Recombinant Salmonella newport Argininosuccinate synthase (argG)

What is argininosuccinate synthase (argG) and what role does it play in Salmonella Newport metabolism?

Argininosuccinate synthase (argG) is a critical enzyme in the arginine biosynthesis pathway of Salmonella Newport that catalyzes the ATP-dependent condensation of citrulline and aspartate to form argininosuccinate. This reaction represents a crucial step in the urea cycle and arginine biosynthesis. In Salmonella species, arginine metabolism has been demonstrated to be essential for virulence and bacterial fitness within host environments. Research has shown that de novo arginine biosynthesis from α-ketoglutarate is necessary for Salmonella to maintain full virulence in host organisms . The arginine biosynthesis pathway works in concert with other metabolic pathways to support bacterial growth and survival under various environmental conditions.

How does arginine metabolism contribute to S. Newport virulence and pathogenesis?

Arginine metabolism significantly enhances Salmonella's resistance to oxidative stress, which represents a key host defense mechanism during infection . Studies demonstrate that Salmonella relies on arginine metabolism both in vivo and in vitro to resist the antimicrobial actions of reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase . Increased arginine concentrations protect the pathogen from peroxide-induced cytoplasmic pH collapse and subsequent killing, thereby preserving pH homeostasis during infection . This metabolic adaptation is particularly important for persistent strains like REPJJP01, which has caused numerous outbreaks and illnesses in the United States since its first detection in 2015 .

How does S. Newport argG compare structurally and functionally with argG from other Salmonella serotypes?

While Salmonella Newport shows relatively low genetic diversity and a more clonal population structure compared to serotypes like S. Typhimurium, its metabolic genes still exhibit important serotype-specific patterns . The relatively conserved nature of argG within the S. Newport lineage (particularly Lineage IIC, to which most antimicrobial-resistant isolates belong) suggests evolutionary pressure to maintain arginine biosynthesis function . Genomic comparisons reveal that antimicrobial-resistant S. Newport isolates tend to show a high degree of profile similarity regardless of source, which may extend to conservation of metabolic enzymes like argG .

What expression systems are most effective for recombinant S. Newport argG production?

Based on research with similar bacterial enzymes, several expression systems can be effectively employed for recombinant S. Newport argG production:

Codon optimization of the S. Newport argG sequence for the expression host may be necessary to overcome potential codon usage bias. Expression conditions should be optimized to prevent inclusion body formation, which can be particularly problematic for metabolic enzymes.

What purification strategies yield highest purity and activity for recombinant S. Newport argG?

A multi-step purification approach typically yields the best results for obtaining highly pure and active recombinant argG:

• Initial capture: Affinity chromatography using His-tag, allowing elution with imidazole gradient

• Intermediate purification: Ion exchange chromatography based on argG's predicted isoelectric point

• Polishing: Size exclusion chromatography to remove aggregates and ensure homogeneity

Buffer optimization is crucial for maintaining enzyme activity throughout purification:

• pH maintenance in the 7.0-7.5 range approximating cytoplasmic conditions

• Inclusion of 10-20% glycerol as a stabilizing agent

• Addition of reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain cysteine residues

• Consideration of substrate analogs or products to stabilize the active site conformation

What are the critical factors affecting the solubility and stability of recombinant S. Newport argG?

Several factors significantly impact the solubility and stability of recombinant S. Newport argG:

Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ) may significantly improve folding and solubility of recombinant argG, especially when expressing at higher levels.

What are the most reliable assays for measuring recombinant S. Newport argG enzymatic activity?

Several complementary approaches can be used to assess argG activity:

• Direct assay: Measurement of argininosuccinate formation via HPLC or LC-MS

• Coupled assay: ATP consumption or AMP production using luciferase-based detection

• Colorimetric assay: Citrulline consumption using colorimetric reagents

• Radiometric assay: Using ¹⁴C-labeled substrates to track product formation

Standard reaction conditions typically include:

• Buffer: 50 mM Tris-HCl or HEPES at pH 7.5

• Substrates: 1-5 mM citrulline, 1-5 mM aspartate, 1-2 mM ATP

• Cofactors: 5-10 mM MgCl₂ (essential for ATP binding)

• Optional additives: 1 mM DTT, 50-100 mM KCl

How can researchers investigate S. Newport argG kinetics and substrate specificity?

