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Microbiology 152 (2006), 343-349; DOI  10.1099/mic.0.28433-0
© 2006 Society for General Microbiology

Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae

Sébastien Guiral, Vincent Hénard, Maria-Halima Laaberki{dagger}, Chantal Granadel, Marc Prudhomme, Bernard Martin and Jean-Pierre Claverys

Laboratoire de Microbiologie et Génétique Moléculaires, UMR 5100 CNRS-Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France

Correspondence
Jean-Pierre Claverys
claverys{at}ibcg.biotoul.fr

In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of Streptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in Escherichia coli, but not in S. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, PM, separated from a kanamycin-resistance gene by NcoI and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of PM can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and NcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.

Abbreviations: CSP, competence-stimulating peptide; CEP, chromosomal expression platform; Ery, erythromycin; Kan, kanamycin; RLU, relative luminescence unit; Sm, streptomycin

{dagger}Present address: UMR1225 Interactions Hôtes-Agents Pathogènes, INRA-Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles BP 87614, 31076 Toulouse Cedex 3, France.




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