Recombinant Staphylococcus aureus Phosphoenolpyruvate-protein phosphotransferase (SAS1019), partial

What is the phosphoenolpyruvate-protein phosphotransferase system in Staphylococcus aureus?

The phosphoenolpyruvate-protein phosphotransferase system (PTS) in S. aureus is a major mechanism for carbohydrate uptake and phosphorylation. It operates through a series of phosphoryl transfers starting from phosphoenolpyruvate (PEP) as the phosphoryl donor. The system involves three essential catalytic entities: Enzyme I, Enzyme II (with various sugar-specific variants), and HPr (heat-stable, histidine-phosphorylatable protein) .

This system is characterized by four successive phosphoryl transfers:

• Initial autophosphorylation of Enzyme I using PEP as substrate

• Transfer of the phosphoryl group from Enzyme I to HPr

• Transfer from HPr to the sugar-specific EIIA component

• Final transfer to the sugar via EIIB during transport through the membrane-bound EIIC

Unlike other transport systems, the PTS simultaneously transports and phosphorylates carbohydrates, coupling these processes to conserve energy .

What are the structural and functional properties of SAS1019?

SAS1019 refers to a partial recombinant form of the phosphoenolpyruvate-protein phosphotransferase from S. aureus. Based on general PTS research, this protein likely represents Enzyme I (EI), the first component in the PTS phosphorylation cascade. The molecular weight of Enzyme I in S. aureus has been estimated at approximately 100,000 ± 15% Da .

Functionally, if SAS1019 is indeed Enzyme I, it catalyzes the initial phosphorylation step in the PTS cascade, using PEP to autophosphorylate at a conserved histidine residue. This phosphoryl group is then transferred to the HPr protein, initiating the cascade that ultimately results in sugar uptake and phosphorylation .

What role does the PTS system play in S. aureus metabolism and virulence?

The PTS system serves several critical functions in S. aureus:

Primary metabolic functions:

Regulatory functions:

• Controls carbon catabolite repression

• Regulates expression of genes involved in central carbon metabolism

• Coordinates carbon and nitrogen metabolism

• Influences cell wall recycling and peptidoglycan metabolism

Connection to virulence:
While specific information on S. aureus is limited in the search results, studies in related bacteria suggest PTS components can influence virulence. For example, in Borrelia burgdorferi, mutations in ptsG (encoding EIIBC components) eliminated mouse infectivity . The PTS is likely involved in S. aureus adaptation to host environments through regulation of metabolism and gene expression.

What analytical methods are used to verify the identity and activity of recombinant PTS proteins?

Identity verification:

• SDS-PAGE to confirm molecular weight (~100,000 Da for EI components)

• Western blotting with anti-His or anti-PTS component antibodies

• Mass spectrometry (MALDI-TOF or LC-MS/MS) for accurate mass determination and peptide mapping

• N-terminal sequencing to confirm protein identity

Activity assays:

• Colorimetric assays based on formation of o-nitrophenyl-β-d-galactoside-6-phosphate and its hydrolysis by 6-phospho-β-galactosidase

• Coupled enzyme assays measuring pyruvate production from PEP

• Direct measurement of phosphoryl transfer using:

  • Radioactive [32P]PEP

  • FRET between fluorescently labeled PTS components

• In vitro reconstitution of the complete PTS cascade with purified components

How do mutations in specific PTS components affect S. aureus physiology and virulence?

Research on PTS mutations has revealed significant impacts on bacterial physiology:

Metabolic effects:

• Accumulation of metabolic intermediates (e.g., MurNAc-GlcNAc in MurP mutants)

• Altered carbon catabolite repression patterns

• Changes in central carbon metabolism flux

Growth phenotypes:

• Mutants in mupG and murQ components show slight growth defects during late exponential and stationary phases

• Sugar utilization profiles are altered in PTS component mutants

Virulence implications:
While specific S. aureus data is limited, research in related bacteria is informative:

• In Borrelia burgdorferi, ptsG mutants lost infectivity in mice but survived normally in ticks

• PTS mutations can affect adaptation to host environments through altered metabolism

Genetic compensation:

• Transcriptome analysis reveals compensatory changes in gene expression following PTS mutations

• PTS mutations trigger expression changes in metabolic and regulatory genes

How does the phosphorylation state of PTS proteins integrate with broader metabolic networks?

