coelicolor, we constructed a cosmid library using a vector, pHAQ31, containing two cos sites, oriT, multiple cloning sites and Streptomyces selection markers tsr/melC (Xia et al., 2009). The insertion sequences of c. 2000 cosmids were determined to construct an ordered cosmid library, which covered 98.5% of the S. coelicolor genome. To determine the lengths to be deleted at the left subtelomeric region of the linear chromosome, for example, two large segments (e.g. 8.7 and 5.2 kb) cut from different cosmids and a kan gene were cloned in pHAQ31.
The resulting plasmid, pFX175, was introduced by conjugation from E. coli GW-572016 nmr into S. coelicolor M145, and thiostrepton-resistant colonies were obtained on MS medium containing thiostrepton. After streaked on MS medium for sporulation, three colonies showed thiostrepton-sensitive and kanamycin-resistant NVP-BGJ398 in vivo phenotypes among 150 screened colonies and indicated the occurrence of intramolecular double crossing over to delete the tsr marker. The deletion and
replacement of a large segment with the kan gene was verified by PCR analysis. Thus, a c. 137-kb segment (65 492–202 631 bp) at the left subtelomere was deleted (designated strain FX16, Fig. 1). Similarly, we constructed plasmids pFX176, pFX219, pFX218, and pFX183 and obtained thiostrepton-sensitive and kanamycin-resistant colonies for pFX176 and pFX219 (yielding strains designated FX17 and FX18, respectively), but failed to obtain such colonies for pFX218 and pFX183 even by screening 200 clones (Fig. 1). Aspartate Thus, a c. 900-kb sequence (65 492–965 740 bp) at the left subtelomeric region was shown to be deletable.
Similarly, four plasmids (pJXY3, pJXY5, pJXY6, and pJXY7) were constructed for the deletion of the right subtelomeric region of the linear chromosome. Thiostrepton-sensitive and kanamycin-resistant colonies were obtained for pJXY3 and pJXY5 (yielding strains designated JXY3 and JXY5, respectively), but we failed to obtain such colonies for pJXY6 and pJXY7 after screening up to 270 clones (Fig. 1). Thus, a c. 313-kb sequence (8 105 685–8 418 406 bp) at the right subtelomeric region was shown to be deletable. We also constructed plasmids (pFX153, pFX171, pFX172, pFX179, pFX186, and pFX180) for circularization of the linear chromosome. As shown in Fig. 1, a c. 1600-kb region [FX15, c. 840-kb (1–840 417 bp) for the left arm of the linear chromosome and a c. 761-kb (7 906 368–8 667 507 bp) for the right arm], including both the subtelomeric and telomere sequences, could be deleted, suggesting that by screening more clones for double crossover (although c. 270 clones screened for pXJY6, see Fig. 1), linear chromosome containing deletion of c. 761-kb sequence at the right subtelomeric region should be obtained. These results confirmed the deletable length (900 kb) on the left arm and indicated that more sequence (761 vs. 313 kb) at the right arm of the linear chromosome could be removed.