To characterize the kinetic properties and substrate specificity of recombinant S. Newport argG:

• Determine Michaelis-Menten parameters (Km, Vmax, kcat) for each substrate:

  • Vary one substrate concentration while keeping others constant

  • Plot reaction velocity versus substrate concentration

  • Fit data to appropriate enzyme kinetic models

• Examine substrate specificity:

  • Test structural analogs of citrulline and aspartate

  • Evaluate alternative nucleotide triphosphates beyond ATP

  • Investigate potential allosteric regulators

• Study the impact of environmental conditions:

  • pH profile (typically pH 6.0-9.0 in 0.5 unit increments)

  • Temperature dependence (15-45°C)

  • Salt concentration effects (0-500 mM NaCl or KCl)

These studies are particularly relevant given the demonstrated importance of arginine metabolism in Salmonella's ability to withstand oxidative stress during infection .

How does argG function contribute to S. Newport's resistance to oxidative stress?

Arginine biosynthesis, including the reaction catalyzed by argG, plays a critical role in Salmonella's defense against oxidative stress . Research demonstrates that arginine metabolism buffers Salmonella's cytoplasm during oxidative stress, preventing peroxide-induced pH collapse and subsequent bacterial death . The mechanisms through which argG specifically contributes to this protection likely include:

These protective mechanisms are particularly relevant for multidrug-resistant strains like REPJJP01, which must overcome both host defenses and antibiotic challenges .

What is the relationship between argG expression/activity and antimicrobial resistance in S. Newport?

While direct evidence linking argG to antimicrobial resistance is not explicitly stated in the available research, several observations suggest potential connections:

• Multidrug-resistant S. Newport strains like REPJJP01 show persistent circulation and outbreaks , suggesting enhanced fitness potentially related to metabolic adaptations.

• The demonstrated role of arginine metabolism in oxidative stress resistance may indirectly contribute to antibiotic tolerance, as many antimicrobials induce oxidative stress as part of their killing mechanism.

• Antimicrobial-resistant S. Newport isolates show distinctive genetic characteristics that may include adaptations in metabolic pathways like arginine biosynthesis.

• The clonal nature of antimicrobial-resistant S. Newport populations suggests that specific genetic configurations, potentially including argG variants, may be selectively advantageous under antimicrobial pressure.

How does argG sequence conservation compare within lineages of S. Newport and between Salmonella serotypes?

Genomic analyses of Salmonella enterica isolates reveal important patterns in gene conservation:

• Approximately 65% of core genes, which likely include metabolic genes like argG, show phylogenetic clustering by serotype . This suggests selective pressures maintaining serotype-specific variants.

• AMR S. Newport isolates demonstrate a relatively low level of diversity and a more clonal population structure compared to serotypes like S. Typhimurium , potentially indicating stronger conservation of core metabolic genes within this lineage.

• Most antimicrobial-resistant S. Newport isolates belong to Lineage IIC , suggesting that this lineage may possess specific genetic adaptations, potentially including optimized metabolic pathways.

• S. Newport isolates show a high degree of AMR profile similarity regardless of source , which may reflect broader genetic conservation including metabolic genes.

What mutations in argG have been identified across S. Newport isolates and how do they affect enzyme function?

While specific mutations in S. Newport argG are not detailed in the available research, several approaches can be used to identify and characterize functional variants:

• Comparative genomics across S. Newport lineages to identify natural variants

• Site-directed mutagenesis of conserved catalytic residues to assess functional impacts

• Random mutagenesis approaches to identify residues affecting stability and activity

• Structure-based predictions of mutations that might alter substrate binding or catalysis

The study of argG variants is particularly relevant given the demonstrated importance of arginine metabolism in Salmonella virulence and the potential for metabolic adaptations to contribute to persistence of strains like REPJJP01 .

How can recombinant S. Newport argG be used to develop novel antimicrobial strategies?