The PTS system functions as a sophisticated signaling network that integrates multiple metabolic inputs:

Integration with carbon metabolism:

• Senses the PEP:pyruvate ratio, reflecting the energy state of the cell

• Responds to glycerol, pyruvate, oxaloacetate, and serine through distinct mechanisms

• Coordinates with glycolysis, TCA cycle, and gluconeogenesis

Cross-talk with nitrogen metabolism:

• In some bacteria, α-ketoglutarate (which accumulates under nitrogen limitation) affects PTS activity, though this interaction varies between species

• PTS components regulate nitrogen-related gene expression in some bacteria

Signaling network properties:

This integration allows the PTS to function as a global nutritional sensor rather than simply a sugar transport system.

What techniques are used to study PTS protein-protein interactions in vitro and in vivo?

In vitro interaction studies:

• Pull-down assays with purified components

• Surface plasmon resonance (SPR) to measure binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Chemical cross-linking followed by mass spectrometry

• In vitro reconstitution of phosphoryl transfer using purified components

In vivo interaction studies:

• FRET analysis with fluorescent protein fusions:

  • Proteins tagged with CFP and YFP allow monitoring of interactions through changes in YFP/CFP emission ratio

  • Studies reveal stimulation-dependent interactions between PTS components upon sugar addition

• Bacterial two-hybrid systems

• Co-immunoprecipitation from cell lysates

• Split-reporter assays (luciferase, GFP)

Key findings from FRET studies:

• Cytoplasmic PTS components are recruited to membrane transporters upon sugar stimulation

• Interactions between different PTS proteins respond similarly to all PTS sugars

• The relative response amplitudes upon stimulation with saturating concentrations of different PTS sugars are equal across different protein pairs

These techniques have revealed that the phosphorylation state of proteins within the network can rapidly equilibrate, suggesting reversibility is a general property of the PTS.

How can site-directed mutagenesis be used to investigate functional residues in phosphoenolpyruvate-protein phosphotransferase?

Key residues for targeted mutagenesis:

• Conserved histidine phosphorylation sites

• PEP binding pocket residues

• Protein-protein interaction interfaces

• Regulatory domains

Mutagenesis strategy:

• Identify conserved residues through sequence alignment of PTS proteins across species

• Design mutagenesis primers introducing substitutions:

  • Histidine → Alanine (eliminates phosphorylation)

  • Histidine → Glutamate (mimics phosphorylation)

  • Conservative substitutions to assess specific chemical properties

Expression and analysis workflow:

• Create mutant constructs using PCR-based methods (QuikChange, overlap extension)

• Express wild-type and mutant proteins under identical conditions

• Purify to homogeneity using affinity tags and chromatography

• Compare structural integrity using circular dichroism or thermal shift assays

• Assess functional impacts through:

  • In vitro phosphoryl transfer assays

  • Protein-protein interaction studies

  • Complementation of PTS-deficient bacterial strains

Validation approaches:

• Complementation studies in S. aureus PTS mutants

• In vivo assessment of sugar uptake and metabolism

• Structural analysis of mutant proteins by X-ray crystallography or NMR

How can isotope labeling be used to track phosphoryl transfer in the PTS cascade?

