title: A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.
abstract: The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Trine
authors.initials: T
authors.lastname: Aakvik
authors.forename: Kristin Fløgstad
authors.initials: KF
authors.lastname: Degnes
authors.forename: Rannveig
authors.initials: R
authors.lastname: Dahlsrud
authors.forename: Frank
authors.initials: F
authors.lastname: Schmidt
authors.forename: Ragnar
authors.initials: R
authors.lastname: Dam
authors.forename: Lihua
authors.initials: L
authors.lastname: Yu
authors.forename: Uwe
authors.initials: U
authors.lastname: Völker
authors.forename: Trond Erling
authors.initials: TE
authors.lastname: Ellingsen
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
mesh_terms: D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004261:DNA Replication; D004783:Environmental Microbiology; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D042822:Genomic Instability; D011551:Pseudomonas fluorescens / Q000235:genetics; D018741:Replication Origin; D014169:Transformation, Bacterial; D016959:Xanthomonas campestris / Q000235:genetics