STREPTOMYCES AKIYOSHIENSIS DIFFERS FROM OTHER GRAM-POSITIVE BACTERIA IN THE ORGANIZATION OF A CORE BIOSYNTHETIC-PATHWAY GENE FOR ASPARTATE FAMILY AMINO-ACIDS

A partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E, coil CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenas e (Asd). One of 39 000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain con ferred prototrophy when used to transform 5, coil CGSC 6212, The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleo tide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd(-)-complementing plasmid pJV36 loc ated three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (M(r) 3548 4), was identified as the gene for Asd by comparing its sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coil CGS C 6212 to prototrophy supported this assignment. Southern hybridizatio n indicated that the sequenced region of the cloned DNA fragment repre sented a continuous segment of the S. akiyoshiensis chromosome. The de duced amino acid sequences of the ORFs adjacent to asd showed no simil arity to sequences for aspartate kinase (Ask); also, transformation wi th plasmids containing asd and adjacent regions from the S. akiyoshien sis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in S. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.