Mol Gen Genet (1984) 196:494-500 OI-GG © Springer-Verlag 1984 Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO Masataka Tsuda, Shigeaki Harayama*, and Tetsuo Iino Laboratory of Genetics, Department of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan Summary. Transposon insertion mutagenesis of the Pseudo- monas aeruginosa PAt chromosome with Tnl and Tn501 was carried out using a mutant plasmid of R68::Tn501 temperature-sensitive for replication and maintenance. This method consists of three steps. Firstly, the temperature- independent, drug-resistant clones were selected from the strain carrying this plasmid. In the temperature-indepen- dent clones, the plasmid was integrated into the chromo- some by Tnl- or Tn5Ol-mediated cointegrate formation. Secondly, such clones were cultivated at a permissive tem- perature to provoke the excision of the integrated plasmid from the chromosome. Excision occurred by the reciprocal recombination between the two copies of Tnl or Tn501 flanking the integrated plasmid, leaving one Tnl or Tn501 insertion on the chromosome. Thirdly, the excised plasmid was cured by cultivating these isolates at a non-permissive temperature without selection for the drug resistance. Using this method, we isolated 1 Tnl-induced and 43 Tn5Ol-in- duced auxotrophic mutations in this organism. Genetic mapping allowed us to identify two new genes, pur-8001 and met-8003. The Tn5Ol-induced auxotrophic mutations were distributed non-randomly among auxotrophic genes, and the reversion of the mutations by precise excision of the Tn501 insertion occurred very rarely. Introduction Bacterial transposons, which usually encode the genes for resistances to antibiotics and/or heavy metal ions, are capa- ble of moving, as discrete units, from one site on a bacterial replicon to another. These elements can provoke various kinds of structural rearrangement of the DNA sequence adjacent to the elements, namely, deletion, inversion, dupli- cation, replicon fusion, and so on (for recent reviews, see Kleckner 1981 ; Shapiro 1983 a). Because of their properties, transposons can be used as powerful tools for in vivo and in vitro genetic manipulation such as mutant isolation, strain construction, and molecular cloning (Kleckner et al. 1977; Shapiro 1983b). Transposon insertion mutagenesis * Present address: Department of Medical Biochemistry, Univer- sity of Geneva, CH-1211 Geneva, Switzerland Offprint requests to." M. Tsuda has been developed to a high level of sophistication in Escheriehia coli and Salmonella typhimurium (Kleckner et al. 1977), and application of this method has been ex- tended to various gram-negative bacteria in recent years (Beringer et al. 1978; Sato et al. 1981; Selvaraj and Iyer 1983). We have previously reported a method of Tnl insertion mutagenesis using a temperature-sensitive mutant of plas- mid RP4 (Harayama et al. 1980, 1981). An E. coli strain carrying this plasmid (pTH10) showed temperature-sensi- tivity for the drug resistances, and its temperature-indepen- dent drug-resistant derivatives contained pTHI0 integrated into the chromosome by means of the Tnl-mediated cointe- grate formation. Curing of the integrated plasmid resulted in generation of clones which carried only Tnl insertions on the chromosome. Since RP4, which is identical to RP1 and R68 (Burkardt et al. 1979), has a broad host-range among the gram-negative bacteria (Datta et al. 1971), it was expected that Tnl insertion mutagenesis using a tem- perature-sensitive mutant of RP4 would be applicable in many gram-negative bacteria. Some preliminary reports have been published on Tnl insertion mutagenesis in a gram-negative soil bacterium Pseudomonas aeruginosa by use of such plasmids (Robinson et al. 1980; Haas et al. 1981 ; Holloway et al. 1982). This paper describes insertion mutagenesis of the P. aeruginosa PAt chromosome with the mercury transposon Tn501 (Bennett et al. 1978) using a temperature-sensitive. mutant of R68: : Tn501 and characterisation of the Tn501- induced auxotrophic mutations isolated. Materials and methods Bacterial strains, plasmids and bacteriophages. All of the strains were derivatives of P. aeruginosa PAt (Holloway 1969), and they are, together with plasmids, listed in Ta- ble 1. Various R-prime plasmids used in this study were derived from R68.45. The Tn5Ol-loaded derivatives of pMO190 and RP4 were obtained in the cross at 30 °C using MT1551(pMO190) or MT2060(RP4) as the donor and PAt1 as the recipient, and selecting the prototrophic trans- conjugants resistant to tetracycline and HgC12. pMT1000 is one of the pMO190::Tn501 derivatives, pMT902 and pMT929 are the RP4::Tn501 derivatives, and restriction endonuclease analysis showed that the Tn501 insertions in