General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum.

We utilized diaminopimelate-lysine mutants of Escherichia coli K12 to clone the genes specifically involved in the Corynebacterium glutamicum diaminopimelate-lysine anabolic pathway. From a cosmid genomic bank of C. glutamicum strain AS019, we isolated cosmids pSM71, pSM61 and pSM531, that are respectively able to complement dapA/dapB, dapD, and lysA mutants of E. coli. DNA hybridization analysis indicates that these complementing genes are located on the chromosome of C. glutamicum in at least three separate transcription units. Subcloning of parental cosmids in dapA, dapD, and lysA mutants of E. coli localized these genes, respectively, within 1.4, 3.4, and 1.8 kb fragments, cloned in an E. coli/C. glutamicum shuttle vector. Enzymatic analysis in C. glutamicum identified the dapA-complementing gene as L-2,3-dihydrodipicolinate synthetase (dapA), and the lysA-complementing gene as meso-diaminopimelate decarboxylase (lysA). In contrast, complementation of E. coli dapD8, presumably lacking L-delta 1-tetrahydrodipicolinate synthetase (dapD), led us to clone a diaminopimelate-lysine anabolic gene of C. glutamicum which does not exist in E. coli: meso-diaminopimelate dehydrogenase. Although meso-diaminopimelate is crucial in lysine formation and in cell wall biosynthesis, expression of the genomic copies of the cloned genes, which encode activities involved at key branching points of the diaminopimelate-lysine pathway of C. glutamicum, appears constitutive with regard to the addition of diaminopimelate and/or lysine during cell growth.