 insertions

  • Alternatively, create a targeted transposon insertion

  • Confirm disruption by PCR and Southern blot

For proper validation of the mutant strain, the following controls are necessary:

• Complementation strain (restoring SAR0883 expression)

• Whole-genome sequencing to rule out off-target mutations

• Growth curve analysis to assess general fitness effects

A complementation strain can be created by integrating a single copy of SAR0883 at a neutral site in the chromosome, using an integrative plasmid with a constitutive or inducible promoter. PCR amplification of resistance markers and Southern blot analysis should be performed to confirm the genetic constructs, as demonstrated in studies of other bacterial genes .

How can protein-protein interactions of SAR0883 be effectively investigated?

Understanding the protein interaction network of SAR0883 can provide insights into its functional role within the S. aureus cellular context. Several complementary approaches are recommended:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express epitope-tagged SAR0883 in S. aureus

  • Perform pull-down experiments under native conditions

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions by reciprocal pull-downs

• Bacterial two-hybrid system:

  • Clone SAR0883 into appropriate vectors

  • Screen against a library of S. aureus proteins

  • Confirm positive interactions with targeted assays

• Proximity-dependent biotinylation (BioID):

  • Fuse SAR0883 to a promiscuous biotin ligase

  • Express in S. aureus and allow in vivo biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

• Co-immunoprecipitation of suspected partners:

  • Based on functional relationships, test specific proteins

  • Focus on oxidative stress response proteins

  • Examine interactions during normal and stress conditions

As SAR0883 functions in oxidative stress response, it likely interacts with other components of this pathway. Potential interaction partners might include regulatory proteins (e.g., SarA, TcaR), other oxidative stress response proteins (e.g., KatA, SodA, SodM), or proteins involved in related metabolic processes . Interactions may be dynamic and stress-dependent, so experimental conditions should mimic relevant physiological states.

What are the critical considerations for designing site-directed mutagenesis studies of SAR0883?

Site-directed mutagenesis studies are essential for understanding structure-function relationships in SAR0883. Based on successful approaches with other NMOs, the following considerations are important:

• Target residue selection:

  • Conserved catalytic histidine (equivalent to H199 in Rv1894c)

  • FMN-binding residues (based on structural modeling)

  • Substrate-binding pocket residues

  • Surface residues potentially involved in protein-protein interactions

• Mutation design strategy:

  • Conservative replacements (e.g., H→N) to test specific chemical properties

  • Non-conservative replacements (e.g., H→A) for complete function elimination

  • Chimeric proteins to test domain functions

• Functional characterization:

  • Verify protein expression and stability

  • Assess FMN binding (spectroscopic analysis)

  • Measure enzymatic activity (nitrite production assay)

  • Determine substrate binding (isothermal titration calorimetry)

• Structural analysis:

  • Circular dichroism to confirm proper folding

  • Thermal stability assays

  • Crystallization attempts for high-resolution analysis

A systematic mutagenesis approach should begin with the most conserved residues. For instance, mutation of the catalytic histidine to alanine in Rv1894c abolished enzymatic activity while maintaining protein folding and flavin binding . Similar mutations in SAR0883 would be expected to produce comparable effects and should be prioritized in initial studies.

How should enzymatic activity data for SAR0883 be properly analyzed and presented?

Proper analysis and presentation of enzymatic activity data are crucial for accurate interpretation and comparison with other studies. The following guidelines are recommended:

• Data collection:

  • Perform reactions in triplicate at minimum

  • Include appropriate controls in each experiment

  • Measure time-dependent activity at multiple substrate concentrations

• Kinetic analysis:

  • Plot initial velocity versus substrate concentration

  • Fit data to appropriate kinetic models (Michaelis-Menten, Hill equation)

  • Calculate and report kinetic parameters (Km, kcat, kcat/Km)

  • Compare with published values for similar enzymes

• Data presentation:

  • Use clear tables for numerical data (see example below)

  • Create graphs showing time course or concentration dependence

  • Include error bars representing standard deviation or standard error

  • Provide representative raw data in supplementary materials

Table 3: Sample presentation of kinetic parameters for SAR0883

For time-course studies, present data as in the Rv1894c study, showing nitrite production over time with appropriate error bars . When presenting complex datasets with multiple variables, consider using heat maps or 3D surface plots to visualize relationships between parameters (e.g., activity as a function of both pH and temperature) .

