Recombinant Salmonella enteritidis PT4 Phosphoserine aminotransferase (serC)

What is phosphoserine aminotransferase (serC) and what is its function in Salmonella enteritidis PT4?

Phosphoserine aminotransferase (serC) is an essential enzyme in the serine biosynthesis pathway that catalyzes the conversion of 3-phosphohydroxypyruvate to 3-phosphoserine. In Salmonella enteritidis PT4, this enzyme plays a fundamental role in amino acid metabolism, which may indirectly influence virulence and pathogenicity. While not classified as a direct virulence factor in S. enteritidis PT4, serC's metabolic functions can affect bacterial fitness during infection.

Recent studies in related bacterial species have demonstrated that serC contributes to biofilm formation and bacterial adaptation to stress conditions . The genome of Salmonella enteritidis PT4 contains approximately 4,506 coding genes, with virulence factors constituting about 3.66% of these genes . SerC falls within the larger group of metabolic genes that support bacterial survival within hosts.

How does the genetic organization of serC differ between Salmonella enteritidis PT4 and other Salmonella serovars?

The serC gene in Salmonella enteritidis PT4 is part of the core genome conserved across Salmonella species. When comparing S. enteritidis PT4 with other serovars like S. Typhimurium, the genetic organization surrounding serC remains largely conserved, reflecting its fundamental metabolic role. The serC gene in S. enteritidis PT4 is distinct from the twelve Salmonella pathogenicity islands (SPIs) that encode specific virulence factors .

Comparative genome analysis between Salmonella Enteritidis and Salmonella Typhimurium strains reveals that most variation between serovars occurs in genes related to metabolism, membrane proteins, and hypothetical proteins . Since serC encodes a metabolic enzyme, strain-specific variations might exist that could influence enzyme efficiency or regulation under different environmental conditions.

What experimental techniques are most effective for creating recombinant Salmonella enteritidis PT4 with modified serC?

Creating recombinant S. enteritidis PT4 with modified serC requires precise genetic manipulation. Based on established protocols, the following approaches are most effective:

• CRISPR interference (CRISPRi) for gene knockdown:

  • Identify PAM sites on the serC gene sequence

  • Design complementary sgRNA primers targeting serC with appropriate restriction sites

  • Clone annealed double-stranded DNA into vectors such as pLJR962

  • Transform into S. enteritidis PT4 via electroporation

  • Induce with anhydrotetracycline to achieve variable levels of knockdown

• Plasmid-based expression systems:

  • Clone serC variants into expression vectors under control of different promoters

  • Create fusion proteins with reporter genes like GFP for tracking expression

  • Transform into attenuated S. enteritidis strains (e.g., aroA mutants)

• Transposon mutagenesis for high-throughput screening:

  • Generate large transposon mutant libraries as described for S. enteritidis PT4 strain P125109

  • Screen for colonization defects using microarray-based negative-selection methods

How can researchers validate serC function in recombinant Salmonella strains?

Validating serC function in recombinant Salmonella strains requires multiple approaches to confirm both expression and enzymatic activity:

• Molecular validation:

  • RT-qPCR to quantify serC transcript levels

  • Western blotting to detect the SerC protein (using tagged versions if antibodies aren't available)

  • Flow cytometry to measure expression of SerC-fluorescent protein fusions at the single-cell level

• Biochemical validation:

  • Enzymatic assays to measure phosphoserine aminotransferase activity in cell lysates

  • Metabolomic analysis to quantify serine pathway intermediates

  • Complementation with wild-type serC to restore function in mutants

• Phenotypic validation:

  • Growth assessment in minimal media with and without serine supplementation

  • Biofilm formation assays (as serC regulates biofilm formation in other bacteria)

  • Stress response testing (oxidative, acid, temperature stress)

• In vivo validation:

  • Colonization assessment in animal models

  • Competitive index assays comparing wild-type and serC-modified strains

  • Tissue-specific bacterial burden quantification

What NIH guidelines must researchers follow when working with recombinant Salmonella enteritidis PT4?

