Development of designer S. meliloti strains and multi-functional vectors.

(a) Schematic of the designer S. meliloti ΔpSymA ΔhsdR strains including the genome reduction and deletion of the restriction-system. Several versions of the designer strains were developed including strains with the pTA-Mob conjugative plasmid, and/or compatible genome engineering vectors (pAGE or pBGE) installed. (b) Vector map of pAGE/pBGE MHS vector with both standard and interchangeable components. Standard components include the BAC and YAC backbone (BAC/YAC), an origin of transfer (oriT), and the selectable marker for P. tricornutum nourseothricin N-acetyl transferase (Ntc). Interchangeable components include selectable markers for S. meliloti: tetracycline (Tet), kanamycin/neomycin (Neo), and spectinomycin (Spec); and origins of replication for S. meliloti: the pSymA origin (repA2B2C2), and pSymB origin (repA1B1C1). Three vectors (pAGE1.0, pAGE2.0, and pAGE3.0) were constructed utilizing the repA2B2C2 origin with Spec, Tet or Neo selection, respectively. Three vectors (pBGE1.0, pBGE2.0, and pBGE3.0) were constructed utilizing the repA1B1C1 origin with Spec, Tet or Neo selection, respectively. (c) Following conjugation of three pAGE plasmids from E. coli ECGE101 conjugative strain to S. meliloti RmP4122 pSymA ∆hsdR Nms, an antibiotic selection test for each plasmid was performed. The pAGE1.0, pAGE2.0, and pAGE3.0 vectors in S. meliloti contain antibiotic resistance markers for Spec, Tet, and Neo, respectively. S. meliloti without a plasmid (control) was plated alongside S. meliloti harbouring pAGE1.0, pAGE2.0, or pAGE3.0 on LBmc agar, and LBmc agar supplemented with either spectinomycin (200 μg mL-1), tetracycline (10 μg mL-1), or neomycin (100 μg mL-1).