A broad host range replicon with different requirements for replication initiation in three bacterial species

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Plasmid RK2 exhibits broad-host-range replication in Gram-negative bacteria.
• Replication requires the origin of replication (oriV) and the TrfA initiation protein.
• Understanding RK2 replication in diverse hosts is crucial for genetic engineering.

Purpose of the Study:

• To investigate the functional compatibility of Pseudomonas DnaB helicases with RK2 replication machinery.
• To determine if accessory proteins are universally required for helicase loading at oriV.

Main Methods:

• Genes for Pseudomonas putida and Pseudomonas aeruginosa DnaB helicases were cloned and expressed.
• Purified helicases were tested for activity at the RK2 origin with TrfA and host DnaA proteins.
• Helicase loading and activation were assessed in vitro.

Main Results:

• Both Pseudomonas DnaB helicases were recruited and activated at RK2 oriV with host DnaA and TrfA.
• P. putida DnaB functioned with both TrfA forms, while P. aeruginosa DnaB required TrfA-44.
• Unlike E. coli DnaB, Pseudomonas helicases loaded at oriV without an accessory ATPase (DnaC-like protein).

Conclusions:

• Pseudomonas DnaB helicases can be activated at the RK2 origin.
• A DnaC-like accessory ATPase is not universally essential for replicative helicase loading at this origin.
• This finding broadens our understanding of DNA replication initiation mechanisms across different bacterial species.