A partial 3Al digest of genomic DNA from was cloned in a shuttle vector, and the recombinant plasmids were used to transform CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd -complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids 35484), was identified as the gene for Asd by comparing its sequence with database sequences of from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the chromosome. The deduced amino acid sequences of the ORFs adjacent to showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing and adjacent regions from the chromosome did not complement the mutant CGSC 5074. It is concluded that and in are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.


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