Radioactive labeling approaches:

• [³²P]PEP as phosphoryl donor:

  • Follow phosphoryl transfer through sequential PTS components

  • Detect radiolabeled intermediates by SDS-PAGE and autoradiography

  • Time-course experiments to determine transfer kinetics

  • Quench-flow experiments for rapid kinetics

• [¹⁴C] or [³H]-labeled sugars:

  • Monitor uptake and phosphorylation in whole cells

  • Measure accumulation of labeled sugar phosphates

  • Compare wild-type versus mutant strains

Stable isotope applications:

• ¹³C-labeled sugars or PEP:

  • Track carbon flow through metabolic pathways

  • Identify branch points and flux distributions

  • Combine with mass spectrometry for metabolite analysis

• ¹⁸O-labeled phosphate:

  • Distinguish newly formed phosphoryl bonds

  • Follow phosphate movement through the system

  • Mass spectrometry analysis of phosphorylated intermediates

NMR applications:

• Real-time monitoring of phosphoryl transfer reactions

• Structural analysis of phosphorylated intermediates

• Investigation of protein conformational changes

Experimental workflow:

• Prepare isotope-labeled substrates or proteins

• Initiate phosphoryl transfer reaction

• Sample at defined timepoints

• Separate components by gel electrophoresis, HPLC, or other methods

• Detect isotope incorporation by appropriate methods

• Analyze kinetics and pathway intermediates

How has our understanding of the PTS system evolved through systems biology approaches?

Recent systems biology studies have transformed our understanding of the PTS from a simple sugar transport system to a sophisticated sensory and regulatory network:

Network sensing properties:

Integration of multiple inputs:

• Beyond PTS sugars, the system responds to metabolites like glycerol, pyruvate, oxaloacetate, and serine

• These signals are propagated through the network in different ways:

  • Some share common signaling pathways (pyruvate, oxaloacetate, serine)

  • Others use distinct mechanisms (glycerol)

Global regulation:

• Transcriptome analysis reveals extensive regulation beyond sugar metabolism

• PTS components modulate expression of genes involved in virulence, stress response, and other functions

Mathematical modeling insights:

• Kinetic models predict optimal regulatory strategies based on carbon source quality

• Default sugar uptake through uninduced PTS correlates with carbon source quality

• This represents an optimal strategy for resource allocation

What emerging technologies are advancing PTS research?

CRISPR-based approaches:

• CRISPR interference (CRISPRi) for tunable repression of PTS components

• CRISPR activation (CRISPRa) for overexpression studies

• CRISPR-Cas9 genome editing for precise chromosomal modifications

Advanced imaging technologies:

• Super-resolution microscopy to visualize PTS component localization

• Single-molecule FRET to observe individual phosphoryl transfer events

• Live-cell imaging with fluorescent sensors for sugar uptake and phosphorylation

Computational approaches:

• Molecular dynamics simulations of PTS component interactions

• Systems-level modeling of PTS regulatory networks

• Machine learning analysis of PTS-dependent gene expression patterns

High-throughput methodologies:

• Multiplexed assays for PTS activity across multiple conditions

• Deep mutational scanning to comprehensively map functional residues

• Automated microfluidic systems for kinetic measurements

Synthetic biology applications:

• Engineering PTS components for non-native sugar utilization

• Creating biosensors based on PTS regulatory mechanisms

• Developing metabolic switches controlled by PTS signaling

What are the implications of PTS research for developing new antimicrobial strategies?

PTS research offers several promising avenues for antimicrobial development:

Targeting PTS components:

• Inhibitors of Enzyme I or HPr could disrupt carbon metabolism broadly

• Sugar-specific EII inhibitors might prevent utilization of specific carbon sources

• Allosteric modulators of PTS protein interactions could disrupt signaling

Metabolic vulnerabilities:

• PTS mutant studies reveal metabolic bottlenecks that could be targeted

• Understanding carbon source utilization in infection environments identifies crucial pathways

• PTS-dependent gene regulation may reveal non-obvious drug targets

Connection to virulence:

• In some bacteria like B. burgdorferi, PTS components (PtsG) are essential for infectivity

• PTS regulation affects expression of virulence factors in some pathogens

• Targeting PTS could attenuate virulence without necessarily killing bacteria (anti-virulence approach)

Potential advantages:

• Novel targets outside traditional antibiotic classes

• Potential for pathogen-specific targeting based on PTS differences

• Metabolic targets may be less susceptible to conventional resistance mechanisms

Challenges:

• Redundant carbon utilization pathways may bypass PTS inhibition

• Human glucose transporters could be inadvertently affected

• Resistance might develop through mutations in target sites or expression of alternative transporters

How does the PTS system contribute to S. aureus pathogenesis in different infection sites?

