Gene, 78 (1989) 19-27 Elsevier 19 GEN 02988 A simple procedure for transferring genes cloned in Esclrerickz zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR coli vectors into other Gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xyZK (Broad-host-range cloning vector; inverse transposition; transposon TnlO; IncW plasmid; allylglycine; Pseudomonas putida; recombinant DNA; transconjugants) Shigeaki Harayama and Monique Rekik Department of Medical Biochemistry, University of Geneva, Geneva (Switzerland) Tel. (22)22.91.49 Received by J. Davison: 3 October 1988 Revised: 16 December 1988 Accepted: 17 January 1989 SUMMARY A simple method to transfer non-conjugative zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC Escherichiu coli plasmids to other Gram-negative bacteria and their maintenance is described. This method involves generation of inverse transposition-mediated cointegrates ofthe non-conjugative E. coliplasmid with a conjugative IncW broad-host-range plasmid, R388, carrying TnlO. Isolation of such cointegrates was readily effected by conjugal transfer from an E. coli donor containing the two plasmids to an E. coli recipient, with selection for transconjugants expressing a marker of the E. coli plasmid. This method is particularly useful when large series of E. coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination. Utility of the method is shown by a functional analysis in Pseudomonas putidu of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0. INTRODUCTION In the past few years, much effort has been devoted toward developing techniques for the genetic mani- pulation of bacteria other than E. coli, particularly those exhibiting interesting biological properties that cannot be functionally expressed in E. coli. As a Correspondence to: Dr. S. Harayama, Department of Medical Biochemistry, University of Geneva, 1, rue Michel-Servet, 1211 Geneva 4 (Switzerland) Tel. (22)22.91.49; Fax (22)47.33.34. Abbreviations: AM3, antibiotic medium 3; Ap, ampicillin; Nal, nalidixic acid; Pip, piperacillin; R, resistant; s, sensitive; Sm, streptomycin; Tc, tetracycline; Tn, transposon; Tp, trimetho- prim; [ 1, designates plasmid-carrier state. result, transposon mutagenesis and gene cloning can now be employed in a variety of Gram-negative bac- teria other than E. coli (Bagdasarian and Timmis, 1982; Haas, 1983; Mermod et al., 1986a; Simon et al., 1983). For gene cloning, a variety of broad- host-range vectors have been developed and suc- cessfully used to clone genes in bacteria other than E. coli. However, such vectors are relatively primitive by comparison with current E. coli narrow-host- range vectors. Their large size (greater than 10 kb) renders tedious the construction of detailed restric- tion maps and many of those vectors exhibit struc- tural instability (Haas, 1983; Mermod et al., 1986a; Davison et al., 1988). While considerable effort is being made to increase 0378-l 119/89/$03.50 0 1989 Else-&r Science Publishers B.V. (Biomedical Division)