Mutations in the Corynebacterium glutamicum Proline Biosynthetic Pathway: a Natural Bypass of the proA Step

Two chromosomal loci containing the Corynebacterium glutamicum ATCC 17965 proB and proC genes were isolated by complementation of Escherichia coli proB and proC auxotrophic mutants. Together with a proA gene described earlier, these new genes describe the major C. glutamicum proline biosynthetic pathway. The proB and proA genes, closely linked in most bacteria, are in C. glutamicum separated by a 304-amino-acid open reading frame (unk) whose predicted sequence resembles that of the 2-hydroxy acid dehydrogenases. C. glutamicum mutants that carry null alleles of proB, proA, and proC were constructed or isolated from mu-tagenized cultures. Single proC mutants are auxotrophic for proline and secrete D1-pyrroline-5-carboxylate, which are the expected phenotypes of bacterial proC mutants. However, the phenotypes of proB and proA mutants are unexpected. A proB mutant has a pleiotropic phenotype, being both proline auxotrophic and affected in cell morphology. Null proA alleles still grow slowly under proline starvation, which suggests that a proA-independent bypass of this metabolic step exists in C. glutamicum. Since proA mutants are complemented by a plasmid that contains the wild-type asd gene of C. glutamicum, the asd gene may play a role in this bypass. Exposure of bacteria from the genus Corynebacterium (8) to hyperosmotic stress is correlated with biosynthesis and accu-mulation of some endogenous substrates, also termed compat