Cryptic plasmid pRK2 from Escherichia coli W: sequence analysis and segregational stability

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Escherichia coli W cryptic plasmid pRK2 is an ancestor to penicillin G acylase production strains.
• Understanding plasmid characteristics is crucial for genetic engineering and strain development.

Purpose of the Study:

• To sequence and characterize the cryptic plasmid pRK2.
• To analyze its genetic elements and potential functions.
• To develop and assess stable recombinant plasmids for biotechnological applications.

Main Methods:

• Plasmid DNA sequencing and bioinformatics analysis.
• Identification of open reading frames (ORFs) and protein homology searches.
• Construction of recombinant plasmids with a Spectinomycin resistance determinant (Spc(R)).
• Assessment of plasmid stability and copy number in E. coli.

Main Results:

• pRK2 was classified as a ColE1-like plasmid based on its replication origin.
• Five ORFs were identified, encoding proteins homologous to those involved in replication control (Rom) and mobilization (Mob A-D).
• Recombinant plasmids (pRS11, pRS12, pRS2, pRS3) containing the Spc(R) determinant were successfully constructed.
• These constructs exhibited stable maintenance in E. coli for 63 generations without selection pressure.
• The copy number ranged from 25 to 40 molecules per chromosomal equivalent.

Conclusions:

• The cryptic plasmid pRK2 possesses genetic features characteristic of ColE1-like plasmids.
• Its sequenced genes provide insights into plasmid replication and mobilization mechanisms.
• The developed Spc(R) recombinant plasmids are stable and suitable for use in E. coli.
• These findings support the utility of pRK2 derivatives in biotechnological applications requiring stable expression systems.