Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae

• Published in: Plasmid 2012, Vol.67 (1), p.53-59
• Main Authors: Ruiz-Masó, José A., López-Aguilar, Celeste, Nieto, Concha, Sanz, Marta, Burón, Patricia, Espinosa, Manuel, del Solar, Gloria
• Format: Article
• Language: English
• Subjects: Aequorea victoria; Bacterial Proteins - genetics; Base Sequence; Cloning vector; Cloning, Molecular; DNA, Bacterial - genetics; Gene Expression Regulation, Bacterial; Genes, Bacterial - genetics; Genetic Vectors; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Maltose - metabolism; Molecular Sequence Data; Plasmid pMV158; Plasmids - genetics; Pneumococcal malR gene; Polymerase Chain Reaction; Promoter Regions, Genetic - genetics; Regulated expression; Replicon - genetics; Repressor Proteins - genetics; Sequence Homology, Nucleic Acid; Streptococcus; Streptococcus pneumoniae; Streptococcus pneumoniae - genetics
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1016/j.plasmid.2011.09.001

► We construct a plasmid vector for regulated gene expression in pneumococcus. ► Expression of target genes is induced by maltose. ► We test plasmid vector by cloning and expressing the gfp reporter gene. ► The expression system is stable under repression and induction conditions. We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter P M of the S. pneumoniae malMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from P M is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive P tet promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniae.