Physical and Genetic Characterization of Chromosomal Copies of the Streptomyces coelicolor Mini-circle

John Innes Institute and AFRC Institute of Plant Science Research, Norwich NR4 7UH, UK ABSTRACT SUMMARY: The two linear, integrated mini-circle copies of Streptomyces coelicolor A3(2) were cloned in Escherichia coli and their positions on the S. coelicolor genetic map were determined. Mini-circle copy A is close to the argA locus , 20 kb upstream of the gyl operon. Mini-circle copy B is close to the cysD locus and is absent from S. coelicolor J1501, which has suffered a chromosomal deletion including mini-circle copy B and possibly associated with the pgl mutation. The mini-circle copies were not involved in any of the several previously identified physical interactions between the plasmid SCP1 and the S. coelicolor chromosome. A new insertion sequence was identified close to the right end of mini-circle copy B in S. coelicolor M145. The free mini-circle of S. coelicolor , when inserted into an attP -deleted derivative of phage °C31, actively integrated this phage into the chromosomes of S. coelicolor and S. lividans at preferred and secondary sites. The resulting prophages were stably inherited and remained physically intact. No precise excision of prophages from S. lividans lysogens carrying insertions at preferred or secondary integration sites was detected: instead, free phages were generated by imprecise excisions. These phages allowed the in vivo cloning of segments (> 3 kb in length) of the chromosomal DNA flanking preferred and secondary integration sites. Attempts to delete the preferred integration site by double homologous recombination with a clone carrying flanking DNA sequences and an antibiotic resistance gene were unsuccessful. Present address: Institute of Plant Science Research, Cambridge Laboratory, Maris Lane, Cambridge CB2 2JB, UK. Present address: Department of Botany, University of Cambridge, Cambridge, UK.