How can transcriptomic and proteomic data be integrated to understand SAR0883 function in the context of S. aureus physiology?

Integrating transcriptomic and proteomic data provides a comprehensive view of SAR0883's role in S. aureus physiology. The following methodology is recommended:

• Multi-omics experimental design:

  • Collect samples under identical conditions for both analyses

  • Include wild-type, SAR0883 mutant, and complemented strains

  • Test multiple stress conditions relevant to S. aureus pathogenesis

• Data integration approaches:

  • Correlation analysis between transcript and protein levels

  • Pathway enrichment analysis for differentially expressed genes/proteins

  • Network analysis to identify functional modules

  • Visualization of integrated data using platforms like Cytoscape

• Specific analyses for SAR0883:

  • Identify co-regulated genes/proteins

  • Map SAR0883 to known stress response pathways

  • Compare expression patterns with other nitronate monooxygenases

  • Identify potential regulatory mechanisms

When examining the role of SAR0883, focus on its relationship with known stress response regulons like σᴮ and CtsR, which have been shown to be upregulated under stress conditions in S. aureus . The table below shows an example of how such integrated data might be presented.

Table 4: Integrated transcriptomic and proteomic data for oxidative stress response genes in S. aureus

Note: X.XX represents values to be determined experimentally. Known values from literature are included .

What statistical approaches are most appropriate for analyzing SAR0883 mutant phenotypes?

• Experimental design considerations:

  • Use biological replicates (minimum n=3, preferably n≥5)

  • Include technical replicates within each biological replicate

  • Randomize sample processing to avoid batch effects

  • Include appropriate positive and negative controls

• Statistical tests for different data types:

  • For continuous data (e.g., growth rates, enzyme activities):

    • Student's t-test (two groups)

    • ANOVA with post-hoc tests (multiple groups)

    • Non-parametric alternatives if normality assumptions are violated

  • For survival/resistance data:

    • Log-rank test for survival curves

    • Cox proportional hazards model for multivariate analysis

  • For gene expression data:

    • Appropriate multiple testing correction (e.g., Benjamini-Hochberg)

• Reporting standards:

  • Include exact p-values rather than thresholds (e.g., p=0.032 not p<0.05)

  • Report effect sizes and confidence intervals

  • Describe all statistical methods in detail

  • Provide raw data in supplementary materials

For complex phenotypic analysis, consider multivariate approaches such as principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) to identify patterns across multiple phenotypic variables. These methods can reveal relationships that might not be apparent from univariate analyses and help generate hypotheses about the functional role of SAR0883 .

What are the key future research directions for understanding SAR0883 function in S. aureus?

Based on current knowledge gaps, several promising research directions would advance understanding of SAR0883 function:

• Structural biology approaches:

  • Determine high-resolution crystal structure of SAR0883

  • Perform structural comparisons with characterized NMOs

  • Use structure to guide rational design of inhibitors

• Physiological role exploration:

  • Identify natural substrates in the S. aureus cellular environment

  • Determine contribution to virulence in various infection models

  • Investigate role in biofilm formation and persistence

• Regulatory network mapping:

  • Characterize transcriptional and post-translational regulation

  • Identify environmental cues that modulate SAR0883 expression/activity

  • Map interactions with other stress response systems

• Therapeutic potential assessment:

  • Evaluate SAR0883 as a potential drug target

  • Screen for selective inhibitors

  • Test combination approaches targeting multiple stress response pathways

The integration of these research directions would provide a comprehensive understanding of SAR0883's role in S. aureus biology and potential applications in addressing antimicrobial resistance. Collaborative approaches combining expertise in biochemistry, structural biology, genetics, and infection models would be particularly valuable for advancing this field.