(44). On the basis of this assay, the parallel function of the succinylase and acetylase variants supposed to operate in corynebacteria (16,44). The situation is even more complex, since the distribution of label from 14C-aspartate (23) and '3C-glucose (14,49) found in secreted * Corresponding author. lysine is inconsistent with the sole function of the succinylase or acetylase variant but requires operation of the dehydrogenase pathway, too. This resulted in the demonstration of diaminopimelate dehydrogenase activity in C. glutamicum, and it was concluded that all three variants could be used in parallel in this organism (16).Recently, the genes converting aspartate to lysine via the dehydrogenase variant have been cloned from C. glutamicum (7,14,18,51). During this cloning, a dapD mutant of E. coli devoid of PDC-N-succinyltransferase activity was also used (15,50

We have developed a system for the genetic manipulation of the amino acid-producing Corynebacterium glutamicum. Gene disruption and replacement were achieved by introducing, via conjugation, Escherichia coli vector plasmids carrying manipulated C. glutamicum DNA fragments. We obtained stable mutants in which the chromosomal lysA gene, encoding meso-diaminopimelate decarboxylase, was interrupted by a chloramphenicol resistance cartridge, or in which an essential internal part of the lysA gene was deleted. The deletion mutants retain neither antibiotic resistance markers nor vector sequences. This strategy is generally applicable to the construction of industrial strains to be used in fermentation processes.

RP4-mediated transfer of mobilizable plasmids in intergeneric conjugation of Escherichia coli donors with Corynebacterium glutamicum ATCC 13032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid transfer that permitted the distinction between restriction-negative and restriction-positive C. gludamicum clones was developed. By using this procedure, clones of the restrictiondeficient mutant strain C. glutamicum RM3 harboring a plasmid library of the wild-type chromosome were checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a plasmid with a 7-kb insertion originating from the wild-type chromosome. This plasmid, termed pRES806, is able to complement the restriction-deficient phenotype of different C. glutamicum mutants. Sequence analysis revealed the presence of two open reading frames (orfl and orfj) on the complementing DNA fragment. The region comprising orfi and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orfi is essential for complementation, but inactivation of o42 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophage CL31 from Corynebacterum lilum ATCC 15990.

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B. lactofermentum ATCC 13869. The hom- and thrB-disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine.

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