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Updated: May 23, 2026
Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG
Published on: May 16, 2017
Identification of the region required for maintaining pHW126 in its monomeric form
Wilfried Rozhon1, Mamoona Khan, Brigitte Poppenberger
1Biotechnology of Horticultural Crops, Technische Universität München, Freising, Germany. wilfried.rozhon@wzw.tum.de
Abstract:
The pHW126-like plasmids are a recently discovered small group of cryptic plasmids replicating by the rolling circle mode. The replication origin of pHW126 consists of a conserved stretch, four perfect direct repeats and a so-called accessory region. The latter increases plasmid stability but is not absolutely necessary for replication. Here, we report that deletion of the accessory region causes rapid multimerization of pHW126. While the number of pHW126-units per cell remains constant, the number of physically independent plasmid molecules is reduced by approximately 40%, rendering random distribution to daughter cells less effective. A conserved inverted repeat within the accessory region could be identified as a sequence necessary for maintaining pHW126 in its monomeric form. A mutant version of pHW126 lacking this inverted repeat could be rescued by placing the single-strand initiation site (ssi) of pHW15 on the plus strand, while including the ssi in the opposite direction had no effect. Thus, our data provide evidence that multimer formation is, besides copy number reduction and ssDNA accumulation, an additional means how loss of a mechanism ensuring efficient lagging strand synthesis may cause destabilization of rolling circle plasmids.
Insights
Deletion of a specific region in pHW126 plasmids causes rapid multimerization. This instability in rolling circle plasmids highlights the importance of accessory regions for maintaining monomeric forms and efficient replication.
Area of Science:
- Molecular Biology
- Genetics
Background:
- pHW126-like plasmids are cryptic plasmids that replicate via rolling circle mechanism.
- Their replication origin includes conserved regions, direct repeats, and an accessory region crucial for stability.
- The accessory region enhances plasmid stability but is not essential for replication itself.
Discussion:
- Deletion of the accessory region in pHW126 leads to rapid plasmid multimerization.
- While the number of plasmid units per cell is constant, the reduction in independent molecules impairs distribution.
- A conserved inverted repeat within the accessory region is identified as critical for maintaining monomeric form.
Key Insights:
- The accessory region, specifically an inverted repeat, is vital for preventing multimerization of pHW126 plasmids.
- Loss of this region destabilizes the plasmid, leading to reduced independent molecules and ineffective distribution.
- Plasmid multimerization is presented as an additional destabilization mechanism linked to impaired lagging strand synthesis.
Outlook:
- Further research can explore the precise mechanism by which the inverted repeat prevents multimerization.
- Investigating the role of single-strand initiation sites (ssi) in other rolling circle plasmids could yield broader insights.
- Understanding these destabilization pathways is crucial for engineering stable rolling circle plasmids for biotechnological applications.