Targeting argG and arginine metabolism represents a promising approach for antimicrobial development:

• High-throughput screening using purified recombinant argG to identify small molecule inhibitors

• Structure-based drug design targeting the ATP-binding site or substrate-binding pockets

• Development of transition-state analog inhibitors specific to bacterial argG

• Exploitation of structural differences between bacterial and human argininosuccinate synthase

The demonstrated importance of arginine metabolism for Salmonella survival during oxidative stress suggests that inhibiting argG could potentially sensitize bacteria to host defense mechanisms and conventional antibiotics, making it a promising adjuvant therapeutic target.

What techniques can be used to study the structure-function relationship of S. Newport argG?

Several complementary approaches can elucidate structure-function relationships in S. Newport argG:

These studies would be particularly valuable given the critical role of arginine metabolism in Salmonella virulence and stress resistance .

What are the most appropriate animal models for studying S. Newport argG function in pathogenesis?

Based on previous research on Salmonella arginine metabolism and pathogenesis, several animal models are suitable for studying S. Newport argG:

• Mouse models:

  • C57BL/6 mice have been successfully used to study the effect of deletions in arginine biosynthesis genes on Salmonella virulence

  • C3H/HeN mice have also been utilized to study the l-arginine deiminase pathway's contribution to Salmonella virulence

• Cell culture models:

  • Macrophage infection models to study intracellular survival

  • Epithelial cell models to examine invasion mechanisms

  • Co-culture systems to investigate interactions with multiple host cell types

• Ex vivo tissue models:

  • Intestinal tissue explants to study local infection dynamics

  • Precision-cut liver slices to examine hepatic infection

The choice of model should be guided by the specific aspect of argG function being investigated, such as its role in oxidative stress resistance, nutrient acquisition, or systemic dissemination.

How can genomic approaches be integrated with functional studies to understand argG's role in S. Newport pathogenesis?

An integrated research approach combining genomic and functional studies offers the most comprehensive understanding of argG's role:

• Genomic approaches:

  • Comparative genomics across S. Newport lineages to identify natural variants

  • Transcriptomic analysis of argG expression during infection

  • ChIP-seq to identify regulators controlling argG expression

  • Whole genome sequencing to contextualize argG within the broader genome

• Functional studies:

  • Construction of argG knockout and complementation strains

  • Site-directed mutagenesis to test specific functional hypotheses

  • In vivo fitness assays using barcoded mutant libraries

  • Metabolomic profiling to assess global impacts of argG disruption

This integrated approach has proven valuable in understanding the complex interplay between Salmonella metabolism and virulence, as demonstrated by research showing how arginine metabolism contributes to oxidative stress resistance and pathogenesis .

How can CRISPR-Cas technologies be applied to study S. Newport argG function?

CRISPR-Cas systems offer powerful approaches for investigating argG function in S. Newport:

• Precise genome editing:

  • Generation of clean deletions or point mutations without antibiotic markers

  • Introduction of reporter fusions at the native locus

  • Creation of conditional expression systems

• Gene regulation:

  • CRISPRi for tunable repression of argG expression

  • CRISPRa for enhanced expression if needed

• High-throughput screening:

  • Genome-wide CRISPR screens to identify genetic interactions with argG

  • Pooled mutant libraries for in vivo fitness assays

These approaches are particularly valuable for studying essential or nearly essential genes like those involved in core metabolism, allowing for more nuanced manipulation than traditional knockout approaches.

What are the emerging research questions regarding argG's role in persistent S. Newport strains like REPJJP01?

Several critical research questions emerge regarding argG in persistent strains like REPJJP01:

• Does argG sequence or expression differ in persistent strains compared to transient isolates?

• How does argG activity respond to antimicrobial exposure, and does this contribute to survival during treatment?

• Are there strain-specific regulatory mechanisms controlling argG expression during infection?

• Does argG contribute to S. Newport's ability to persist in environmental reservoirs between human infections?

• Could targeting argG or the broader arginine biosynthesis pathway help control persistent strains like REPJJP01?

These questions are particularly relevant given that REPJJP01 has caused over 3,100 reported illnesses since its first detection in 2015 , with a concerning 31% hospitalization rate .