Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria

Area of Science:

• Microbiology
• Synthetic Biology
• Molecular Biology

Background:

• Rhodobacterales are abundant, metabolically diverse marine alphaproteobacteria with biotechnological potential.
• Limited molecular tools hinder genetic engineering of Rhodobacterales strains.
• Versatile plasmids and secondary metabolite production make them attractive for marine biotechnology.

Purpose of the Study:

• To establish standardized, modular repABC-based plasmid vectors for Rhodobacterales.
• To expand the genetic toolbox for engineering this important bacterial lineage.
• To facilitate genetic construct shuttling across alphaproteobacterial orders.

Main Methods:

• Construction of standardized, modular repABC-based plasmid vectors from four compatibility groups.
• Verification of plasmid replication in Rhodobacterales, Hyphomicrobiales, Rhodospirillales, and Caulobacterales.
• Demonstration of vector applicability via functional complementation of a nitrogenase mutant.

Main Results:

• Successfully established and validated repABC-based plasmid vectors (pABC vectors) in Rhodobacterales and Hyphomicrobiales.
• Confirmed plasmid replication across multiple alphaproteobacterial orders, including Rhodospirillales and Caulobacterales.
• Demonstrated the utility of modular pABC vectors for synthetic pathway implementation through complementation assays.

Conclusions:

• The new pABC vectors significantly expand the genetic engineering capabilities for Rhodobacterales and related alphaproteobacteria.
• The modular design and compatible replication systems facilitate complex genetic manipulations and synthetic biology applications.
• These tools enable efficient genetic construct transfer between different alphaproteobacterial genera and orders.