The PTS system plays multiple roles in S. aureus pathogenesis across different infection environments:

Blood and tissue infections:

• PTS components facilitate adaptation to glucose-limited environments

• Mutations in PTS genes can affect survival in human blood

• Sugar sensing via PTS may regulate expression of immune evasion factors

Biofilm formation:

• PTS-dependent carbon metabolism influences biofilm development

• Altered metabolism in biofilms may rely on alternative PTS substrates

• Cell wall recycling mediated by MurP and MupG affects peptidoglycan remodeling in biofilms

Intracellular survival:

• Experimental evolution of S. aureus in macrophages produces mutations in metabolic genes

• Small colony variants (SCVs) with altered metabolism show enhanced intracellular survival

• These variants exhibit increased survival in macrophages and human blood, along with vancomycin resistance

Host-specific adaptation:

• PTS system helps sense available nutrients in different host niches

• Functions as a global nutrient sensor rather than detecting specific sugars

• Coordinates metabolic adaptation to changing host environments

Research in other pathogens supports these roles - for example, in Borrelia burgdorferi, the PtsG component is essential for establishing mouse infection but not for survival in ticks .

What role do PTS proteins play in S. aureus antibiotic resistance?

PTS proteins contribute to antibiotic resistance through several mechanisms:

Metabolic adaptation:

• Altered carbon metabolism affects susceptibility to antibiotics targeting active growth

• PTS regulation influences cell wall synthesis and turnover pathways

• The recovery of peptidoglycan turnover products via MurP affects cell wall homeostasis

Regulatory connections:

• PTS-dependent gene regulation affects expression of resistance determinants

• Metabolic state sensed by PTS influences antibiotic tolerance

• Small colony variants (SCVs) with altered metabolism show increased resistance to vancomycin

Cell wall metabolism:

• The PTS component MurP is involved in recovery of the cell wall turnover product MurNAc-GlcNAc

• MupG hydrolyzes MurNAc 6-phosphate-GlcNAc intracellularly

• These processes affect peptidoglycan recycling and potentially antibiotic susceptibility

Stress response coordination:

• PTS functions as a sensor for nutrient availability

• This sensing capability helps coordinate stress responses that contribute to antibiotic tolerance

• Metabolic adaptations sensed through PTS may trigger persistence mechanisms

Experimental evolution studies have shown that S. aureus adaptation to intracellular environments can produce variants with both enhanced survival in macrophages and increased antibiotic resistance .

What computational tools are valuable for analyzing PTS protein structure and function?

Structural analysis tools:

• Homology modeling servers (SWISS-MODEL, I-TASSER, Phyre2)

• Molecular dynamics simulations (GROMACS, AMBER, NAMD)

• Protein-protein docking (HADDOCK, ClusPro, ZDOCK)

• Binding site prediction (SiteMap, FTMap, CASTp)

• Molecular visualization (PyMOL, Chimera, VMD)

Sequence analysis tools:

• Multiple sequence alignment (Clustal Omega, MUSCLE, T-Coffee)

• Conservation analysis (ConSurf, SIFT, PolyPhen)

• Domain prediction (SMART, Pfam, InterPro)

• Phosphorylation site prediction (NetPhos, GPS, PPSP)

• Transmembrane topology prediction (TMHMM, Phobius)

Systems biology approaches:

• Network analysis (Cytoscape, STRING, InnateDB)

• Metabolic modeling (COBRA Toolbox, PathwayTools)

• Gene expression analysis (DESeq2, EdgeR, GSEA)

• Kinetic modeling (COPASI, CellDesigner)

• Flux balance analysis (BiGG, COBRA)

Application examples:

• Homology modeling of S. aureus PTS components based on solved structures

• Molecular dynamics simulations of phosphoryl transfer mechanisms

• Systems models of PTS regulation integrating transcriptomic data

• Prediction of critical residues for mutagenesis studies

• Virtual screening for potential PTS inhibitors

How can network analysis reveal emergent properties of the PTS system?

