Recombinant Xylella fastidiosa Phosphoserine aminotransferase (serC)

What is the role of phosphoserine aminotransferase (SerC) in Xylella fastidiosa metabolism?

SerC in X. fastidiosa, like in other organisms, functions as a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 3-phosphohydroxypyruvate (3-PHP) to 3-phosphoserine (PSer) in an L-glutamate-linked reversible transamination reaction. This represents the second step in the phosphorylated pathway of serine biosynthesis . The process typically proceeds through a bimolecular ping-pong mechanism.

In bacterial metabolism, SerC participates in:

• The three-step phosphorylated serine biosynthesis pathway, converting 3-phosphoglycerate (3-PGA) to serine

• Potentially in the DXP-dependent vitamin B6 biosynthetic pathway (based on evidence from related bacteria)

The importance of SerC in X. fastidiosa metabolism likely extends beyond serine biosynthesis, as it may exhibit substrate promiscuity similar to that observed in other bacterial species.

How does the structure of X. fastidiosa SerC compare to characterized SerC proteins from other organisms?

While the specific crystal structure of X. fastidiosa SerC has not been reported in the provided literature, we can infer its likely characteristics based on conserved features of SerC proteins:

• It likely contains a PLP binding site, essential for its aminotransferase activity

• It may possess a similar domain organization to E. coli SerC, which has been extensively characterized

• The active site architecture would accommodate both 3-PHP and glutamate as substrates

Structural analysis would be crucial for understanding:

• Potential substrate binding specificities unique to X. fastidiosa SerC

• Conformational changes during catalysis

• Opportunities for selective inhibition

Homology modeling using related SerC structures could provide preliminary insights into X. fastidiosa SerC structural features prior to experimental determination.

What expression systems yield optimal recombinant X. fastidiosa SerC production?

Based on successful recombinant protein expression strategies for X. fastidiosa proteins and general approaches for SerC expression:

E. coli Expression System:

• BL21(DE3) or similar E. coli strains are suitable hosts for initial expression trials

• Expression can be optimized using the protocol described for other recombinant enzymes, involving:

  • IPTG induction (typically 0.5 mM) when culture reaches OD600 of ~1.0

  • Temperature reduction to 18°C post-induction for 16-18 hours

  • Addition of K2HPO4 (40 mM) at induction to enhance protein stability

Vector Selection:

• pET vectors containing a 6xHis tag facilitate purification

• Codon optimization may be necessary due to potential codon usage differences between X. fastidiosa and E. coli

Considerations for Activity:

• Co-expression with chaperones may improve folding

• Addition of PLP (20-100 μM) to the culture medium can enhance cofactor incorporation

What purification strategies yield the highest purity and activity for recombinant X. fastidiosa SerC?

A multi-step purification process would typically include:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged SerC

  • Binding buffer: 50 mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 1 mM TCEP

  • Elution with imidazole gradient (50-300 mM)

• Secondary Purification: Ion exchange chromatography

  • Resource Q or similar anion exchanger at pH 8.0

  • Salt gradient elution (0-500 mM NaCl)

• Polishing Step: Size exclusion chromatography

  • Superdex 200 column in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

Activity Retention:

• Include PLP (0.1-0.2 mM) in all purification buffers

• Add reducing agents (1-5 mM DTT or TCEP) to prevent oxidation of cysteine residues

• Avoid freeze-thaw cycles; store at -80°C in small aliquots with 15-20% glycerol

What assays can be used to measure X. fastidiosa SerC activity?

Direct Activity Assays:

• Spectrophotometric Coupled Assay:

  • Measure the decrease in NADH absorbance at 340 nm

  • Couple SerC reaction with 3-phosphoserine phosphatase and serine dehydrogenase

  • Reaction mixture: 50 mM HEPES pH 7.5, 5 mM α-ketoglutarate, 0.2 mM NADH, 0.5 mM 3-PHP, coupling enzymes, and purified SerC

• HPLC-Based Assay:

  • Quantify 3-phosphoserine formation by HPLC after derivatization

  • Reaction conditions: 50 mM potassium phosphate pH 7.6, 5 mM glutamate, 1 mM 3-PHP, 50 μM PLP, and purified SerC

  • Terminate reaction with TCA and derivatize products with o-phthalaldehyde

Indirect Assessment:

• Complementation Studies:

  • Transform E. coli serC deletion mutants with X. fastidiosa serC

  • Assess growth restoration on minimal medium without serine supplementation

How do temperature, pH, and metal ions affect X. fastidiosa SerC activity?

While specific data for X. fastidiosa SerC are not available in the provided literature, general characterization would include:

Temperature Optimization:

• Assay enzymatic activity across 20-50°C range

• X. fastidiosa's optimal growth temperature is 26-28°C, suggesting SerC may have maximal activity in this range

pH Profile:

• Test activity across pH 6.0-9.0 using appropriate buffers

• PLP-dependent enzymes typically show optimal activity between pH 7.0-8.0

Metal Ion Effects:

• Evaluate divalent cations (Mg2+, Mn2+, Ca2+, Zn2+) at 1-5 mM concentrations

• Some aminotransferases require metal ions for structural stability or catalytic enhancement

Substrate Specificity:

• Compare activity with various α-keto acids and amino acids

• Determine kinetic parameters (Km, kcat, kcat/Km) for primary substrates

How can serC mutants in X. fastidiosa be generated and characterized?

