Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1 replicon

Abstract

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 10(1) to 1.0 x 10(5) transformants microg(-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 lacZalpha 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 alpha-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in alpha-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.

FIG. 1.

Physical maps of the plasmids utilized and the constructs obtained from the pBC1 replicon in this work. The key tracks the origins, lengths, and direction of the open reading frames and other structures, as indicated. Only relevant restriction enzyme sites are indicated. Molecules are proportional but not drawn to scale.

FIG. 2.

Hybridization experiments aimed to analyze pBC1 replication intermediates using internal segments of repB genes from pBC1 from B. catenulatum and pBM02 from Lactococcus lactis (positive control for the detection of ssDNA) as probes (36). (A and C) Ethidium bromide-stained gels showing total DNA preparations from B. pseudocatenulatum M115 harboring construct pBC1.2 (8.0 kbp) (lanes 1 and 5), B. pseudocatenulatum M115 carrying construct pBC1.5 (8.7 kbp) (lanes 2 and 6), B. catenulatum L48 containing the original pBC1 plasmid (2.5 kbp) (lanes 3 and 7), and E. coli DH5α carrying construct p210 from L. lactis (3.8 kbp) (lanes 4 and 8). M, molecular weight marker. Plasmids were isolated before (N) and after incubation of the cells for 1 h with both rifampin and chloramphenicol or erythromycin (R). (B) Autoradiogram after hybridization of a gel transferred under nondenaturing conditions (which favors the transfer of ssDNA). (D) Autoradiogram after hybridization of a gel transferred under denaturing conditions. The position of ssDNA of plasmid p210 from L. lactis in the sample treated with rifampin and chloramphenicol (B and D, lanes 8) is indicated.

FIG. 3.

Amplification and cloning of the α-l-arabinofuranosidase gene abfB from Bifidobacterium longum B667 and cloning in pAM1. Molecules are proportional but not drawn to scale.