Improved Cloning Vectors for Bifidobacteria, Based on the Bifidobacterium catenulatum pBC1 Replicon

• Published in: Applied and Environmental Microbiology 2008-08, Vol.74 (15), p.4656-4665
• Main Authors: Álvarez-Martín, Pablo, Belén Flórez, Ana, Margolles, Abelardo, del Solar, Gloria, Mayo, Baltasar
• Format: Article
• Language: English
• Subjects: Antibiotic resistance; Antibiotics; Bacteria; Bacterial Proteins - genetics; Bacteriology; Bifidobacterium - genetics; Bifidobacterium - growth & development; Bifidobacterium catenulatum; Cloning; Cloning, Molecular - methods; DNA Primers; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; DNA, Single-Stranded - genetics; Drug Resistance, Bacterial; Genes; Glycoside Hydrolases - genetics; In Situ Hybridization; Meeting Presentations; Plasmids; Replicon - genetics
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.00074-08

This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 x 10¹ to 1.0 x 10⁵ transformants μg⁻¹ but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZα peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an α-L-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in α-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.