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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 4896. bp 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. © 2012 Elsevier Inc., Funding was obtained from projects from the Spanish Ministry of Science and Innovation (MICINN) (Ref. AGL2011-24300-ALI) and the Institute of Biochemistry and Biophysics-PAN (IBB-PAN).

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures. © 2012, American Society for Microbiology., This work was done in the framework of scientific cooperation between the Polish Academy of Sciences (PAN) and the Consejo Superior Investigaciones Científicas (CSIC) and was partly supported by the Spanish Ministry of Science and Innovation (MICINN) (AGL2007-61869-ALI) and the Institute of Biochemistry and Biophysics, PAN (IBB PAN). Á. Alegría was supported by a grant from FICYT (BP08-053).