RRC ID 33371
Author Merriman TR, Lamont IL.
Title Construction and use of a self-cloning promoter probe vector for gram-negative bacteria.
Journal Gene
Abstract Transposon Tn5 has been used extensively for the genetic analysis of Gram- bacteria. We describe here the construction and use of a Tn5 derivative which contains the ColE1 origin of DNA replication, thereby allowing the cloning of DNA adjacent to the Tn without the need for construction of genomic libraries. The Tn is derived from Tn5-B21 [Simon et al., Gene 80 (1989) 161-169] and contains a promoter-probe lacZ gene and genes encoding resistance to tetracycline and beta-lactams. It is housed within a mobilisable suicide plasmid which can be transferred to a wide range of Gram- bacteria. The Tn was tested using pyoverdine siderophore-synthesis genes (pvd) from Pseudomonas aeruginosa. The simple cloning procedure allowed 15.9 kb of pvd-associated DNA to be cloned; in addition, the lacZ reporter gene allowed the transcription of pvd genes to be studied. The bacteria were resistant to carbenicillin only if the Tn (and hence the beta-lactamase-encoding gene) was downstream from an active promoter.
Volume 126(1)
Pages 17-23
Published 1993-4-15
PII 0378-1119(93)90585-Q
PMID 8386128
MeSH Blotting, Southern Cloning, Molecular DNA Transposable Elements* Drug Resistance, Microbial / genetics Genetic Vectors* Iron / metabolism Lac Operon Mutagenesis, Insertional Oligopeptides* Pigments, Biological / biosynthesis Pigments, Biological / genetics* Promoter Regions, Genetic* Pseudomonas aeruginosa / genetics* Restriction Mapping Siderophores / biosynthesis Siderophores / genetics*
IF 2.498
Times Cited 44
Prokaryotes E. coli pOT182