Coryneform bacteria are rod-shaped, nonsporulating, grampositive bacteria with a high GC content. The nonpathogenic species, Corynebacterium glutamicum, Brevibacterium lactofermentum, and Brevibacterium flavum, are used for the fermentative production of nucleotides and amino acids, especially L-glutamate. Recently, B. lactofermentum and B. flavum have been reclassified as C. glutamicum (22), and the genome of C. glutamicum ATCC 13032 has been sequenced (12, 30). C. glutamicum was originally isolated as an L-glutamate-producing bacterium (20,36), although the wild type does not overproduce L-glutamate under ordinary culture conditions. Significant L-glutamate production is induced by incubating the biotin-auxotrophic wild type in a biotin-limited medium (32). Although L-glutamate overproduction is suppressed in the presence of an excess of biotin, the addition of certain fatty acid ester surfactants (7, 35) or penicillin (27) to the medium also induces L-glutamate overproduction. For example, as much as 220 mM L-glutamate is produced from 360 mM glucose by wild-type C. glutamicum cells under the L-glutamateproducing condition of adding 1 g of Tween 40 liter Ϫ1 , whereas only 22 mM L-glutamate is produced without Tween 40 addition (Table 1). This means that at least 10-fold more L-glutamate is metabolically synthesized under L-glutamate-producing conditions than under non-L-glutamate-producing conditions. From this perspective, metabolic flux change is expected to be crucial for L-glutamate overproduction in C. glutamicum.The molecular basis of the induction of L-glutamate overproduction remains unclear. One widely held view is that Lglutamate efflux is caused by an alteration in the chemical or physical characteristics of the cell membrane; this is because several changes in the bacterial membrane have been observed under L-glutamate-producing conditions, including a decrease in lipid content, a change in the amount of phospholipid (35), and altered fatty acid composition (31). Recently, alterations of the chemical and physical properties of the cytoplasmic membrane caused by biotin limitation and by genetic modification of lipid biosynthesis were shown to be necessary, but not sufficient, to achieve high L-glutamate efflux (11,25).Changes in metabolic flux have not been discussed as often as the basis of L-glutamate overproduction, although there have been suggestions that this process is important. The 2-oxoglutarate dehydrogenase complex (ODHC) catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A (succinyl-CoA), and glutamate dehydrogenase (GDH) catalyzes ammonia assimilation of 2-oxoglutarate to form Lglutamate (Fig. 1). These two enzymes compete for 2-oxoglutarate at the branch point of the tricarboxylic acid (TCA) cycle and L-glutamate biosynthesis. It has been reported that ODHC activities are decreased under L-glutamate-producing conditions (14, 33). We and others previously compared ODHC and GDH activities and found a reduction in ODHC activity relative to GDH activity during...

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The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs. The characteristic enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to exist outside methylotrophs. However, the presumedyckG gene product (YckG) of Bacillus subtilisshows a primary structure similar to that of methylotroph HPS (F. Kunst et al., Nature 390:249–256, 1997). We have also investigated the sequence similarity between the yckF gene product (YckF) and methylotroph PHI (Y. Sakai, R. Mitsui, Y. Katayama, H. Yanase, and N. Kato, FEMS Microbiol. Lett. 176:125–130, 1999) and found that the yckG and yckF genes of B. subtilis express enzymatic activities of HPS and PHI, respectively. Both of these activities were concomitantly induced inB. subtilis by formaldehyde, with induction showing dependence on the yckH gene, but were not induced by methanol, formate, or methylamine. Disruption of either gene caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system for formaldehyde in B. subtilis. In conclusion, we found an active yckG (for HPS)-yckF (for PHI) gene structure (now namedhxlA-hxlB) in a nonmethylotroph, B. subtilis, which inherently preserves the RuMP pathway.