Network analysis approaches uncover system-level properties of the PTS:

Network representation approaches:

• Protein-protein interaction networks:

  • Nodes represent PTS components

  • Edges represent physical interactions or phosphoryl transfer

  • Weighted by interaction strength or kinetic parameters

• Regulatory networks:

  • Include PTS-regulated genes and proteins

  • Capture transcriptional and post-translational regulation

  • Represent feedback loops and cross-regulation

• Metabolic networks:

  • Integrate PTS with broader metabolic pathways

  • Capture substrate flows and energy coupling

  • Predict metabolic responses to perturbations

Emergent properties revealed:

Network analysis techniques:

• Topological analysis to identify key nodes and bottlenecks

• Dynamic analysis to predict system responses to perturbations

• Constraint-based analysis to predict feasible metabolic states

• Comparative analysis across species to identify evolutionary patterns

What are promising areas for future research on S. aureus phosphoenolpyruvate-protein phosphotransferase?

Several promising research directions emerge from current knowledge:

Structural biology:

• Determining high-resolution structures of complete S. aureus PTS complexes

• Characterizing dynamic conformational changes during phosphoryl transfer

• Mapping interaction interfaces between PTS components and regulatory targets

• Structural basis for sugar specificity in different EII transporters

Systems biology:

• Comprehensive mapping of the PTS interactome in S. aureus

• Integration of transcriptomic, proteomic, and metabolomic responses to PTS perturbations

• Development of predictive models for PTS regulation during infection

• Comparative analysis of PTS networks across S. aureus strains with different virulence properties

Host-pathogen interactions:

• Role of PTS in sensing host-specific nutritional environments

• Influence of PTS on immune evasion mechanisms

• Contribution to formation of antibiotic-tolerant persister cells

• Role in biofilm development and chronic infection

Therapeutic applications:

• High-throughput screening for inhibitors of key PTS components

• Development of PTS-targeted antimicrobial peptides

• Exploration of PTS components as vaccine targets

• Design of metabolic adjuvants to enhance antibiotic efficacy

Technological innovations:

• Development of real-time sensors for PTS activity in living cells

• Application of synthetic biology approaches to reprogram PTS regulation

• Creation of cellular diagnostics based on PTS function

• Single-cell analysis of PTS activity during infection

What interdisciplinary approaches could accelerate discoveries in PTS research?

Interdisciplinary approaches offer powerful ways to advance PTS research:

Integrative structural biology:

• Combining X-ray crystallography, cryo-EM, and NMR spectroscopy

• Integrating computational modeling with experimental data

• Using hydrogen-deuterium exchange mass spectrometry (HDX-MS)

• Applying single-molecule techniques to observe dynamic processes

Synthetic biology:

• Engineering minimal PTS systems with defined components

• Creating biosensors based on PTS signaling mechanisms

• Developing orthogonal PTS pathways for biotechnology applications

• Rewiring PTS regulation to create new cellular behaviors

Advanced imaging:

• Super-resolution microscopy to visualize PTS component localization

• Live-cell imaging with fluorescent biosensors

• Correlative light and electron microscopy

• Label-free imaging techniques (Raman microscopy, FTIR)

Machine learning applications:

• Prediction of PTS component interactions

• Analysis of complex phenotypic data from PTS mutants

• Virtual screening for PTS inhibitors

• Modeling evolutionary trajectories of PTS components

Host-pathogen systems biology:

• Dual RNA-seq of infected host cells

• Metabolic modeling of host-pathogen nutrient competition

• Multi-omics integration across infection stages

• Agent-based modeling of infection dynamics

These interdisciplinary approaches can reveal new insights about how the PTS functions as a global sensor of sugar influx and integrator of multiple metabolic signals, rather than just a sugar transport system .