Sequence analysis of plasmid pSP02 from Bifidobacterium longum M62 and construction of pSP02-derived cloning vectors

Replicons from bifidobacteria species are required for the construction of general- and special-purpose vectors that would allow the undertaking of molecular studies of these bacteria. In this work, pSP02, a cryptic plasmid from Bifidobacterium longum M62, was cloned, sequenced and characterized. pSP02 was found to consist of 4896bp with four ORFs coding for proteins over 50 amino acids long. Among the deduced protein sequences only a replicase (RepA) and a mobilization-like protein (MobA) showed known functional domains. Similar to previously described bifidobacterial plasmids, the organization of the putative ori region of pSP02 resembles that of the theta-replicating plasmids of Gram-positives. In spite of this, hybridization experiments detected single stranded (ss)-DNA as an intermediate product in the pSP02 replication, demonstrating it follows the rolling-circle (RC) replication mode. The ori region of pSP02 was used to construct a series of first generation cloning vectors able to replicate in many bifidobacterial species. Real time quantitative PCR established the copy number of pSP02 and its derived vectors to be around 12 copies per chromosome equivalent. pSP02-derivatives showed full segregational and structural stability even in the absence of antibiotic selection.