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

Members of the alphaproteobacterial order Rhodobacterales
are metabolically
diverse and highly abundant in the ocean. They are becoming increasingly
interesting for marine biotechnology, due to their ecological adaptability,
wealth of versatile low-copy-number plasmids, and their ability to
produce secondary metabolites. However, molecular tools for engineering
strains of this bacterial lineage are limited. Here, we expand the
genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups
from the Roseobacter group applicable in the Rhodobacterales, and
likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales,
Caulobacterales). We confirmed replication of these newly constructed
pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members
of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and “Agrobacterium fabrum” C58). Maintenance of
the pABC vectors in the biotechnologically valuable orders Rhodobacterales
and Hyphomicrobiales facilitates the shuttling of genetic constructs
between alphaproteobacterial genera and orders. Additionally, plasmid
replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member
of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of
four compatible replication systems, which allows their coexistence
in a host cell, are advantageous features for future implementations
of newly designed synthetic pathways. The vector applicability was
demonstrated by functional complementation of a nitrogenase mutant
phenotype by two complementary pABC-based plasmids in R. capsulatus.