A phasmid shuttle vector for the cloning of complex operons in Salmonella.
Phasmid (phage plasmid hybrid) P4 vir1 can be propagated in Escherichia coli as a helper-dependent lytic phage, as a plasmid, or as a prophage. On the basis of an understanding of these modes of propagation, derivatives of P4 have been constructed for use as cloning vectors. In this report we demonstrate that phasmid P4 (i) will propagate as a helper-dependent lytic phage and as a plasmid in Salmonella spp. and (ii) can be used as a high efficiency phage shuttle vector for the reversible transfer of cloned genes between Salmonella spp. and E. coli. For both E. coli and Salmonella spp., P4 phage-mediated gene transfer proved to be only 10-fold lower than plaquing efficiency. For the case of Salmonella spp., this frequency is ca. 10(4)-fold more efficient than is typically found for the transformation of DNA molecules. The usefulness of this cloning vector system for analyses of pathogenic virulence factors is demonstrated by the cloning and expression of both the P pilus adhesin operon and the hemolysin operon of uropathogenic E. coli.
Gutmann
L
L
Maxwell Finland Laboratory For Infectious Diseases, Boston University, School of Medicine, Massachusetts 02118.
Agarwal
M
M
Arthur
M
M
Campanelli
C
C
Goldstein
R
R
D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000235:genetics; D009876:Operon; D010957:Plasmids; D015183:Restriction Mapping; D012475:Salmonella / Q000235:genetics*; D012482:Salmonella paratyphi A / Q000235:genetics; D012486:Salmonella typhimurium / Q000235:genetics; D014161:Transduction, Genetic; D014169:Transformation, Bacterial; D010948:Viral Plaque Assay
D006460:Hemolysin Proteins