Characterization of the cryptic plasmid pBGR1 from Bartonella grahamii and construction of a versatile Escherichia coli-Bartonella spp. shuttle cloning vector

We report herein the isolation and molecular characterization of pBGR1, the first native plasmid isolated from the genus Bartonella. Cloning and sequencing revealed a 2725-base pair (bp) cryptic plasmid comprising two open reading frames of considerable length, which were designated rep and mob. The regions containing rep and mob are separated by 140-bp inverted repeat sequences and display a difference in G + C content from one another. A 1435-bp SacI-BclI fragment containing the rep gene is sufficient to mediate replication in the species Bartonella henselae and Bartonella tribocorum, while this replicon does not appear to be functional in Escherichia coli. The Rep protein of 190 amino acids (aa) shares homology to putative replication proteins of cryptic plasmids of Gram-negative origin, which form a subgroup of the rolling-circle replication proteins of the pSN2 plasmid superfamily of Gram-positive bacteria. The Mob protein of 333 aa is related to mobilization proteins of several cryptic plasmids and is associated with a conserved recombination site A. The tra functions of RP4 can mobilize pBGR1 derivatives in a mob-dependent manner. Mobilizable pBGR1-based E. coli-Bartonella spp. shuttle vectors were constructed and were shown to be maintained in B. tribocorum during in vivo passage in a rat model in the absence of antibiotic selection. The small size and stability of these shuttle cloning vectors should render them particularly valuable for genetic studies in Bartonella spp.