Construction and Characterization of a Versatile Broad Host Range DNA Cloning System for Gram–Negative Bacteria

Abstract

A set of broad host range cloning vectors has been constructed from the IncW plasmid pSa. These vectors have been constructed from the transfer defective deletion derivative pSa151, which encodes resistance to kanamycin and spectinomycin–streptomycin. Two of the vectors also contain the chloramphenicol resistance gene of Tn9, and a third contains the chloramphenicol resistance gene of pSa. One of the vectors contains the cos sequence of bacteriophage λ and can be used with in vitro packaging systems in the construction of large recombinant plasmids. Two of these vectors can be mobilized and transferred in the presence of a pBR322 derivative containing the transfer genes of pSa. Together these vectors contain cloning sites for SstII, HindIII, EcoRI, KpnI, PvuII, BamHI, SmaI, and Bg1II, and recombinants at certain of these sites can be detected by insertional inactivation of a drug resistance phenotype. The broad host range properties of the origin of replication of pSa allow the use of these vectors in a variety of gram–negative bacteria.