Identification and Characterization of the Dicarboxylate Uptake System DccT inCorynebacterium glutamicum

Abstract

Many bacteria can utilize C 4 -carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and L-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and L-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and L-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na ؉ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, L-malate, and likely also oxaloacetate in a sodium-dependent manner.Corynebacterium glutamicum is a predominantly aerobic nonsporulating and biotin-auxotrophic gram-positive bacterium which is used for the industrial production of amino acids, mainly of L-lysine (Ͼ750,000 tons/year) (61) and glutamate (Ͼ1,500,000 tons/year) (50). C. glutamicum is well studied not only with respect to amino acid biosynthesis but also regarding carbon metabolism and its regulation (2, 6, 57). C. glutamicum grows aerobically on a variety of carbohydrates and organic acids as the sole sources of carbon and energy, e.g., on sugars like glucose, fructose, and sucrose and on organic acids like gluconate, acetate, propionate, pyruvate, and L-lactate, but also on ethanol, glutamate, vanillate, 4-hydroxybenzoate, and protocatechuate (1, 7-9, 19, 31, 35, 59). In general, growth on substrate mixtures is characterized by coutilization, e.g., of glucose with acetate (59), and even a nongrowth substrate like serine can be utilized simultaneously with glucose (35). Rarely, preferential utilization of glucose before glutamate or ethanol, e.g., has been observed (1, 28).Citrate is the only tricarboxylic acid (TCA) cycle intermediate described as supporting the growth of C. glutamicum (42). C. glutamicum can grow on citrate as the sole carbon source and coutilizes citrate and glucose (42). In a combined DNA microarray and proteome analysis, it was revealed that the expression of genes for two citrate uptake systems, CitM and TctABC, was induced when citrate was present in the medium (42). Although succinate uptake by C. glutamicum has been observed in biochemical assays, growth on succinate has not been reported (11,41). Other bacteria, e.g., Bacillus subtilis, are able to grow not only with citrate but also with the dicarboxylic TCA cycle intermediates succinate, fumarate, and Lmalate (55). The transporter YdbH in B. subtilis is responsible for the uptake of fumarate and succinate (3) but not for that of L-malate, which is taken up via MaeN (52) or the malate/ lactate antipo...

Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403

“…The 2HCT family is believed to be distantly related to the divalent anion/Na ϩ symporter family (DASS) (TC classification 2.A.47) (15,24). The dicarboxylate transporters DccT of Corynebacterium glutamicum (31) and SdcL of Bacillus licheniformis (29) are found in the latter family and were shown previously to be competitively inhibited by oxaloacetate.…”

“…Transporters for oxaloacetate are rarely found in bacteria. The dicarboxylate transporters DccT of Corynebacterium glutamicum (31) and SdcL of Bacillus licheniformis (29) as well as the DctA transporters of C. glutamicum (32) and Bacillus subtilis (10) were shown to be competitively inhibited by oxaloacetate. In the present study, the physiological response of the oxaloacetatedeficient mutant to the potentially toxic accumulation of oxaloacetate in the cytoplasm is demonstrated.…”

Transcription of malP is subject to phosphotransferase system-dependent regulation in Corynebacterium glutamicum

“…Data represent mean values and SDs from three independent cultivations with three technical replicates. Uhde et al, 2013;Youn et al, 2008Youn et al, , 2009. No mutations were observed in sequence analyses of the malP promoter region, as well as of the malP gene itself, in DNA preparations from C. glutamicum HSM; thus, no evidence for cis-acting mutations was found.…”

A Na+-coupled C4-dicarboxylate transporter (Asuc_0304) and aerobic growth of Actinobacillus succinogenes on C4-dicarboxylates

“…7a). DccT of C. glutamicum catalysed the uptake of succinate, fumarate, L-malate and oxaloacetate in a Na + -dependent manner under aerobic conditions (Youn et al, 2008). SdcS of S. aureus is an electroneutral symporter for Na + and succinate (2 : 1 ratio), and the succinate uptake was inhibited competitively by succinate, fumarate, malate and presumably also oxaloacetate (Hall & Pajor, 2007).…”

“…More recently, transporters of the DASS (divalent anion : sodium symporter) family have also been shown to catalyse C 4 -dicarboxylate uptake by C 4 -dicarboxylate : Na + symport. Transporters of this type, which are different from the members of the CitT subgroup, are DccT of Corynebacterium glutamicum, SdcS of Staphylococcus aureus, SdcL of Bacillus licheniformis and VcINDY of Vibrio cholera (Hall & Pajor, 2007;Youn et al, 2008;Strickler et al, 2009;Mancusso et al, 2012). A. succinogenes contains 11 transporters (30 genes) of the DASS, TDT (tellurite resistance dicarboxylate transporter) and TRAP-T families that are candidates for aerobic C 4 -dicarboxylate transport (Table S1).…”