The gene encoding the alternative thymidylate synthase ThyX is regulated by sigma factor SigB in Corynebacterium glutamicum ATCC 13032

Both ThyA and ThyX proteins catalyze the transfer of the methyl group from methylenetetrahydrofolate (CH(2) H(4) -folate) to dUMP, forming dTMP. To estimate the relative steady state expression levels of ThyA and ThyX, Western blot analysis was performed using ThyA or ThyX antiserum on total protein from the wild-type, ΔthyX, and thyX-complemented strains of Corynebacterium glutamicum. The level of ThyA decreased gradually during the stationary growth phase but that of ThyX was maintained steadily. Whereas the expression level of ThyA in a ΔsigB strain was comparable to that of the wild-type, the level of ThyX was significantly diminished in the deletion mutant and was restored to that of the wild-type in the complemented strain, indicating that the level of ThyX was regulated by SigB. Growth of the C.?glutamicum ΔsigB strain was dependent upon coupling activity of dihydrofolate reductase (DHFR) with ThyA for the synthesis of thymidine, and thus showed sensitivity to the inhibition of DHFR by the experimental inhibitor, WR99210-HCl. These results suggested that the relative levels of ThyA and ThyX differ in response to different growth phases and that SigB is necessary for maintenance of the level of ThyX during transition into the stationary growth phase.     

Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product

By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa. A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably form transmembrane α-helices. A C. glutamicum mutant strain harboring a defined deletion of brnQ in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min^–1 mg (dry mass)^–1 as compared to the wild-type strain rate of 1.2 nmol min^–1 mg (dry mass)^–1. Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min^–1 mg (dry mass)^–1. Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake. Received: 16 October 1997 / Accepted: 27 November 1997     

Metabolic engineering of Corynebacterium glutamicum ATCC13032 to produce S-adenosyl-l-methionine

As an important biological methyl group donor, S-adenosyl-l-methionine is used as nutritional supplement or drug for various diseases, but bacterial strains that can efficiently produce S-adenosyl-l-methionine are not available. In this study, Corynebacterium glutamicum strain HW104 which can accumulate S-adenosyl-l-methionine was constructed from C. glutamicum ATCC13032 by deleting four genes thrB, metB, mcbR and Ncgl2640, and six genes metK, vgb, lysC^m, hom^m, metX and metY were overexpressed in HW104 in different combinations, forming strains HW104/pJYW-4-metK-vgb, HW104/pJYW-4-SAM2C-vgb, HW104/pJYW-4-metK-vgb-metYX, and HW104/pJYW-4-metK-vgb-metYX-hom^m-lysC^m. Fermentation experiments showed that HW104/pJYW-4-metK-vgb produced more S-adenosyl-l-methionine than other strains, and the yield achieved 196.7 mg/L (12.15 mg/g DCW) after 48 h. The results demonstrate the potential application of C. glutamicum for production of S-adenosyl-l-methionine without addition of l-methionine.     

Population Heterogeneity in Corynebacterium glutamicum ATCC 13032 caused by prophage CGP3

The genome of Corynebacterium glutamicum type strain ATCC 13032 (accession number BX927147) contains three prophages, CGP1, CGP2, and CGP3. We recently observed that many genes within the CGP3 prophage region have increased mRNA levels in a dtxR deletion mutant that lacks the master regulator of iron homeostasis (J. Wennerhold and M. Bott, J. Bacteriol. 188:2907-2918, 2006). Here, we provide evidence that this effect is due to the increased induction of the prophage CGP3 in the dtxR mutant, possibly triggered by DNA damage caused by elevated intracellular iron concentrations. Upon induction, the CGP3 prophage region is excised from the genome and forms a circular double-stranded DNA molecule. Using quantitative real-time PCR, an average copy number of about 0.1 per chromosome was determined for circular CGP3 DNA in wild-type C. glutamicum. This copy number increased about 15-fold in the dtxR mutant. In order to visualize the CGP3 DNA within single cells, a derivative of the wild type was constructed that contained an array of tet operators integrated within the CGP3 region and a plasmid-encoded YFP-TetR fusion protein. As expected, one to two fluorescent foci that represented the chromosomally integrated CGP3 prophage were detected in the majority of cells. However, in a small fraction (2 to 4%) of the population, 4 to 10 CGP3 DNA molecules could be observed in a single cell. Interestingly, the presence of many CGP3 copies in a cell often was accompanied by an efflux of chromosomal DNA, indicating the lysis of the corresponding cell. However, evidence for the formation of functional infective CGP3 phage particles could not be obtained.     