Research involving recombinant Salmonella enteritidis PT4 must adhere to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Key requirements include:

• Institutional oversight:

  • Approval from an Institutional Biosafety Committee (IBC) before initiating any experiments

  • Appropriate risk assessment and containment measures based on the risk group classification

• Compliance scope:

  • All research conducted at institutions receiving any NIH support for recombinant research

  • Research involving materials containing recombinant nucleic acids developed with NIH funds

  • International research supported by NIH funds or involving testing in humans of NIH-funded materials

• Biocontainment requirements:

  • Minimum of Biosafety Level 2 (BSL-2) practices for work with Salmonella enteritidis

  • Enhanced BSL-2 or BSL-3 practices for large-scale culture or particularly hazardous modifications

• Documentation and reporting:

  • Maintenance of detailed records of experimental protocols and risk assessments

  • Reporting of any significant research-related incidents or violations

  • Regular review and updating of safety protocols

Researchers must ensure that safety practices are "reasonably consistent with the NIH Guidelines" regardless of where the research is conducted .

How can researchers accurately measure serC expression during in vivo infection with Salmonella enteritidis PT4?

Measuring gene expression during in vivo infection presents significant technical challenges. For serC expression in Salmonella enteritidis PT4, the following advanced approaches provide the most reliable data:

• Reporter gene systems:

  • Create transcriptional fusions between the serC promoter and fluorescent proteins (GFP, mCherry)

  • Recover bacteria from infected tissues and analyze by flow cytometry

  • Quantify reporter protein molecules per bacterium using calibration standards

  • Account for plasmid loss by replica plating on selective and non-selective media

• Direct RNA analysis from infected tissues:

  • Selective capture of transcribed sequences (SCOTS) to enrich bacterial RNA

  • RNAseq with specific alignment to the Salmonella genome

  • RT-qPCR targeting serC with appropriate housekeeping gene controls

  • Dual RNA-seq to simultaneously profile host and pathogen transcriptomes

• In situ visualization:

  • RNA fluorescence in situ hybridization (RNA-FISH) targeting serC transcripts

  • Immunohistochemistry using antibodies against SerC or epitope-tagged versions

  • Multiphoton intravital microscopy for real-time tracking in live animals

What experimental approaches best elucidate the impact of serC on Salmonella enteritidis PT4 virulence mechanisms?

To comprehensively evaluate the role of serC in S. enteritidis PT4 virulence, researchers should implement a multi-faceted experimental strategy:

• Construction of defined genetic systems:

  • Complete serC deletion mutant with complementation controls

  • Point mutations in catalytic residues to distinguish enzymatic from structural roles

  • Conditional expression systems for temporal control of serC expression

  • Strain-specific variants to assess serC contributions across different isolates

• In vitro virulence assays:

  • Epithelial cell invasion assays

  • Macrophage survival studies with primary and cultured cells

  • Biofilm formation quantification

  • Stress response profiling (oxidative, acid, antimicrobial peptides)

• In vivo infection models:

  • BALB/c mouse systemic infection model as used for S. enteritidis PT4 colonization studies

  • Competitive index assays comparing wild-type and serC mutants

  • Organ-specific bacterial burden determination

  • Temporal tracking of infection progression

• Host response analysis:

  • Flow cytometric assessment of immune cell recruitment and activation

  • Cytokine profiling in infected tissues

  • Histopathological evaluation of tissue damage

  • T-cell activation assessment using methods similar to those for ovalbumin-specific responses

• Systems biology approach:

  • Transcriptomic analysis of serC mutants versus wild-type during infection

  • Metabolomic profiling to identify downstream effects of serC disruption

  • Network analysis integrating multiple data types to identify virulence mechanisms

How does serC expression influence biofilm formation in Salmonella enteritidis PT4?

While direct evidence for serC's role in S. enteritidis PT4 biofilm formation is limited, research in other bacterial species provides a framework for investigation. In Mycobacterium smegmatis, serC positively regulates biofilm formation, with serC knockdown significantly impairing biofilm development . For S. enteritidis PT4, researchers should consider the following approaches:

• Biofilm phenotype characterization:

  • Compare biofilm formation between wild-type and serC-modified strains

  • Quantify biofilm biomass using crystal violet staining

  • Assess biofilm architecture via confocal microscopy

  • Measure extracellular matrix components (exopolysaccharides, proteins, eDNA)

• Regulatory pathway investigation:

  • Identify potential transcription factors that regulate both serC and biofilm-associated genes

  • Similar to NapR in mycobacteria, search for Salmonella regulators that bind the serC promoter

