Recombinant Pseudomonas syringae pv. tomato Orotate phosphoribosyltransferase (pyrE)

Basic Research Questions

• What is the pyrE gene in Pseudomonas syringae pv. tomato DC3000?

  The pyrE gene (locus tag PSPTO_0080) in Pseudomonas syringae pv. tomato DC3000 encodes orotate phosphoribosyltransferase (OPRTase), an essential enzyme in the pyrimidine biosynthetic pathway. It is located on the chromosome at position 101265-101906 on the positive strand . The gene encodes a 23.2 kDa protein with an isoelectric point of 5.30 and a Kyte-Doolittle hydrophobicity value of 0.062 . Unlike in mammals where this function is part of a bifunctional enzyme (UMP synthase), in bacteria like P. syringae, OPRTase is an independent enzyme with a unique gene coding for the protein .

  Key genomic and protein details:

  Feature	Information
  Locus Tag	PSPTO_0080
  NCBI Old Locus Tag	PSPTO0080
  Genomic location	101265 - 101906 (+ strand)
  RefSeq	NP_789939.1
  UniProtKB Acc	Q88BD7
  Molecular Weight	23.2 kDa
  Isoelectric Point	5.30
  Charge (pH 7)	-4.07

• What is the function of orotate phosphoribosyltransferase in P. syringae metabolism?

  Orotate phosphoribosyltransferase (OPRTase) catalyzes a critical step in the pyrimidine biosynthesis pathway. Specifically, it catalyzes the reversible phosphoribosyl transfer from 5'-phospho-alpha-D-ribose 1'-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5'-monophosphate (OMP) . This reaction represents the fifth step in the de novo pyrimidine synthesis pathway in bacteria .

  The reaction can be represented as:

  Orotate + PRPP → OMP + PPi

  This enzymatic step is essential for bacterial growth and survival, as pyrimidine nucleotides are fundamental components of nucleic acids and other cellular processes. In P. syringae, the pathway begins in the cytosol with the synthesis of carbamoyl phosphate, followed by several enzymatic steps to form dihydroorotate, which is then oxidized to orotate. The orotate is subsequently converted to OMP by OPRTase, and finally, OMP is decarboxylated to form UMP, the precursor for all pyrimidine nucleotides .

• How does bacterial OPRTase differ from mammalian OPRTase in structure and function?

  The key difference between bacterial and mammalian OPRTase lies in their protein organization:

  • In bacteria (including P. syringae): OPRTase is an independent enzyme with a dedicated gene (pyrE) encoding only the OPRTase function .

  • In mammals and other multicellular organisms: The OPRTase catalytic function is carried out by the N-terminal domain of a bifunctional enzyme called UMP synthase (UMPS). The C-terminal domain of this enzyme contains orotidylate decarboxylase activity, which catalyzes the subsequent step in the pathway .

  This structural difference has implications for metabolic regulation and potentially for drug targeting, as bacterial OPRTase can be targeted specifically without directly affecting the human enzyme's structure. The bacterial enzyme's independent nature means it may have different regulatory mechanisms and kinetic properties compared to the mammalian bifunctional enzyme.

• What experimental approaches are recommended for genetically manipulating the pyrE gene in P. syringae?

  Several approaches can be used for manipulating the pyrE gene in P. syringae, with recombineering being particularly effective. Based on recent advances, the following methods are recommended:

  1. RecTE-mediated recombineering: P. syringae has endogenous RecT and RecE homologs that can be leveraged for genetic manipulation. The RecT protein alone is sufficient for recombination of single-stranded DNA oligonucleotides, while efficient recombination of double-stranded DNA requires both RecT and RecE .

  2. Optimizing homology length: For successful RecTE-mediated recombination, the length of flanking homologies significantly impacts efficiency. Research indicates that longer homology arms increase recombination frequency .

  3. Substrate design considerations: The length of sequences being inserted or deleted affects recombination efficiency. Consider these factors when designing your recombineering strategy .

  Recommended workflow:

  • Express RecTE proteins from P. syringae pv. syringae B728a in your target strain

  • Design oligonucleotides or dsDNA with appropriate homology arms (50-100 bp recommended)

  • Electroporate the DNA into cells expressing RecTE

  • Select for successful recombinants using appropriate markers

  This approach has been successfully used to make targeted gene disruptions in the P. syringae chromosome .

Intermediate Research Questions