Mutant Generation:

• Homologous Recombination Strategy:

  • Create a suicide vector containing antibiotic resistance cassette flanked by ~1000 bp genomic fragments adjacent to serC

  • Transform X. fastidiosa with the construct using natural competence

  • Select transformants on appropriate antibiotic-containing media

  • Confirm deletion by PCR and Southern blotting

• Protocol Example:

  • Transform X. fastidiosa strain with a pUC19-based suicide vector harboring kanamycin resistance gene flanked by genomic fragments

  • Culture transformants on PWG plates with kanamycin for 2-3 weeks at 28°C

  • Screen colonies by PCR to confirm deletion

Phenotypic Characterization:

• Growth Analysis:

  • Compare growth rates in standard media and minimal media with/without serine

  • Evaluate biofilm formation using crystal violet staining

  • Assess cell morphology by microscopy

• Virulence Assessment:

  • Inoculate host plants and monitor disease development

  • Quantify bacterial populations in planta over time

  • Evaluate expression of virulence factors in the mutant

Does SerC expression in X. fastidiosa vary under different environmental conditions?

To investigate SerC expression patterns:

Transcriptional Analysis:

• qRT-PCR to quantify serC mRNA levels under various conditions:

  • Different growth phases (log vs. stationary)

  • Nutrient limitation stress

  • Oxidative stress

  • Host plant extracts or xylem sap

  • Biofilm vs. planktonic cells

Proteomic Approach:

• Western blot analysis using anti-SerC antibodies

• MS-based proteomics to quantify SerC protein levels in different growth conditions

Regulation Assessment:

• Identify potential regulatory elements in the serC promoter region

• Determine if serC is regulated by quorum sensing via DSF (diffusible signaling factor)

• Investigate if rpfF or rpfC mutations affect serC expression

How does SerC vary across different X. fastidiosa subspecies and what might this reveal about host adaptation?

X. fastidiosa subspecies (fastidiosa, multiplex, pauca, morus, and sandyi) show differences in host range and virulence . Comparative analysis of SerC could reveal:

Sequence Variation:

• Perform multiple sequence alignment of SerC from different subspecies

• Identify conserved regions essential for catalytic function

• Detect subspecies-specific variations that might correlate with host specificity

Evidence from Genome Analysis:

• Recombination between subspecies has been documented in X. fastidiosa

• The r/m value (relative effect of recombination vs. mutation) is 2.259

• Subspecies-specific alleles might result from horizontal gene transfer or recombination events

Functional Implications:

• Subspecies-specific SerC variants might exhibit different substrate preferences

• Activity differences could contribute to metabolic adaptations to specific host environments

• Changes in SerC sequence might affect interaction with other metabolic enzymes

What evidence exists for horizontal gene transfer or recombination affecting serC in X. fastidiosa populations?

While specific information about serC recombination is not provided in the search results, the general pattern of recombination in X. fastidiosa is well-documented :

Recombination Dynamics:

• Type I restriction-modification systems influence horizontal gene transfer in X. fastidiosa

• Different subspecies show varying levels of recombination:

  • X. fastidiosa subsp. fastidiosa strains in the US show low recombination (average 3.22 of 622 core genes)

  • X. fastidiosa subsp. multiplex shows higher recombination (average 9.60 recombining genes)

  • X. fastidiosa subsp. morus exhibits intersubspecies recombination reaching 15.30% in the core genome

Detection Methods:

• Compare serC sequences across diverse X. fastidiosa isolates

• Use statistical approaches like GARD, RDP4, or ClonalFrameML to detect recombination events

• Perform phylogenetic analysis to identify incongruencies suggestive of horizontal gene transfer

Potential Consequences:

• Recombination in serC could impact enzyme efficiency in different hosts

• Horizontal acquisition of serC variants might contribute to adaptation to new plant hosts

• Monitoring serC variation could provide insights into X. fastidiosa population dynamics

Could recombinant X. fastidiosa SerC serve as a target for antimicrobial development?

SerC could be a promising antimicrobial target for several reasons:

Target Validation:

• SerC is involved in essential amino acid biosynthesis

• A serC deletion would likely be auxotrophic for serine

• Targeting enzymes in amino acid biosynthesis pathways has precedent in antimicrobial development

Rational Inhibitor Design:

• Structure-based drug design using homology models or experimentally determined structures

• Virtual screening against SerC binding pocket

• Fragment-based approaches to identify initial hit compounds

Considerations for Specificity:

• Compare X. fastidiosa SerC to plant and human homologs to identify divergent features

• Target X. fastidiosa-specific residues or conformations

• Design inhibitors that exploit differences in substrate binding or catalytic mechanism

Delivery Challenges:

• X. fastidiosa resides in plant xylem, requiring inhibitors compatible with xylem transport

• Antimicrobial compounds would need to be stable in planta

• Potential for transgenic expression of SerC inhibitors in susceptible crops

How does SerC interact with other metabolic pathways in X. fastidiosa, particularly in relation to virulence?

Understanding SerC's role in X. fastidiosa metabolism requires examining its connections to other pathways:

Metabolic Integration:

• SerC links glycolysis/Calvin cycle (via 3-PGA) to amino acid biosynthesis

• May interact with vitamin B6 biosynthesis, as seen in other bacteria

• Could affect cellular PLP levels, impacting numerous PLP-dependent enzymes

Connection to Virulence Mechanisms:

• Biofilm formation is crucial for X. fastidiosa virulence and is influenced by metabolic status

• Cell-cell signaling via DSF regulates virulence traits

• Proteases contribute to X. fastidiosa pathogenicity , and their activity might be influenced by amino acid availability

Experimental Approaches:

• Metabolomics analysis comparing wild-type and serC mutants

• Interactome studies to identify SerC protein-protein interactions

• Transcriptomics to detect global expression changes resulting from serC mutation