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

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, P-M, separated from a kanamycin-resistance gene by Ncol and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of P-M can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and Ncol/BamHl-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.