  • Evaluate serC expression in various biofilm mutants

• Metabolic connections:

  • Investigate how serine availability affects biofilm formation

  • Assess whether serC-dependent metabolites contribute to extracellular matrix

  • Monitor amino acid flux during biofilm development

• Genetic complementation studies:

  • Test if serC from other species (like M. smegmatis) can restore biofilm function

  • Create chimeric SerC proteins to identify domains critical for biofilm regulation

  • Implement domain swapping between species to identify functional differences

Experimental findings suggest that in M. smegmatis, serC knockdown strains exhibit smooth colony morphology compared to the rough surface of wild-type strains, and show significantly impaired biofilm formation at the air-liquid interface when the knockdown is induced with anhydrotetracycline . Similar phenotypic analyses should be performed with S. enteritidis PT4.

What molecular mechanisms regulate serC expression in Salmonella enteritidis PT4 during different growth conditions?

Understanding serC regulation in S. enteritidis PT4 requires investigation of both genetic elements and environmental factors. Based on studies in related bacteria, the following regulatory mechanisms likely control serC expression:

• Transcriptional regulation:

  • Promoter characterization using reporter fusions under various conditions

  • Identification of transcription factor binding sites through DNase I footprinting

  • Electrophoretic Mobility Shift Assays (EMSA) to detect protein-DNA interactions

  • Implementation of the EMSA protocol with 8% native polyacrylamide gels as described for mycobacterial studies

• Environmental responsive elements:

  • Nutrient availability (especially amino acid limitation)

  • Oxygen tension and redox state

  • pH changes encountered during intestinal passage

  • Host-derived antimicrobial factors

• Global regulatory networks:

  • Integration with stress response systems

  • Coordination with virulence gene expression

  • Growth phase-dependent regulation

  • Quorum sensing effects

• Post-transcriptional control:

  • RNA thermosensors or riboswitches

  • Small regulatory RNAs targeting serC mRNA

  • RNA stability determinants

  • Translational efficiency factors

For experimental verification, researchers should construct a series of serC promoter truncations fused to reporter genes and test their activity under various conditions relevant to Salmonella infection cycles. This approach will help map the regulatory elements controlling serC expression and identify the environmental signals that modulate its transcription.

How can immunological responses to recombinant Salmonella expressing modified serC be characterized and optimized?

Characterizing immune responses to recombinant Salmonella expressing modified serC requires comprehensive immunological analyses. For vaccine development or immunotherapy applications, researchers should implement the following methodological approaches:

• Antigen design and delivery optimization:

  • Create SerC fusion proteins with known immunogenic epitopes

  • Test different promoters for optimal in vivo expression

  • Evaluate attenuated Salmonella strains (e.g., aroA mutants like SL3261) as delivery vectors

  • Determine optimal dose and route for immune response induction

• T-cell response characterization:

  • Flow cytometric analysis of antigen-specific T cells

  • Assessment of activation markers (CD69, CD25, CD44)

  • Intracellular cytokine staining for Th1/Th2/Th17 profiling

  • In vivo CTL assays for cytotoxic T lymphocyte function

  • Implement methods similar to those described for tracking ovalbumin-specific T-cell responses

• Antibody response evaluation:

  • Isotype-specific ELISAs for SerC-specific antibodies

  • Functional assays (opsonization, neutralization)

  • B-cell ELISpot for antibody-secreting cell quantification

  • Affinity maturation assessment through avidity testing

• Mucosal immunity assessment:

  • IgA measurement in intestinal lavage and fecal samples

  • Tissue-resident memory T-cell characterization

  • Mucosal lymphoid tissue analysis (Peyer's patches, mesenteric lymph nodes)

• Protective efficacy determination:

  • Challenge studies with virulent Salmonella

  • Bacterial burden quantification in tissues

  • Survival analysis and clinical scoring

  • Correlates of protection identification

What is the optimal protocol for creating and validating serC knockdown in Salmonella enteritidis PT4?

