A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vector

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Pseudomonas putida is a versatile bacterium with a complex plasmid landscape.
• Cryptic plasmids, lacking apparent function, are common but poorly understood.
• Understanding plasmid replication mechanisms is crucial for bacterial genetics.

Purpose of the Study:

• To characterize a novel cryptic circular plasmid, pPP8-1, from Pseudomonas putida P8.
• To elucidate the replication mechanism of pPP8-1.
• To develop pPP8-1 as a potential cloning vector for pseudomonads.

Main Methods:

• Plasmid isolation and sequencing.
• Bioinformatic analysis of open reading frames (ORFs).
• Identification of replication origins (double-strand and single-strand).
• Confirmation of replication intermediates via Southern analysis and nuclease digestion.
• Cloning of a kanamycin-resistance gene and transformation experiments.

Main Results:

• Three cryptic plasmids (pPP8-1, pPP8-2, pPP8-3) were detected in P. putida P8.
• pPP8-1 (2534 bp) contains four ORFs; ORF A shows homology to rolling circle (RC) replication proteins.
• A double-strand origin and a putative single-strand origin characteristic of RC replication (RCR) were identified in pPP8-1.
• Single-stranded DNA replication intermediates were confirmed.
• pPP8-1 was successfully engineered into a functional cloning vector for P. putida and Escherichia coli.

Conclusions:

• pPP8-1 represents the first identified rolling circle replicating plasmid in pseudomonads.
• The characterized replication mechanism provides insights into plasmid diversity in this genus.
• The engineered pPP8-1 vector is a valuable new tool for genetic manipulation of Pseudomonas species.