title: Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae.
abstract: 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. pneumoniaemalMP 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.
authors.affiliation: Molecular Microbiology and Infection Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E28040 Madrid, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Ruiz-Masó
authors.forename: Celeste
authors.initials: C
authors.lastname: López-Aguilar
authors.forename: Concha
authors.initials: C
authors.lastname: Nieto
authors.forename: Marta
authors.initials: M
authors.lastname: Sanz
authors.forename: Patricia
authors.initials: P
authors.lastname: Burón
authors.forename: Manuel
authors.initials: M
authors.lastname: Espinosa
authors.forename: Gloria
authors.initials: G
authors.lastname: del Solar
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012093:Replicon / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013296:Streptococcus pneumoniae / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; C084528:malR protein, bacteria; D049452:Green Fluorescent Proteins; D008320:Maltose