Creating effective serC knockdown strains requires careful design and validation. Based on successful approaches in related bacteria, the following protocol is recommended:

• CRISPR interference (CRISPRi) system design:

  • Identify optimal PAM sites within the serC coding sequence

  • Design sgRNA primers with appropriate restriction sites (e.g., BsmBI)

  • Clone annealed primers into vectors appropriate for Salmonella (similar to pLJR962)

  • Transform into S. enteritidis PT4 via electroporation at 2500V

  • Select transformants on appropriate antibiotic media

  • Verify constructs by sequencing and PCR

• Induction and titration of knockdown:

  • Culture transformants in media with varying concentrations of anhydrotetracycline

  • Establish dose-response relationship between inducer concentration and knockdown level

  • Determine optimal conditions that achieve significant knockdown without severe growth defects

  • Create growth curves to document any changes in bacterial fitness

• Comprehensive validation approach:

  • Quantify serC mRNA levels via RT-qPCR (aim for >80% reduction)

  • Measure phosphoserine aminotransferase activity in cell lysates

  • Assess metabolic consequences via targeted metabolomics

  • Verify phenotype reversal with complementation constructs

• Phenotypic characterization:

  • Growth in minimal media with/without serine supplementation

  • Survival under various stress conditions

  • Biofilm formation capability

  • Virulence-associated phenotypes in cellular models

This protocol ensures the creation of reliable knockdown strains with minimal off-target effects and well-characterized phenotypes suitable for detailed functional studies of serC.

How should researchers optimize flow cytometry protocols for analyzing recombinant Salmonella expressing SerC-fluorescent protein fusions?

Flow cytometric analysis of bacteria expressing fluorescent fusion proteins requires specific optimization. For SerC-fluorescent protein fusions in Salmonella, researchers should follow these guidelines:

• Sample preparation:

  • For in vitro cultures:

    • Harvest at specific growth phases

    • Wash to remove media autofluorescence

    • Fix if necessary (note: fixation may impact some fluorescent proteins)

  • For in vivo samples:

    • Homogenize infected tissues (spleen, mesenteric lymph nodes, Peyer's patches)

    • Treat with 0.1% Triton X-100 to lyse host cells while preserving bacteria

    • Filter through 5μm filters to remove tissue debris

• Instrument setup and optimization:

  • Use appropriate laser lines for the specific fluorescent protein

  • Optimize forward/side scatter settings for bacterial detection

  • Implement threshold settings to exclude debris

  • Include single-stained controls for compensation

  • Use bacterial size beads for instrument calibration

• Data acquisition and analysis:

  • Collect sufficient events (minimum 50,000 bacterial events)

  • Gate based on scatter properties to identify bacterial population

  • Quantify fluorescence intensity and convert to molecules per cell using standards

  • Assess population heterogeneity through histogram analysis

  • Measure plasmid retention by comparing to non-selective cultures

• Advanced applications:

  • Sort different expression level populations for subsequent analysis

  • Combine with viability dyes for live/dead discrimination

  • Implement dual-reporter systems for correlation studies

  • Use imaging flow cytometry for morphological assessment

This optimized flow cytometry approach enables precise quantification of SerC-fusion protein expression at the single-cell level and reveals population heterogeneity that might be missed by bulk measurement techniques.

What experimental design is most effective for identifying transcription factors that regulate serC in Salmonella enteritidis PT4?

Identifying transcription factors that regulate serC requires a systematic experimental approach. Based on successful strategies in related systems, the following experimental design is recommended:

• Promoter mapping and characterization:

  • Identify the serC transcription start site using 5' RACE

  • Create a series of promoter deletion constructs fused to reporters

  • Test activity under various conditions to identify regulatory regions

  • Perform site-directed mutagenesis of predicted binding sites

• Protein-DNA interaction studies:

  • Conduct Electrophoretic Mobility Shift Assays (EMSA) using the following procedure:

    • Amplify the serC promoter fragment by PCR

    • Prepare 8% native polyacrylamide gels

    • Mix the promoter fragment with various protein concentrations

    • Include appropriate buffers (H buffer) and 50% glycerol

    • Incubate for 15 minutes before loading

    • Run electrophoresis at 150V for 1 hour in TBE buffer

    • Stain with ethidium bromide and image

  • Perform DNase I footprinting to precisely map binding sites

  • Conduct chromatin immunoprecipitation (ChIP) for in vivo confirmation

• Transcription factor identification:

  • DNA affinity chromatography using immobilized serC promoter

  • Mass spectrometry of bound proteins

  • Candidate approach testing known regulators of metabolic genes

  • Screening transcription factor mutant libraries for altered serC expression

• Functional validation:

  • Create deletion mutants of identified factors

  • Perform complementation studies

  • Assess serC expression in regulatory mutants

  • Analyze direct binding using purified proteins

  • Evaluate physiological relevance under infection-relevant conditions

This comprehensive approach will identify specific transcription factors that regulate serC expression and characterize their binding sites and regulatory mechanisms.

What is the optimal protocol for assessing the impact of serC modifications on Salmonella survival within macrophages?

Evaluating how serC affects Salmonella survival within macrophages requires rigorous infection models and quantification methods. The following protocol provides a comprehensive assessment:

• Bacterial strain preparation:

  • Culture wild-type, serC mutant, and complemented strains to mid-log phase

  • Wash and resuspend in serum-free media

  • Opsonize with normal mouse serum if desired

  • Adjust concentration for consistent multiplicity of infection (MOI)

• Macrophage infection:

  • Prepare primary bone marrow-derived macrophages or macrophage cell lines

  • Seed 5×10^5 cells per well in 24-well plates

  • Add bacteria at MOI 10:1 (bacteria:macrophage)

  • Synchronize infection by centrifugation (5 min, 1000×g)

  • Incubate for 30 minutes to allow phagocytosis

  • Wash and add media containing gentamicin (100 μg/ml) for 1 hour to kill extracellular bacteria

  • Reduce gentamicin to 10 μg/ml for remaining incubation

• Quantification of intracellular survival:

  • At designated time points (0, 2, 8, 24 hours), lyse macrophages with 0.1% Triton X-100

  • Perform serial dilutions and plate for CFU enumeration

  • Calculate percent survival relative to initial uptake

  • Compare survival kinetics between wild-type and serC-modified strains

  • For fluorescent strains, perform flow cytometry to assess bacterial numbers and expression levels

• Microscopy-based analysis:

  • Infect macrophages on coverslips

  • Fix and stain at various time points

  • Perform immunofluorescence using antibodies against Salmonella and lysosomal markers

  • Quantify bacterial numbers per cell and co-localization with lysosomal markers

  • For live imaging, use time-lapse microscopy with fluorescent reporter strains

• Host response assessment:

  • Measure cytokine production (TNF-α, IL-1β, IL-6)

  • Assess reactive oxygen and nitrogen species production

  • Evaluate macrophage viability and mode of cell death

  • Analyze activation markers by flow cytometry

This protocol enables quantitative assessment of how serC modifications affect Salmonella survival within macrophages, providing insights into its role in virulence.

What approaches should researchers use to study the effects of environmental conditions on serC expression and function in Salmonella enteritidis PT4?

Environmental conditions significantly impact bacterial gene expression and protein function. To comprehensively evaluate how various environments affect serC in S. enteritidis PT4, researchers should implement the following approaches:

• Reporter systems for expression analysis:

  • Create transcriptional and translational fusions between serC and reporter genes

  • Test expression under conditions relevant to Salmonella lifestyle:

    • Nutrient limitation (carbon, nitrogen, phosphorus)

    • pH stress (pH 4.5-7.5)

    • Oxidative stress (H₂O₂, paraquat)

    • Antimicrobial peptide exposure

    • Bile salts

    • Various temperatures (37°C, 42°C)

    • Anaerobic vs. aerobic growth

  • Quantify expression using plate readers, flow cytometry, or in-plate imaging systems

• Protein function assessment:

  • Develop enzymatic assays for SerC activity under various conditions

  • Express and purify SerC for in vitro characterization

  • Determine enzyme kinetics parameters under different pH, temperature, and salt conditions

  • Assess protein stability using thermal shift assays

  • Examine post-translational modifications that might regulate activity

• Metabolic impact analysis:

  • Perform metabolomic profiling under various conditions

  • Quantify serine pathway intermediates

  • Conduct 13C flux analysis to track carbon flow

  • Compare metabolic profiles between wild-type and serC mutants

  • Correlate metabolic changes with virulence phenotypes

• In vivo expression profiling:

  • Use animal infection models to recover bacteria from different host niches

  • Implement flow cytometry to measure serC-reporter expression in bacteria isolated from tissues

  • Compare expression levels between different host compartments (intestine, liver, spleen)

  • Assess temporal changes in expression throughout infection