title: New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces.
abstract: Integrative plasmids are one of the best options to introduce genes in low copy and in a stable form into bacteria. The ΦC31-derived plasmids constitute the most common integrative vectors used in Streptomyces. They integrate at different positions (attB and pseudo-attB sites) generating different mutations. The less common ΦBT1-derived vectors integrate at the unique attB site localized in the SCO4848 gene (S. coelicolor genome) or their orthologues in other streptomycetes. This work demonstrates that disruption of SCO4848 generates a delay in spore germination. SCO4848 is co-transcribed with SCO4849, and the spore germination phenotype is complemented by SCO4849. Plasmids pNG1-4 were created by modifying the ΦBT1 integrative vector pMS82 by introducing a copy of SCO4849 under the control of the promoter region of SCO4848. pNG2 and pNG4 also included a copy of the P ermE * in order to facilitate gene overexpression. pNG3 and pNG4 harboured a copy of the bla gene (ampicillin resistance) to facilitate selection in E. coli. pNG1-4 are the only integrative vectors designed to produce a neutral phenotype when they are integrated into the Streptomyces genome. The experimental approach developed in this work can be applied to create phenotypically neutral integrative plasmids in other bacteria.
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional e IUOPA, Facultad de Medicina, Universidad de Oviedo, 33006, Oviedo, Spain.
authors.forename: Nathaly
authors.initials: N
authors.lastname: Gonzalez-Quiñonez
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional e IUOPA, Facultad de Medicina, Universidad de Oviedo, 33006, Oviedo, Spain.
authors.forename: María Teresa
authors.initials: MT
authors.lastname: López-García
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional e IUOPA, Facultad de Medicina, Universidad de Oviedo, 33006, Oviedo, Spain.
authors.forename: Paula
authors.initials: P
authors.lastname: Yagüe
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional e IUOPA, Facultad de Medicina, Universidad de Oviedo, 33006, Oviedo, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Rioseras
authors.affiliation: Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pisciotta
authors.affiliation: Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Alduina
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional e IUOPA, Facultad de Medicina, Universidad de Oviedo, 33006, Oviedo, Spain. mantecaangel@uniovi.es.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Manteca
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D010641:Phenotype; D010957:Plasmids; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics* / Q000821:virology*
keywords: Heterologous expression; Neutral phenotype; Streptomyces; ΦBT1 integrative vector