A new replicon suitable for cloning and gene expression was successfully introduced into Streptococcus pneumoniae. The non-integrative lactococcal vectors pIL253 (higher-copy) and pIL252 (lower-copy), which are based on the promiscuous theta-replicating plasmid pAM beta 1, were established in pneumococcus. The stability and the small size of these plasmids, together with the presence of a helpful multi-cloning site make them a useful genetic tool for gene expression in this bacterium. The functionality of the system was tested by cloning and expressing the pneumococcal RNase R gene in pIL253. Full constitutive expression of the cloned gene was observed, clearly demonstrating that this plasmid can be used as an expression vector in S. pneumoniae. Moreover, gene expression can be regulated by the use of the lower- or higher-copy number vector versions. The existence of other replicative plasmids based on this family, which are also probably functional in pneumococcus, further broadens the cloning possibilities. We also show that S. pneumoniae cells can accommodate simultaneously pIL252 or pIL253 together with pLS1, a pMV158 derivative, which replicates via a rolling circle mechanism. This fact greatly increases the ability to manipulate this bacterium. The availability of a new family of replicative vectors for genetic manipulation in S. pneumoniae is an important contribution to the study of this pathogenic microorganism. (C) 2013 Elsevier Inc. All rights reserved.