Physical Map Of Streptomyces Hygroscopicus 10-22 And The Localization And Cloning Trails For Genes Involved In Antibiotic 5101-Ⅳ Production

Streptomyces species has a linear chromosome with a size of c.8Mb, which is almost one and half times that of E. coli, and terminal proteins at its ends. Genome mapping and structure of Streptomyces hygroscopicus 10-22 is the prime interest of the present study. PFG1E (Pulsed Field Gel Electrophoresis) revealed a linear chromosome with a size of 7.35Mb. and 11 fragments could be generated with Asel digestion. No obvious difference couM be observed with SDS-treated and PK-treated 10-22 samples, which seems to be opposite of 5. coelicolor A3(2) and S. lividans 1326 (which were known to possess terminal proteins and thus will retard in the slot during PFGE), indicating that the terminal structure of 10-22 might be different from the ones described so far in Streptomyces, whose exact property still remains unknown.Oae of the common features of Streptomyces is its instability, which was manifested by frequently occurring chromosomal deletions, as is the same case with 10-22. In 10-22, the unstable region encompasses the 170-, 210-A, 210-B, 280-, 300-, 500-, 680-, 900- and 1300-A Asel fragments. By the analysis of the restriction banding patterns of 10-22 and its variant mutants N-103, T157, Q303, N-1011, T106, Q1513, T155, WH-14, WH-27, etc, the above mentioned fragments could be located on chromosome and thus make it possible to draw an almost complete chromosomal physical map of 10-22, though stil! circuilar. The attempt of detecting the terminal fragments was failed as there is no difference observed with SDS and PK treated samples digested with Asel.Normally, the changes of phenotype are closely related to the genome instabilities. The sarnss phenomenon could also be observed in S. hygroscopicus 10-22. Notable changes in spotre color, pigments production and antibiotics biosynthesis have been observed in 10-22 derivatives in which large chromosomal deletions had occurred. 30 blocked strains for 5102-IV antibiotic production were obtained in a screening project. PFGE analysis of these 30 blocked strains revealed a common chromosomal deletion in the 300-kb Asel fragment which might be responsible for 5102-IV antibiotic biosynthesis. So this 300kb fragment was recovered and used as probe to hybridize with 10-22 genomic library. Cosmids within this region were aligned and suitable fragments in this region were selected and used further for the construction of plasmids for gene replacement. At last, 16 mespmcement plasmids for two different parts were obtained and have been introduced intou 1022. but final results had not obtained yet.