367 Gene, 34 (1985) 367-370 Elsevier GENE 1236 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Clonipg of a multicopy plasmid from the actinorhizal nitrogen-ftxing bacterium hzakia sp.and determination of its restriction map (Recombinant DNA; Streptomyces; symbiosis; Actinomycetes) Philippe Normand, J. Allan Downie *, Andrew W.B. Johnston *, Tobias Kieser * and Maurice Lalonde D$artement des Sciences Forestit+es, Vniversitt? Laval, Q&bee, PQ (Canada GlK 7P4) Tel. (418) 656-5944, and *JohnInnes Institute, Colney Lane, Norwich NR4 7VH (U.K.) Tel. (603) 5.2571 (Received November lSth, 1984) (Revision received and accepted December 12th, 1984) SUMMARY An 8.3-kb multicopy plasmid, pFQ31, from the nitrogen-fixing Frankia sp. strain ArI3, was cloned into Escherichia coli plasmid vectors and analysed physically. pFQ31 has no detectable sequence homology with another Frankia plasmid, pFQ32, which is present in the same host. Derivatives of pFQ31 with an ant&&c resistance marker were introduced into ~~~tornyces ~jvi~ans, which is taxonomically related to Frankia, but no stable replication could be achieved. INTRODUCTION The actinomycete Frankia lives in symbiosis with certain trees, inducing nitrogen-f&g root nodules, which enables the host plants to thrive on poor soils (Lechevalier, 1983). All Frankia strains grow very slowly with generation times of at least two days, making direct analysis very tedious. The cloning of Frankia genes and their expression in heterologous hosts may be a quicker route towards the under- standing of the natural biology and genetics of these economically important bacteria. Several plasmids which might be useful for the development of cloning vectors have been identified in Frankia strains (Nor- mand et al., 1983; Simonet et al., 1984). We describe Abbreviations: Ap, ampicillin; bp, base pairs; EtBr, ethidium bromide; kb, kilobase pairs; Km, kanamycin; R, resistance; Ts, thiostrepton; tsr, tbiostrepton resistance gene. 0378-l 119/85~$03.30 0 1985 Elsevier Science Publishers here the cloning and physical analysis of such a plasmid, called pFQ31. EXPERIMENTAL (a) Physical cbaracterisation of pFQ31 Frankia sp. strain ArI3, previously isolated from root nodules of A fnus ra&ra (Berry and Torrey, 1979) contains two plasmids, pFQ31 (8.3 kb) and pFQ32 (18.3 kb) (Normand et al., 1983). To estimate their copy number, plasmids were isolated (Normand et al., 1983) and separated on agarose gels, and the DNA was visual&d by staining with EtBr. By scanning the peak areas with a laser densitometer, the copy numbers of pFQ31 and pFQ32 were esti- mated to be 200 and 50 per chromosome, respec-