Nucleic Acids Research Completion of the nucleotide sequence of the central region of Ta5 confirms the presence of three resistance genes

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5_ up to the right inverted repeat TS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes : the kanamycin-neomycin resistance gene, a gene coding for resistance to CT.990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in J!. coli. A Tn5* mutant able to express streptomycin resistance in Ĵ . coli was isolated. With this mutant, it was demonstrated that in 15. coli the expression of the three resistance genes is coordinated in a single operon. INTRODUCTION Transposon Tn5 (recently reviewed by Berg and Berg(l)) was originally isolated as a kanamycin-neomycin resistance determinant present in a Klebslella resistance factor (2). It is composed of a central region of 2.7 kilobase pairs (Kb) flanked by two inverted repeats of 1.5 kb, the insertion sequences IS50, denoted respectively IS50L (left) and IS50R (right). IS50R codes for two proteins translated from a single reading frame. One of these is a transposase, the other an inhibitor of transposition (3,4). IS50L differs by one base pair from IS50R. This single change has two consequences: (i) it produces an ochre mutation that truncates both proteins which then, become inactive; (ii) it creates a good promoter P for the kanamycin resistance gene located in the adjacent region of the central part of Tn_5 (5,6). Only the kanamycin resistance function was known for Tn5 until 1981 when a streptomycin resistance unexpressed in j!. coli was evidenced in Methylobacterium organophilum (7) and later confirmed in Rhizobium (8) and in other non-enteric bacteria (9). The streptomycin resistance gene was located in a 1000 bp segment of the central region, 300 bp downstream from the kanamycin resistance gene (10). The direction of transcription of © IRL Press Limited, Oxford, England. 195 Downloaded from https://academic.oup.com/nar/article-abstract/13/1/195/2385243 by guest on 25 January 2018 Nucleic Acids Research kanamycin and streptomycin resistance genes was shown to be identical (8,10,11). In addition, two different aminoglycoside phosphotransferase activities were separated in crude extracts of M. organophilum (Tn5) (10). One corresponded to the aminoglycoside phosphotransferase (3') type II (APH(3')II) the only aminoglycoside modifying enzyme expressed by Tn5_ in Ĵ. coli. The other was a streptomycin phosphotransferase (SPH). The two activities almost coeluted from a gel filtration column, the SPH being slightly retarded. Recently G. Tiraby (Universite Paul Sabatier, Toulouse, France) showed that Tn!̂ codes also for a resistance against a glycopeptide of the bleomycin family, called compound CL990 (to be patented). This new resistance gene, which is expressed in J5. coli, was located in a 400 bp DNA segment immediately downstream from the kanamycin resistance gene (G. Tiraby, personal communication). Independently, Genilloud et^ a\_ found that Tn^ encoded bleomycin resistance in the aforementioned region (12). DNA sequence of the IS50s was achieved by Auerswald et^ a_l̂ (13). In the central region, the kanamycin resistance gene and 360 nucleotides downstream from it were sequenced by Beck ej^ a\_ (14). The completion of the sequence of the central region presented in this paper provides information on the precise location of the resistance genes mentioned above. Moreover, their expression is demonstrated to be organized in a single operon with the aid of a Tn5_* mutant able to express streptomycin resistance in Ê . coli. MATERIALS AND METHODS Source of DNA : Recombinant plasmids pPMlll, pPM116 and pPM122, previously constructed to localize the streptomycin resistance gene in Tn^ (10), were used as sources of DNA. They contained fragments of Tn5̂ cloned into plasmid pBR322. The initial source of Tn_5 was plasmid pRZ102 (15). Plasmids were purified from E. coli HB101 (16) and IE. coli FC1 (10). Purification of plasmid DNA : Plasmid DNA, extracted by the clear lysate method, was purified on cesium chloride gradients as described by Humphreys et al. (17). Nucleotide sequence determination : Fragments were 3'-end-labeled with {a-P} dXTP using DNA polymerase (large fragment) (18). Restriction fragments were separated on thin polyacrylamide gels at 6% or 8% and then eluted overnight by diffusion