Transposable elements for efficient manipulation of a wide range of Gram-negative bacteria: promoter probes and vectors for foreign genes

• Published in: Gene Vol. 85; no. 1; pp. 83 - 89
• Main Authors: Joseph-Liauzun, Evelyne, Fellay, Rémy, Chandler, Michael
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 21.12.1989; Amsterdam Elsevier; New York, NY
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA Probes; DNA Transposable Elements; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes; Genetic engineering; Genetic technics; Genetic Vectors; Gram-Negative Bacteria - genetics; inducible promoters; insertion sequence; IS1; Methods. Procedures. Technologies; Molecular Sequence Data; Mutation; omegon-Km; Paracoccus denitrificans - genetics; plant pathogen; Plasmids; Promoter Regions, Genetic; Pseudomonas - genetics; Recombinant DNA; Restriction Mapping; Rhizobium - genetics; transposition; Xanthomonas - genetics; Xanthomonas campestris; Zymomonas mobilis; Km; omegon-Km; oriV; IS1; nt; Ks; Cm; Recombinant DNA; bp; Tc; transposition; R; IRL and IRR; cat; kb; X; oligo; oriT; Z; Zymomonas mobilis; RBS; plant pathogen; inducible promoters; IS; Rif; Xanthomonas campestris; TEA; insertion sequence; XGal; PolIk; Sm; Chromosomal aberration; Pseudomonadales; Heterologous system; Chromosome insertion; Transcription promoter; Transposon; Site directed mutagenesis; Transposable element; Method; DNA; Abnormal chromosome; Pseudomonadaceae; Bacteria; Genetic engineering; Molecular cloning; Vector; Hybrid gene; Gram negative bacteria
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(89)90467-8

We describe here the construction and use of a series of modified transposons based on the insertion sequence IS1. Like their parent, omegon-Km [Fellay et al., Gene 76 (1989) 215–226], these elements permit efficient insertional mutagenesis of a variety of Gram-negative bacteria. The presence of a functional pBR322 origin of replication within the transposable element facilitates subsequent cloning of the mutated gene. The omegon-Km system was previously shown to function in Pseudomonas putida, Rhizobium leguminosarum and Paracoccus denitrificans. The results we present here demonstrate that its use can be extended to Xanthomonas campestris, a plant pathogen, and to the microaeroduric Zymomonas mobilis. Derivative transposons carrying unique restriction sites for ScaI, NdeI, XbaI and XhoI have been constructed, allowing the cloning and introduction of foreign genes. We have also constructed two derivatives which can be used to generate operon fusions upon insertion and are thus useful for isolating and characterising indigenous promoters. One carries a promoterless chloramphenicol acetyl-transferase (CAT)-encoding gene ( cat) and the second, the entire promoterless Escherichia coli lac operon. We demonstrate the utility of the cat promoter probe in X. campestris to target conditional promoters inducible by high salt or subject to repression by glucose.