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Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in

Shuhei Hashiro1, Hisashi Yasueda1

  • 1a Institute for Innovation , Ajinomoto Co., Inc ., Kawasaki , Japan.

Bioscience, Biotechnology, and Biochemistry
|August 21, 2018
PubMed

Insights

Researchers discovered a mutation in the RepA initiator protein of the cryptic plasmid pHM1519. This genetic change significantly increased the plasmid copy number in Corynebacterium glutamicum, offering new insights into plasmid replication control.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Cryptic plasmid pHM1519 belongs to the pCG1 family and utilizes rolling-circle replication in coryneform bacteria.
  • The shuttle vector pPK4, derived from pHM1519, exhibits a low copy number (40-50 copies/chromosome) in Corynebacterium glutamicum 2256.

Purpose of the Study:

  • To investigate the genetic basis for copy number control in the pHM1519 cryptic plasmid system.
  • To identify mutations affecting the replication initiator protein (RepA) and their impact on plasmid stability and replication.

Main Methods:

  • Site-directed mutagenesis was used to introduce a specific mutation (copA1) in the repA gene.
  • Plasmid copy number was quantified in Corynebacterium glutamicum using the derived shuttle vector pPK4.
  • In silico analysis was performed to predict the secondary structure of the RepA protein.

Main Results:

  • A single G to A base transition in the repA gene (copA1 mutation) resulted in a Glycine to Glutamic acid substitution at position 429.
  • The copA1 mutation dramatically increased the copy number of the mutant plasmid to approximately 800 copies per chromosome.
  • Structural prediction indicated Gly429 is in a disordered region within a helix-turn-helix DNA-binding motif of RepA.

Conclusions:

  • This study presents the first instance of achieving a high copy number for a Corynebacterium glutamicum cryptic plasmid through modification of its replication initiator protein.
  • The altered RepA protein, due to the copA1 mutation, likely influences plasmid replication initiation, leading to increased copy numbers.
  • Findings provide a novel mechanism for manipulating plasmid copy number in C. glutamicum, with potential applications in genetic engineering.
Keywords:
Corynebacterium glutamicumRepA initiator proteinhigh copy number mutationpHM1519shuttle vector

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