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Applied and Environmental Microbiology, August 2008, p. 4656-4665, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00074-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Pablo Álvarez-Martín,1 Ana Belén Flórez,1 Abelardo Margolles,1 Gloria del Solar,2 and Baltasar Mayo1*

Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (CSIC), 33300 Villaviciosa, Asturias, Spain,1 Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maetzu 9, 28040 Madrid, Spain2

Received 10 January 2008/ Accepted 7 April 2008

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 lacZ{alpha} 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.

* Corresponding author. Mailing address: Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain. Phone: 34 985 89 21 31. Fax: 34 985 89 22 33. E-mail: baltasar.mayo{at}ipla.csic.es

{triangledown} Published ahead of print on 6 June 2008.

Applied and Environmental Microbiology, August 2008, p. 4656-4665, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00074-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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