Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1 replicon.
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.
Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain.
Pablo
P
Alvarez-Martín
Ana
A
Belén Flórez
Abelardo
A
Margolles
Gloria
G
del Solar
Baltasar
B
Mayo
D001426:Bacterial Proteins / Q000235:genetics; D001644:Bifidobacterium / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics; D017403:In Situ Hybridization; D010957:Plasmids; D012093:Replicon / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase