Electron Microscopic Observation of New Transposable Elements Inserted into P22 Phage Genome from R Plasmids

Abstract

By using phage P22spl, a deletion mutant of phage P22, the structures of two new transposons on P22 genomes were studied by the electron microscopic heteroduplex method.One of these was the Cm (chloramphenicol) transposon derived from an R plasmid, NR1, and the other the Km (kanamycin) transposon from pNR502.The heteroduplex between P22 phage DNAs with and without the Cm transposon revealed that the Cm transposon was similar in structure to the Tn9 element, a well-known Cm transposon derived from the R plasmid pMS14.On the other hand, the Km transposon of pNR502 was quite different in structure from other Km transposons reported previously.This transposon consists of a 6.8 kilobase (kb) segment of DNA, in which a short inverted repeat is contained.The heteroduplex experiments showed that a 4.5 kb segment of DNA was deleted from the P22 genome in the P22spl genome.Because of a shorter unit length of the genome, phage P22spl is considered to be useful for assaying various kinds of transposable elements.Various kinds of P22-R hybrid phages have been isolated and characterized in our laboratories. These hybrid phages transduced the relevant resistance genes at frequencies higher than 10-3 per plaque-forming unit (PFU). Several genetical observations indicated that the hybrid phages carried the resistance genes translocated from R plasmids to the phage genome without loss of essential phage genes (18,20).By using these phages, we investigated the structure of the translocated resistance genes on the P22-R genome by the electron microscopic heteroduplex method. The results of this investigation are described in this paper. MATERIALS AND METHODSBacterial and phage strains. Nomenclature of drug resistance genes followed the recommendations of Novick et al (22). Salmonella typhimurium strains used were LT2 wild type, CC4415 (met, trp, val), DB47 (strs, recA) and 5J1868 (hisB).

Chloramphenicol transposons found in Salmonella naestved and Escherichia coli of domestic animal origin

“…20,1981 Tn9 (20). Other transposable Cmr genes and transposable multiple resistance genes with Cmr have been so far demonstrated on R plasmids originating from various bacteria, including Shigella (5,11,24), Salmonella ordonez (21), Haemophilus influenzae (13), and Pseudomonas aeruginosa (12,22 …”

“…ture were then plated on L broth agar supplemented with chloramphenicol (25 jg/ml) and kanamycin (25 pug/ml) and incubated at 370C for 24 h.…”