PHYSICAL-GENETIC MAP OF THE ERYTHROMYCIN-PRODUCING ORGANISM SACCHAROPOLYSPORA-ERYTHRAEA

A physical map of the chromosome of the erythromycin-producing actinom ycete Saccharopolyspora erythraea NRRL 2338 has been constructed using the restriction enzymes AseI and DraI. The map was constructed by a v ariety of methods including linking clone analysis, cross-hybridizatio ns using labelled macrorestriction fragments, gene probing, two-dimens ional PFGE and restriction enzyme site generation. Analysis of the ind ividual macrorestriction patterns of the 17 AseI-, 6 DraI- and 22 AseI /DraI-digested fragments indicated a chromosome size of about 8 Mb. Li nking clones for five contiguous Asel fragments were obtained, coverin g 32% of the chromosome. The linkage of an additional eight AseI fragm ents was aided by the finding that the rRNA operons of S. erythraea co ntain an AseI site within the 16S (rrs) gene. Generation of 5. erythra ea strains that contain additional DraI sites within selected AseI fra gments, followed by PFGE analysis and Southern hybridization to determ ine specific linkages, facilitated the completion of the AseI map. The entire DraI map was constructed by gene probing and cross-hybridizati ons. PFGE analysis of agarose-embedded DNA prepared in either the pres ence or absence of proteinase K suggested that the 5. erythraea NRRL 2 338 chromosome is linear. A total of 15 genes or gene clusters were ma pped to specific AseI and DraI fragments, including the erythromycin-b iosynthetic gene cluster and the rRNA operons.