Recombinant Bdellovibrio bacteriovorus Serine hydroxymethyltransferase (glyA)

Basic Research Questions

• What is the primary enzymatic function of GlyA in B. bacteriovorus, and how is it validated experimentally?
  GlyA encodes serine hydroxymethyltransferase (SHMT), which catalyzes the reversible conversion of serine to glycine using tetrahydrofolate as a one-carbon carrier . In B. bacteriovorus, recombinant GlyA may also exhibit alanine racemase activity, as demonstrated in homologous systems like Chlamydia pneumoniae .

  • Methodology:

    • Enzymatic assays: Use D-amino acid oxidase (DAAO) to quantify D-alanine production from L-alanine .

    • Inhibition studies: Test sensitivity to D-cycloserine, a competitive inhibitor of alanine racemases .

    • Genetic complementation: Express recombinant GlyA in E. coli racemase mutants to restore D-alanine synthesis .

• How is recombinant GlyA purified for functional studies?
  Recombinant GlyA is typically expressed in E. coli with affinity tags (e.g., Strep-tag) and purified via column chromatography .

  • Key steps:

    • Expression: Use IPTG-inducible promoters in E. coli BL21(DE3) .

    • Purification: Strep-Tactin® columns for tag-specific elution .

    • Activity validation: Compare racemase activity to positive controls (e.g., Bacillus stearothermophilus alanine racemase) .

Advanced Research Questions

• How does GlyA’s dual functionality (SHMT and alanine racemase) impact B. bacteriovorus predation?
  GlyA may enable B. bacteriovorus to synthesize D-alanine, a critical component of bacterial peptidoglycan, during intraperiplasmic growth . This self-sufficiency could bypass reliance on prey-derived D-alanine, enhancing predation efficiency.

  • Experimental evidence:

    • Gene knockout studies: Compare predation rates of wild-type and ΔglyA mutants in biofilms vs. planktonic prey .

    • Metabolic profiling: Quantify glycine and D-alanine levels in bdelloplasts using LC-MS .

• What structural features of GlyA enable its broad substrate specificity?
  GlyA’s pyridoxal 5′-phosphate (PLP) binding site facilitates diverse reactions, including transamination and retroaldol cleavage .

  • Approaches for analysis:

    • Homology modeling: Compare B. bacteriovorus GlyA to solved structures (e.g., E. coli SHMT, PDB: 1LS3) .

    • Site-directed mutagenesis: Target residues in the PLP-binding domain (e.g., Lys229, Ser254) to disrupt activity .

Data Contradictions and Technical Challenges

• Why do conflicting reports exist about GlyA’s role in biofilm predation?
  Discrepancies arise from differences in experimental conditions (e.g., prey species, biofilm matrix composition) .

  • Key findings:

    Condition	Wild-Type Predation Efficiency	ΔpilT2 Mutant Efficiency
    Planktonic	100% prey lysed	95% prey lysed
    Biofilm	35.4% biofilm remaining	92.5% biofilm remaining

  • Resolution: Use standardized biofilm models (e.g., E. coli MG1655 in flow cells) .

• How does D-cycloserine inhibition affect GlyA’s role in predation?
  D-cycloserine competitively inhibits alanine racemase activity but not SHMT function .

  • Experimental design:

    • Treat B. bacteriovorus with 10 mM D-cycloserine and monitor bdelloplast formation .

    • Rescue assays: Supplement with 5 mM D-alanine to reverse inhibition .

Methodological Recommendations

• What strategies optimize heterologous GlyA expression in E. coli?

  • Vector design: Use low-copy plasmids (e.g., pET-28a) with T7 promoters to reduce toxicity .

  • Codon optimization: Adapt B. bacteriovorus glyA codons for E. coli preference .

  • Induction conditions: 0.1 mM IPTG at 18°C for 16 hours to enhance soluble protein yield .

• How to assess GlyA’s regulatory role in B. bacteriovorus life cycle transitions?

  • Transcriptomics: Compare ΔglyA and wild-type gene expression during attack vs. growth phases .

  • C-di-GMP profiling: Link GlyA activity to secondary messenger signaling using LC-MS/MS .