Detection of D-ornithine extracellularly produced by Corynebacterium glutamicum ATCC 13032::argF

We found that Corynebacterium glutamicum ATCC 13032::argF extracellularly produced a large amount of D-ornithine when cultivated in a CGXII medium containing 1 mM L-arginine. This is the first report that C. glutamicum ATCC 13032 or its mutant produces a D-amino acid extracellularly. C. glutamicum ATCC 13032::argF produced 13 mM D-ornithine in 45 h of cultivation.     

Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032

To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum.     

Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032

As a first step in determining the importance of the anaplerotic function of phosphoenolpyruvate carboxylase (PEPC) in amino acid biosynthesis, the ppc gene coding for PEPC of Corynebacterium glutamicum ATCC13032 has been cloned by complementation of an Escherichia coli ppc mutant strain. PEPC activity encoded by the cloned gene is not affected by acetyl-CoA under conditions where the E. coli enzyme is strongly activated, whereas acetyl-CoA is able to relieve inhibition by L-aspartate used singly or in combination with alpha-ketoglutarate. Amplification of the ppc gene in a C. glutamicum lysine-excreting strain resulted in increased PEPC-specific activity and lysine productivity. The nucleotide sequence of a DNA fragment of 4885 bp encompassing the ppc gene has been determined. At the amino acid level, PEPC from C. glutamicum presents overall a high degree of similarity with corresponding enzymes from three different organisms. The location of some strictly conserved regions may have important implications for PEPC activity and allostery.     

Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.     

A TatABC-Type Tat Translocase Is Required for Unimpaired Aerobic Growth of Corynebacterium glutamicum ATCC13032

The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Escherichia coli and other Gram-negative bacteria possess a TatABC-type Tat translocase in which each of the three inner membrane proteins TatA, TatB, and TatC performs a mechanistically distinct function. In contrast, low-GC Gram-positive bacteria, such as Bacillus subtilis, use a TatAC-type minimal Tat translocase in which the TatB function is carried out by a bifunctional TatA. In high-GC Gram-positive Actinobacteria, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, tatA, tatB, and tatC genes can be identified, suggesting that these organisms, just like E. coli, might use TatABC-type Tat translocases as well. However, since contrary to this view a previous study has suggested that C. glutamicum might in fact use a TatAC translocase with TatB only playing a minor role, we reexamined the requirement of TatB for Tat-dependent protein translocation in this microorganism. Under aerobic conditions, the misassembly of the Rieske iron-sulfur protein QcrA was identified as a major reason for the severe growth defect of Tat-defective C. glutamicum mutant strains. Furthermore, our results clearly show that TatB, besides TatA and TatC, is strictly required for unimpaired aerobic growth. In addition, TatB was also found to be essential for the secretion of a heterologous Tat-dependent model protein into the C. glutamicum culture supernatant. Together with our finding that expression of the C. glutamicum TatB in an E. coli ΔtatB mutant strain resulted in the formation of an active Tat translocase, our results clearly indicate that a TatABC translocase is used as the physiologically relevant functional unit for Tat-dependent protein translocation in C. glutamicum and, most likely, also in other TatB-containing Actinobacteria.     

Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032

Genome analysis of C. glutamicum ATCC 13032 has showed one putative adenylate cyclase gene, cyaB (cg0375) which encodes membrane protein belonging to class III adenylate cyclases. To characterize the function of cyaB, a deletion mutant was constructed, and the mutant showed decreased level of intracellular cyclic AMP compared to that of wild-type. Interestingly, the cyaB mutant displayed growth defect on acetate medium, and this effect was reversed by complementation with cyaB gene. Similarly, it showed growth defect on glucose-acetate mixture minimal medium, and the utilization of glucose was retarded in the presence of acetate. The deletion mutant retained the activity of glyoxylate bypass enzymes. Additionally, the mutant could grow on ethanol but not on propionate medium. The data obtained from this study suggests that adenylate cyclase plays an essential role in the acetate metabolism of C. glutamicum, even though detailed regulatory mechanisms involving cAMP are not yet clearly defined. The observation that glyoxylate bypass enzymes are derepressed in cyaB mutant indicates the involvement of cAMP in the repression of aceB and aceA.